research papers\(\def\hfill{\hskip 5em}\def\hfil{\hskip 3em}\def\eqno#1{\hfil {#1}}\)

IUCrJ
Volume 6| Part 4| July 2019| Pages 649-664
ISSN: 2052-2525

Conservation of the structure and function of bacterial tryptophan synthases

aCenter for Structural Genomics of Infectious Diseases, University of Chicago, Chicago, IL 60367, USA, bStructural Biology Center, Biosciences Division, Argonne National Laboratory, Argonne, IL 60439, USA, cMidwest Center for Structural Genomics, Biosciences Division, Argonne National Laboratory, Argonne, IL 60439, USA, dBroad Institute of MIT and Harvard, Cambridge, MA 02141, USA, and eDepartment of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60367, USA
*Correspondence e-mail: andrzejj@anl.gov

Edited by Z.-J. Liu, Chinese Academy of Sciences, China (Received 25 January 2019; accepted 29 April 2019; online 29 May 2019)

Tryptophan biosynthesis is one of the most characterized processes in bacteria, in which the enzymes from Salmonella typhimurium and Escherichia coli serve as model systems. Tryptophan synthase (TrpAB) catalyzes the final two steps of tryptophan biosynthesis in plants, fungi and bacteria. This pyridoxal 5′-phosphate (PLP)-dependent enzyme consists of two protein chains, α (TrpA) and β (TrpB), functioning as a linear αββα heterotetrameric complex containing two TrpAB units. The reaction has a complicated, multistep mechanism resulting in the β-replacement of the hydroxyl group of L-serine with an indole moiety. Recent studies have shown that functional TrpAB is required for the survival of pathogenic bacteria in macrophages and for evading host defense. Therefore, TrpAB is a promising target for drug discovery, as its orthologs include enzymes from the important human pathogens Streptococcus pneumoniae, Legionella pneumophila and Francisella tularensis, the causative agents of pneumonia, legionnaires' disease and tularemia, respectively. However, specific biochemical and structural properties of the TrpABs from these organisms have not been investigated. To fill the important phylogenetic gaps in the understanding of TrpABs and to uncover unique features of TrpAB orthologs to spearhead future drug-discovery efforts, the TrpABs from L. pneumophila, F. tularensis and S. pneumoniae have been characterized. In addition to kinetic properties and inhibitor-sensitivity data, structural information gathered using X-ray crystallo­graphy is presented. The enzymes show remarkable structural conservation, but at the same time display local differences in both their catalytic and allosteric sites that may be responsible for the observed differences in catalysis and inhibitor binding. This functional dissimilarity may be exploited in the design of species-specific enzyme inhibitors.

1. Introduction

Tryptophan synthase (TrpAB) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that participates in the final two steps of tryptophan synthesis in plants, fungi and bacteria (reviewed in Dunn, 2012[Dunn, M. F. (2012). Arch. Biochem. Biophys. 519, 154-166.]; Raboni et al., 2003[Raboni, S., Pioselli, B., Bettati, S. & Mozzarelli, A. (2003). Biochim. Biophys. Acta, 1647, 157-160.], 2009[Raboni, S., Bettati, S. & Mozzarelli, A. (2009). Cell. Mol. Life Sci. 66, 2391-2403.]; Dunn et al., 2008[Dunn, M. F., Niks, D., Ngo, H., Barends, T. R. M. & Schlichting, I. (2008). Trends Biochem. Sci. 33, 254-264.]). The enzyme consists of two protein chains, α (TrpA) and β (TrpB) (Crawford & Yanofsky, 1958[Crawford, I. P. & Yanofsky, C. (1958). Proc. Natl Acad. Sci. USA, 44, 1161-1170.]), that operate as a linear αββα heterotetrameric complex containing two functional TrpAB units (Fig. 1[link]). In bacteria, TrpA and TrpB are encoded by usually adjacent trpA and trpB genes that belong to the highly regulated tryptophan-biosynthesis operon (reviewed in Merino et al., 2008[Merino, E., Jensen, R. A. & Yanofsky, C. (2008). Curr. Opin. Microbiol. 11, 78-86.]). The TrpA subunit converts indole-3-glycerol phosphate (IGP) into glyceraldehyde 3-phosphate (G3P) and indole (IND) (Fig. 2[link]). Subsequently, the latter product is utilized by TrpB, where it reacts with the L-serine (L-Ser) substrate to generate L-tryptophan (L-Trp). The reaction has a complicated, multistep mechanism involving enzyme–cofactor and substrate covalent adducts and results in the β-replacement of the hydroxyl group of L-Ser with the indole moiety (Fig. 2[link]) (reviewed in Raboni et al., 2009[Raboni, S., Bettati, S. & Mozzarelli, A. (2009). Cell. Mol. Life Sci. 66, 2391-2403.]).

[Figure 1]
Figure 1
Overall structure of the tryptophan synthase αββα heterotetramer from S. pneumoniae. TrpA is shown in yellow and TrpB is shown in cyan, with the COMM domain shown in orange and the PLP cofactor depicted in a sphere representation.
[Figure 2]
Figure 2
Enzymatic reaction of TrpAB. For TrpB, intermediate steps are shown (EA,int, internal aldimine; EGD1, geminal diamine; EA,ex1, external aldimine; EQ1, quinonoid; EAA, aminoacrylate; EQ2, quinonoid; EA,ex2, external aldimine; EGD2, geminal diamine).

As originally shown for TrpAB from the Gram-negative Salmonella typhimurium (StTrpAB), TrpA adopts a canonical (β/α)8-barrel fold (also known as a TIM barrel) with numerous additional elements (Hyde et al., 1988[Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W. & Davies, D. R. (1988). J. Biol. Chem. 263, 17857-17871.]; Figs. 1[link] and 3[link]). The active site is located at the top of the central β-barrel, with two acidic residues involved in catalysis: StGlu49 belonging to the αS2 strand and StAsp60 originating from loop αL2. Another structural element, loop αL6, serves as a lid closing over the binding pocket. TrpB represents a type II PLP-dependent enzyme with two domains, the N- and C-terminal domains, with the active site located in a cleft between them and carrying the covalently attached PLP cofactor. The N-terminal domain encompasses the so-called communication domain (COMM) that plays a key role in coordinating the activity of the two active sites (Schneider et al., 1998[Schneider, T. R., Gerhardt, E., Lee, M., Liang, P. H., Anderson, K. S. & Schlichting, I. (1998). Biochemistry, 37, 5394-5406.]). In the tetrameric arrangement, the TrpA and TrpB catalytic sites of the adjoining subunits are connected by a 25 Å long hydrophobic channel that facilities indole transport from TrpA to TrpB.

[Figure 3]
Figure 3
Sequence alignment of TrpA (top) and TrpB (bottom). Sequences are shown for S. pneumoniae, F. tularensis, L. pneumophila Philadelphia, M. tuberculosis and S. typhimurium. The depicted secondary-structure elements are derived from the SpTrpAB structure.

The TrpA- and TrpB-catalyzed chemical transformations are highly controlled by allosteric effects and other factors, for instance the binding of monovalent cations to TrpB, linked to substrate channeling. These molecular measures, together with other bacterial regulatory mechanisms (Merino et al., 2008[Merino, E., Jensen, R. A. & Yanofsky, C. (2008). Curr. Opin. Microbiol. 11, 78-86.]), are in place to ensure that the cellular resources are efficiently utilized to produce L-Trp, which is a scarce and most energetically expensive amino acid to biosynthesize (Akashi & Gojobori, 2002[Akashi, H. & Gojobori, T. (2002). Proc. Natl Acad. Sci. USA, 99, 3695-3700.]). The well documented ligand-induced reciprocal communication between subunits leading to the mutual activation involves conformational rearrangements. During the catalytic process, both TrpA and TrpB cycle between a low-activity open conformation (αO or βO) and a high-activity closed state (αC or βC) (Dunn, 2012[Dunn, M. F. (2012). Arch. Biochem. Biophys. 519, 154-166.]), depending on the reaction state. The formation of the amino­acrylate Schiff-base intermediate, EAA, from L-Ser and PLP in TrpB triggers movement of the TrpB COMM domain towards a closed state (βC), which subsequently activates TrpA by closure of the αL6 loop (αC). In a reciprocal process, IGP substrate binding to TrpA promotes an αC state, which in turn activates TrpB (βC). The two protein chains convert back to their open states when the L-Trp external aldimine, EA,ex2, is produced.

The availability of L-Trp, either supplied by the environment or synthesized in cellulo, is a prerequisite for bacterial survival. Some species rely heavily on external sources and maintain either no or only limited functionality of the L-Trp operon, while others preserve the complete system for de novo biosynthesis. The absence of the L-Trp biosynthetic pathway in animals and humans makes it a potentially attractive drug target for the treatment of bacterial diseases, even though the enzymes involved are only essential under certain conditions; that is, when exogenous L-Trp becomes depleted. Recent studies exploring these avenues showed that anthranilate syn­thase component I, TrpE (Zhang et al., 2013[Zhang, Y. J., Reddy, M. C., Ioerger, T. R., Rothchild, A. C., Dartois, V., Schuster, B. M., Trauner, A., Wallis, D., Galaviz, S., Huttenhower, C., Sacchettini, J. C., Behar, S. M. & Rubin, E. J. (2013). Cell, 155, 1296-1308.]), as well as functional tryptophan synthase are required for the survival of Mycobacterium tuberculosis in macrophage and mouse infection models, when an adaptive immune response triggers the expression of host indoleamine 2,3-dioxygenase (IDO-1), an enzyme responsible for L-Trp breakdown, or possibly even before this defense mechanism is mounted (Wellington et al., 2017[Wellington, S., Nag, P. P., Michalska, K., Johnston, S. E., Jedrzejczak, R. P., Kaushik, V. K., Clatworthy, A. E., Siddiqi, N., McCarren, P., Bajrami, B., Maltseva, N. I., Combs, S., Fisher, S. L., Joachimiak, A., Schreiber, S. L. & Hung, D. T. (2017). Nature Chem. Biol. 13, 943-950.]). Similar mechanisms inducing L-Trp starvation also function in lung-specific mouse infections with Streptococcus pneumoniae and Francisella tularensis, which are Gram-positive and Gram-negative bacteria, respectively. Under such conditions, the latter organism also requires TrpAB for growth (Peng & Monack, 2010[Peng, K. & Monack, D. M. (2010). Infect. Immun. 78, 2723-2733.]). Other pathogens that utilize tryptophan biosynthesis to evade host defenses or even to highjack it for their own purposes include urogenital serovars of Chlamydia trachomatis (a Gram-negative obligate intracellular parasite), which employ a partly dysfunctional TrpAB to produce L-Trp from external sources of indole provided by coexisting bacteria (Caldwell et al., 2003[Caldwell, H. D., Wood, H., Crane, D., Bailey, R., Jones, R. B., Mabey, D., Maclean, I., Mohammed, Z., Peeling, R., Roshick, C., Schachter, J., Solomon, A. W., Stamm, W. E., Suchland, R. J., Taylor, L., West, S. K., Quinn, T. C., Belland, R. J. & McClarty, G. (2003). J. Clin. Invest. 111, 1757-1769.]; Bonner et al., 2014[Bonner, C. A., Byrne, G. I. & Jensen, R. A. (2014). Front. Cell. Infect. Microbiol. 4, 17.]). The growing list of human pathogens in which the L-Trp biosynthetic pathway plays an important role extends beyond prokaryotes. For example, Cryptosporidium species (parasitic protozoa) inhabiting intestines encode bacteria-derived TrpB, which potentially acts in a similar fashion as it does in C. trachomatis (Sateriale & Striepen, 2016[Sateriale, A. & Striepen, B. (2016). PLoS Pathog. 12, e1005429.]).

Specific biochemical and structural traits of the tryptophan synthases from these organisms have not been explored, with the recent exception of the M. tuberculosis ortholog. The structural and functional information gathered over the past 60 years has helped to explain the roles of individual residues in catalysis and allosteric regulation of the two active sites. Research has focused primarily on a prototypic tryptophan synthase from S. typhimurium (StTrpAB) and to a lesser extent those from E. coli (Heilmann, 1978[Heilmann, H.-D. (1978). Biochim. Biophys. Acta, 522, 614-624.]; Lane & Kirschner, 1983[Lane, A. N. & Kirschner, K. (1983). Eur. J. Biochem. 129, 571-582.]; Drewe & Dunn, 1985[Drewe, W. F. Jr & Dunn, M. F. (1985). Biochemistry, 24, 3977-3987.], 1986[Drewe, W. F. Jr & Dunn, M. F. (1986). Biochemistry, 25, 2494-2501.]; Houben & Dunn, 1990[Houben, K. F. & Dunn, M. F. (1990). Biochemistry, 29, 2421-2429.]; Lim et al., 1991[Lim, W. K., Sarkar, S. K. & Hardman, J. K. (1991). J. Biol. Chem. 266, 20205-20212.]) and Pyrococcus furiosus (Yamagata et al., 2001[Yamagata, Y., Ogasahara, K., Hioki, Y., Lee, S. J., Nakagawa, A., Nakamura, H., Ishida, M., Kuramitsu, S. & Yutani, K. (2001). J. Biol. Chem. 276, 11062-11071.]; Ogasahara et al., 2003[Ogasahara, K., Ishida, M. & Yutani, K. (2003). J. Biol. Chem. 278, 8922-8928.]; Hioki et al., 2004[Hioki, Y., Ogasahara, K., Lee, S. J., Ma, J., Ishida, M., Yamagata, Y., Matsuura, Y., Ota, M., Ikeguchi, M., Kuramitsu, S. & Yutani, K. (2004). Eur. J. Biochem. 271, 2624-2635.]; Lee et al., 2005[Lee, S. J., Ogasahara, K., Ma, J., Nishio, K., Ishida, M., Yamagata, Y., Tsukihara, T. & Yutani, K. (2005). Biochemistry, 44, 11417-11427.]; Buller et al., 2015[Buller, A. R., Brinkmann-Chen, S., Romney, D. K., Herger, M., Murciano-Calles, J. & Arnold, F. H. (2015). Proc. Natl Acad. Sci. USA, 112, 14599-14604.]; Heilmann, 1978[Heilmann, H.-D. (1978). Biochim. Biophys. Acta, 522, 614-624.]; Lane & Kirschner, 1983[Lane, A. N. & Kirschner, K. (1983). Eur. J. Biochem. 129, 571-582.]; Drewe & Dunn, 1985[Drewe, W. F. Jr & Dunn, M. F. (1985). Biochemistry, 24, 3977-3987.], 1986[Drewe, W. F. Jr & Dunn, M. F. (1986). Biochemistry, 25, 2494-2501.]; Houben & Dunn, 1990[Houben, K. F. & Dunn, M. F. (1990). Biochemistry, 29, 2421-2429.]; Lim et al., 1991[Lim, W. K., Sarkar, S. K. & Hardman, J. K. (1991). J. Biol. Chem. 266, 20205-20212.]). Tryptophan synthase has become a prototype system to study the peculiarities of allostery and substrate channeling (Hilario et al., 2016[Hilario, E., Caulkins, B. G., Huang, Y.-M. M., You, W., Chang, C.-E. A., Mueller, L. J., Dunn, M. F. & Fan, L. (2016). Biochim. Biophys. Acta, 1864, 268-279.]; Ngo, Harris et al., 2007[Ngo, H., Harris, R., Kimmich, N., Casino, P., Niks, D., Blumenstein, L., Barends, T. R., Kulik, V., Weyand, M., Schlichting, I. & Dunn, M. F. (2007). Biochemistry, 46, 7713-7727.]; Ngo, Kimmich et al., 2007[Ngo, H., Kimmich, N., Harris, R., Niks, D., Blumenstein, L., Kulik, V., Barends, T. R., Schlichting, I. & Dunn, M. F. (2007). Biochemistry, 46, 7740-7753.]; Niks et al., 2013[Niks, D., Hilario, E., Dierkers, A., Ngo, H., Borchardt, D., Neubauer, T. J., Fan, L., Mueller, L. J. & Dunn, M. F. (2013). Biochemistry, 52, 6396-6411.]; Rhee et al., 1996[Rhee, S., Parris, K. D., Ahmed, S. A., Miles, E. W. & Davies, D. R. (1996). Biochemistry, 35, 4211-4221.]; Rowlett et al., 1998[Rowlett, R., Yang, L.-H., Ahmed, S. A., McPhie, P., Jhee, K.-H. & Miles, E. W. (1998). Biochemistry, 37, 2961-2968.]; Spyrakis et al., 2006[Spyrakis, F., Raboni, S., Cozzini, P., Bettati, S. & Mozzarelli, A. (2006). Biochim. Biophys. Acta, 1764, 1102-1109.]). TrpA is also one of the model proteins that have been used to investigate protein-folding mechanisms (Wu & Matthews, 2002[Wu, Y. & Matthews, C. R. (2002). J. Mol. Biol. 322, 7-13.]; Bilsel et al., 1999[Bilsel, O., Zitzewitz, J. A., Bowers, K. E. & Matthews, C. R. (1999). Biochemistry, 38, 1018-1029.]; Yang et al., 2007[Yang, X., Vadrevu, R., Wu, Y. & Matthews, C. R. (2007). Protein Sci. 16, 1398-1409.]; Vadrevu et al., 2008[Vadrevu, R., Wu, Y. & Matthews, C. R. (2008). J. Mol. Biol. 377, 294-306.]; Wu et al., 2007[Wu, Y., Vadrevu, R., Kathuria, S., Yang, X. & Matthews, C. R. (2007). J. Mol. Biol. 366, 1624-1638.]; Michalska et al., 2015[Michalska, K., Bigelow, L., Endres, M. & Joachimiak, A. (2015). FASEB J. 29, LB215.]). The sparsity of biochemical/structural investigations of other orthologs possibly stems from challenges in obtaining high-quality TrpAB samples and also from interest being focused on very detailed mechanistic aspects rather than on species-specific variations. Importantly, though, as shown by our recent study of M. tuberculosis TrpAB (MtTrpAB; Wellington et al., 2017[Wellington, S., Nag, P. P., Michalska, K., Johnston, S. E., Jedrzejczak, R. P., Kaushik, V. K., Clatworthy, A. E., Siddiqi, N., McCarren, P., Bajrami, B., Maltseva, N. I., Combs, S., Fisher, S. L., Joachimiak, A., Schreiber, S. L. & Hung, D. T. (2017). Nature Chem. Biol. 13, 943-950.]), these so-far ignored differences, especially within the non­conserved tunnel lining, may have profound consequences for the discovery and design of new allosteric inhibitors.

Therefore, to fill the important phylogenetic gaps in our understanding of TrpABs and to uncover potential unique features of other orthologs to facilitate future drug-discovery efforts, we biochemically characterized three TrpABs from Gram-positive and Gram-negative pathogens: Legionella pneumophila Philadelphia, F. tularensis and S. pneumoniae (LpPhTrpAB, FtTrpAB and SpTrpAB, respectively). In addition to kinetic properties and inhibitor-binding capabilities, we also provide high-resolution structural information gathered using X-ray crystallography for the FtTrpAB and SpTrpAB complexes and for two α subunits: LpPhTrpA and that from L. pneumophila Paris (LpPaTrpA).

2. Experimental procedures

2.1. TrpAB gene cloning

The gene cloning was performed as reported previously (Kim et al., 2011[Kim, Y., Babnigg, G., Jedrzejczak, R., Eschenfeldt, W. H., Li, H., Maltseva, N., Hatzos-Skintges, C., Gu, M., Makowska-Grzyska, M., Wu, R., An, H., Chhor, G. & Joachimiak, A. (2011). Methods, 55, 12-28.]). Briefly, F. tularensis Schu 4, L. pneumophila Philadelphia, L. pneumophila Paris and S. pneumoniae TIGR4 genomic DNAs were used as templates for PCR of the genes coding for the TrpA and TrpB subunits of tryptophan synthase. Vector-compatible primers for the amplification of the DNA fragments coding for the subunits were designed using an online tool (https://bioinformatics.anl.gov/targets/public_tools.aspx; Yoon et al., 2002[Yoon, J. R., Laible, P. D., Gu, M., Scott, H. N. & Collart, F. R. (2002). Biotechniques, 33, 1328-1333.]). The TrpA subunit peptides that were cloned were as follows: 1–269 for FtTrpA, 1–272 for LpPhTrpA and LpPaTrpA, and 1–258 for SpTrpA. The TrpB subunit peptides that were cloned were as follows: 1–396 for FtTrpB, 13–396 for LpPhTrpB and 4–407 for SpTrpB. Purified PCR products were treated with T4 DNA polymerase in the presence of dCTP (Eschenfeldt et al., 2010[Eschenfeldt, W. H., Maltseva, N., Stols, L., Donnelly, M. I., Gu, M., Nocek, B., Tan, K., Kim, Y. & Joachimiak, A. (2010). J. Struct. Funct. Genomics, 11, 31-39.]) according to the vendor's specification (New England Biolabs, Ipswich, Massachusetts, USA). The protruded DNA fragment for each of the TrpA subunits was mixed with T4 DNA polymerase-treated vector pMCSG68 (PSI:Biology-Materials Repository) to allow ligation-independent cloning (Aslanidis & Jong, 1990[Aslanidis, C. & de Jong, P. J. (1990). Nucleic Acids Res. 18, 6069-6074.]; Eschenfeldt et al., 2009[Eschenfeldt, W. H., Stols, L., Millard, C. S., Joachimiak, A. & Mark, I. D. (2009). Methods Mol. Biol. 498, 105-115.]). Similarly, the protruded DNA fragment for each of the TrpB subunits was mixed with T4 DNA polymerase-treated vector pRSF with kanamycin resistance, which had an identical ligand-independent cloning site to pMCSG68. Both subunits from each genomic DNA were individually transformed into E. coli BL21-Gold (DE3) cells and grown in the presence of the corresponding antibiotic. A single colony of each transformant was picked, grown and induced with isopropyl β-D-1-thiogalactopyranoside (IPTG). The cell lysate was analyzed to confirm a protein of the correct molecular weight. The solubility of the TrpA subunit was analyzed via small-scale Ni2+-affinity purification and overnight TEV protease cleavage. Once the DNA sequences of the TrpA and TrpB subunits had been verified, both subunit plasmids from each genomic DNA were co-transformed into E. coli BL21-Gold (DE3) cells in LB medium containing ampicillin (150 µg ml−1) and kanamycin (25 µg ml−1). Co-transformed colonies were analyzed using Ni2+-affinity purification, and overnight TEV protease cleavage was performed to verify that the complex was soluble and stable.

2.2. Expression of TrpAB and purification for crystallization

To express SpTrpAB and FtTrpAB, starter cultures were grown overnight at 37°C and 200 rev min−1 in LB medium with ampicillin (100 µg ml−1) and kanamycin (30 µg ml−1) supplemented with 40 mM K2HPO4. The following morning, LB–PO4–glucose (2 g per litre) medium with antibiotics was inoculated with the overnight cultures. After reaching an OD600 of 1.0 at 37°C, the SpTrpAB cultures were transferred to 4°C and, after 1 h, to 18°C. After a subsequent 15 min incubation, the cultures were induced with 0.5 mM IPTG and incubated at 18°C overnight to produce the native protein. FtTrpAB cultures were treated differently to produce selenomethionine (SeMet)-labeled protein. At an OD600 of 1.0, the FtTrpAB cultures were spun down for 30 min at 4000 rev min−1. The supernatant was then decanted. LB–PO4–glucose pellets (from 4 l culture) were resuspended in 1 l M9 medium (Orion Enterprises, Wheeling, Illinois, USA) supplemented with 0.4%(w/v) glucose, 13 mM NaCl, 0.1 mM CaCl2, 2 mM MgSO4, 1%(w/v) thiamine and antibiotics (Stols et al., 2004[Stols, L., Millard, C. S., Dementieva, I. & Donnelly, M. I. (2004). J. Struct. Funct. Genomics, 5, 95-102.]). 0.01%(w/v) each of L-leucine, L-isoleucine, L-lysine, L-phenylalanine, L-threonine and L-valine were added to inhibit the metabolic pathway of methionine synthesis and encourage SeMet incorporation. The culture was transferred to 18°C, and at an OD600 of 1.0 SeMet (90 mg; Orion Enterprises, Wheeling, Illinois, USA) was added. After 15 min, protein expression was induced with 0.5 mM IPTG. The cells were incubated at 18°C overnight. The cells were then harvested at 4500 rev min−1 for 20 min at 4°C and resuspended in lysis buffer [500 mM NaCl, 5%(w/v) glycerol, 50 mM HEPES pH 8.0, 20 mM imidazole, 10 mM β-mercaptoethanol and protease inhibitor (one tablet per 50 ml of extract; Roche, Mannheim, Germany)] supplemented with 1 mM pyridoxal 5′-phosphate (PLP) and stored at −80°C.

SeMet-labeled FtTrpAB and native SpTrpAB were purified using the procedure described previously (Kim et al., 2004[Kim, Y., Dementieva, I., Zhou, M., Wu, R., Lezondra, L., Quartey, P., Joachimiak, G., Korolev, O., Li, H. & Joachimiak, A. (2004). J. Struct. Funct. Genomics, 5, 111-118.]). The harvested cells were thawed and 1 mg ml−1 lysozyme was added. This mixture was kept on ice for 20 min with gentle shaking and was then sonicated. The lysate was clarified by centrifugation at 36 000g for 1 h and filtered through a 0.45 µm membrane. The clarified lysate was applied onto a 5 ml nickel HisTrap HP column (GE Healthcare Life Sciences) and the His6-tagged protein was released with elution buffer (500 mM NaCl, 5% glycerol, 50 mM HEPES pH 8.0, 250 mM imidazole, 10 mM β-mercaptoethanol). This was followed by a buffer-exchange step using a customized desalting column (Sephadex G-25 Fine XK 26/20, GE Healthcare Life Sciences) equilibrated with buffer consisting of 20 mM Tris–HCl pH 7.5, 500 mM NaCl, 2 mM DTT. All of these steps were performed using an ÄKTAxpress system (GE Healthcare Life Sciences). The fusion tag was removed by treatment with recombinant His7-tagged Tobacco etch virus (TEV) protease. Nickel-affinity chromatography was used to remove the His6 tag, uncut protein and His7-tagged TEV protease (Blommel & Fox, 2007[Blommel, P. G. & Fox, B. G. (2007). Protein Expr. Purif. 55, 53-68.]). The SpTrpAB ortholog was subjected to an extra purification step via size-exclusion chromatography on a Superdex 200 HiLoad 26/60 column (GE Healthcare Life Sciences) in crystallization buffer (200 mM NaCl, 20 mM HEPES pH 8.0, 2 mM DTT). The FtTrpAB protein was dialyzed against crystallization buffer consisting of 250 mM NaCl, 20 mM HEPES pH 8.0, 2 mM dithiothreitol (DTT) and the proteins were then concentrated to 68 mg ml−1 (FtTrpAB) and 33.6 mg ml−1 (SpTrpAB) using an Amicon Ultra centrifugal filter device with a 10 000 molecular-weight cutoff (Millipore, Billerica, Massachusetts, USA), flash-cooled in liquid nitrogen and stored at −80°C.

The TrpAB protein concentration was determined spectrophotometrically by measuring the absorbance at 280 nm on a NanoDrop ND-1000 spectrophotometer (Thermo Scientific) against buffer containing an equimolar concentration of PLP. The concentration was calculated using extinction coefficients of 34185 and 39435 M−1 cm−1, respectively, computed from the amino-acid sequence.

2.3. Expression of TrpA and purification for crystallization

An LB medium starter culture was supplemented with 40 mM K2HPO4 and ampicillin (150 µg ml−1) for LpPhTrpA and LpPaTrpA, grown and shaken overnight at 37°C and 200 rev min−1. The starter cultures were used to inoculate 1 l of enriched M9 medium for large-scale SeMet-labeled protein production, which was carried out as described above. From each litre of cell culture, 8 g of cell pellet containing SeMet-labeled LpPhTrpA or LpPaTrpA protein was obtained and was consequently resuspended in lysis buffer and stored at −80°C.

SeMet-labeled LpPhTrpA and LpPaTrpA were purified in the same manner as SeMet-labeled FtTrpAB. However, instead of dialyzing these proteins against crystallization buffer, they were buffer-exchanged using an Amicon Ultra centrifugal filter device with a 10 000 molecular-weight cutoff (Millipore, Billerica, Massachetts, USA) with 250 mM NaCl, 20 mM HEPES pH 8.0, 2 mM DTT, flash-cooled in liquid nitrogen and stored at −80°C. Protein concentrations were also determined with a NanoDrop ND-1000 using extinction coefficients of 24870 and 23505 M−1 cm−1, respectively, computed from the amino-acid sequence.

2.4. Expression and purification for enzymatic assays

For each ortholog, a starter culture was grown overnight at 37°C and 200 rev min−1 in LB medium with ampicillin (100 µg ml−1) and kanamycin (30 µg ml−1) and supplemented with 40 mM K2HPO4. The following morning, 4 l LB–PO4–glucose (2 g per litre) medium with antibiotics was inoculated with 30 ml of the overnight culture and was grown at 37°C and 200 rev min−1. After reaching an OD600 of 1.0 the cultures were transferred to 4°C to cool, and after 1 h the temperature was increased to 18°C. After 15 min, protein expression was induced with 0.5 mM IPTG. The cells were incubated at 18°C overnight. The harvested cells containing TrpAB were resuspended in lysis buffer [500 mM NaCl, 5%(w/v) glycerol, 50 mM HEPES pH 8.0, 20 mM imidazole, 10 mM β-mercaptoethanol, protease inhibitor (one tablet per 50 ml of extract), 1 mM PLP] and stored at −80°C. All three native proteins were purified using the procedure described above for FtTrpAB. The samples were concentrated to 40 mg ml−1 (LpPhTrpAB), 40 mg ml−1 (SpTrpAB) and 140 mg ml−1 (FtTrpAB), flash-cooled in liquid nitrogen in 35 µl droplets and subsequently used in enzymatic assays. MtTrpAB was purified as described previously (Wellington et al., 2017[Wellington, S., Nag, P. P., Michalska, K., Johnston, S. E., Jedrzejczak, R. P., Kaushik, V. K., Clatworthy, A. E., Siddiqi, N., McCarren, P., Bajrami, B., Maltseva, N. I., Combs, S., Fisher, S. L., Joachimiak, A., Schreiber, S. L. & Hung, D. T. (2017). Nature Chem. Biol. 13, 943-950.]).

2.5. Crystallization

The FtTrpAB and SpTrpAB proteins were crystallized using sitting-drop vapor diffusion at 16 and 24°C, respectively, in a CrystalQuick 96-well round-bottom plate (Greiner Bio-One North America, Monroe, North Carolina, USA). A 400 nl droplet of the protein (35 or 34 mg ml−1) with 1 mM PLP and 1 mM L-Ser (FtTrpAB) or 0.5 mM PLP (SpTrpAB) was mixed with a 200 nl droplet and 400 nl crystallization reagent and allowed to equilibrate against 135 µl crystallization reagent. The nanopipetting was performed using a Mosquito nanolitre liquid-handling system (TTP Labtech, Cambridge, Massachusetts, USA). The plates were then incubated within a RoboIncubator automated plate-storage system (Rigaku). Automated crystal visualization (Minstrel III, Rigaku) was utilized to locate several crystals. The best crystals of SeMet-labeled FtTrpAB were obtained from 0.2 M calcium acetate, 0.1 M imidazole–HCl pH 8.0, 10%(w/v) PEG 8000. The SpTrpAB crystals grew from 0.2 M ammonium acetate, 0.1 M Tris–HCl pH 8.5, 25% PEG 3350.

LpPhTrpA (at 25 mg ml−1) and LpPaTrpA (at 62.5 mg ml−1) were screened in the same manner, but without the addition of extra ligands, using a droplet consisting of 400 nl protein solution and 400 nl crystallization reagent that was allowed to equilibrate over 135 µl of the respective reservoir condition. The proteins were screened against the MCSG 1–4 screens (Microlytic) and the Index screen (Hampton Research) at 16°C. The best crystals of SeMet-labeled LpPhTrpA were obtained from 0.01 M sodium citrate, 33%(w/v) PEG 6000. The SeMet-labeled LpPaTrpA crystals grew from 0.2 M sodium chloride, 0.1 M bis-Tris pH 6.5, 25%(w/v) PEG 3350.

2.6. Data collection

The crystals were cryoprotected in their respective mother liquors supplemented with 10% (SpTrpAB, LpPhTrpA and LpPaTrpA) or 25% (FtTrpAB) glycerol and were subsequently flash-cooled in liquid nitrogen. X-ray diffraction data were collected on the Structural Biology Center 19-ID beamline at the Advanced Photon Source, Argonne National Laboratory. The images were recorded on an ADSC Q315r detector. The data sets were processed with the HKL-3000 suite (Minor et al., 2006[Minor, W., Cymborowski, M., Otwinowski, Z. & Chruszcz, M. (2006). Acta Cryst. D62, 859-866.]). Intensities were converted to structure-factor amplitudes in the CTRUNCATE program (French & Wilson, 1978[French, S. & Wilson, K. (1978). Acta Cryst. A34, 517-525.]; Padilla & Yeates, 2003[Padilla, J. E. & Yeates, T. O. (2003). Acta Cryst. D59, 1124-1130.]) from the CCP4 package (Winn et al., 2011[Winn, M. D., Ballard, C. C., Cowtan, K. D., Dodson, E. J., Emsley, P., Evans, P. R., Keegan, R. M., Krissinel, E. B., Leslie, A. G. W., McCoy, A., McNicholas, S. J., Murshudov, G. N., Pannu, N. S., Potterton, E. A., Powell, H. R., Read, R. J., Vagin, A. & Wilson, K. S. (2011). Acta Cryst. D67, 235-242.]). The data-collection and processing statistics are given in Table 1[link].

Table 1
Data-processing and refinement statistics

Values in parentheses are for the highest resolution shell.

Structure SpTrpAB FtTrpAB LpPhTrpA LpPaTrpA
Data processing
 Wavelength (Å) 0.9793 0.9793 0.9792 0.9786
 Resolution range (Å) 50.00–2.45 (2.49–2.45) 30.00–2.80 (2.85–2.80) 50.00–2.00 (2.03–2.00) 40.00–1.91 (1.93–1.91)
 Space group P21 C2221 P212121 P212121
 Unit-cell parameters
  a (Å) 67.70 111.18 47.02 43.71
  b (Å) 71.16 171.99 71.05 69.65
  c (Å) 138.68 76.11 71.82 75.08
  β (°) 101.69      
 Unique reflections 48447 18264 16344 18317
 Merged reflections 2386 902 751 858
 Multiplicity 3.6 (3.3) 4.8 (4.9) 11.3 (8.4) 4.2 (3.1)
 Completeness (%) 99.7 (99.8) 100.0 (100.0) 99.4 (93.1) 99.7 (96.9)
 Mean I/σ(I) 14.3 (1.6) 5.8 (1.5) 38.0 (1.9) 20.0 (2.0)
 Wilson B factor (Å2) 38.97 57.25 24.07 17.55
Rmerge 0.141 (0.740) 0.132 (0.981) 0.058 (0.893) 0.062 (0.490)
 CC1/2 0.683 0.566 0.793 0.703
Refinement
 Resolution range (Å) 49.13–2.45 29.50–2.80 39.21–2.02 37.54–1.91
 Reflections (work/test) 45342/2264 32767/1689 13868/1411 30107/1521
Rwork/Rfree§ 0.181/0.228 0.183/0.235 0.191/0.238 0.176/0.207
 No. of non-H atoms
  Total 10029 4987 2184 2245
  Macromolecules 9902 4971 2054 2041
  Ligands 18 6 0 0
  Solvent 109 10 130 204
 No. of protein residues 1297 655 262 262
 R.m.s.d., bonds (Å) 0.002 0.003 0.003 0.005
 R.m.s.d., angles (°) 0.51 0.57 0.66 0.90
 Ramachandran statistics
  Favored (%) 97.03 94.89 98.47 98.45
  Allowed (%) 2.81 4.95 1.53 1.55
  Outliers (%) 0.16 0.15 0.0 0.0
 Rotamer outliers (%) 1.28 1.17 0.0 3.64
 Clashscore 2.53 5.12 1.70 4.40
 Average B factor (Å2)
  Overall 49.14 56.09 34.33 22.30
  Macromolecules 49.21 56.10 33.83 21.30
  Ligands 63.01 75.38    
  Solvent 40.13 38.11 41.43 32.32
 No. of TLS groups 20 9 6
 PDB entry 5kin 5kzm 5k9x 5kmy
Rmerge = [\textstyle \sum_{hkl}\sum_{i}|I_{i}(hkl)- \langle I(hkl)\rangle|/][\textstyle \sum_{hkl}\sum_{i}I_{i}(hkl)], where Ii(hkl) is the intensity of observation i of reflection hkl.
‡As defined by Karplus & Diederichs (2012[Karplus, P. A. & Diederichs, K. (2012). Science, 336, 1030-1033.]).
§R = [\textstyle \sum_{hkl}\big ||F_{\rm obs}|-|F_{\rm calc}|\big |/][\textstyle \sum_{hkl}|F_{\rm obs}|] for all reflections, where Fobs and Fcalc are the observed and calculated structure factors, respectively. Rfree is calculated analogously for the test reflections, which wre randomly selected and excluded from the refinement.
¶As defined by MolProbity (Chen et al., 2010[Chen, V. B., Arendall, W. B., Headd, J. J., Keedy, D. A., Immormino, R. M., Kapral, G. J., Murray, L. W., Richardson, J. S. & Richardson, D. C. (2010). Acta Cryst. D66, 12-21.]).

2.7. Structure solution and refinement

The SpTrpAB structure was solved by molecular replacement in Phaser (McCoy, 2007[McCoy, A. J. (2007). Acta Cryst. D63, 32-41.]) using the structures of SpTrpA (PDB entry 6qky; unpublished work) and of TrpB from Bacillus anthracis (PDB entry 4neg; Center for Structural Genomics of Infectious Diseases, unpublished work). The initial model was autobuilt in PHENIX (Adams et al., 2013[Adams, P. D., Baker, D., Brunger, A. T., Das, R., DiMaio, F., Read, R. J., Richardson, D. C., Richardson, J. S. & Terwilliger, T. C. (2013). Annu. Rev. Biophys. 42, 265-287.]) and was further improved by manual correction in Coot (Emsley & Cowtan, 2004[Emsley, P. & Cowtan, K. (2004). Acta Cryst. D60, 2126-2132.]) and crystallographic refinement in PHENIX (Afonine et al., 2012[Afonine, P. V., Grosse-Kunstleve, R. W., Echols, N., Headd, J. J., Moriarty, N. W., Mustyakimov, M., Terwilliger, T. C., Urzhumtsev, A., Zwart, P. H. & Adams, P. D. (2012). Acta Cryst. D68, 352-367.]). The FtTrpAB, LpPhTrpA and LpPaTrpA structures were solved by the SAD method using selenium absorption peak data in SHARP (Vonrhein et al., 2007[Vonrhein, C., Blanc, E., Roversi, P. & Bricogne, G. (2007). Methods Mol. Biol. 364, 215-230.]) or HKL-3000 (for LpTrpA; Minor et al., 2006[Minor, W., Cymborowski, M., Otwinowski, Z. & Chruszcz, M. (2006). Acta Cryst. D62, 859-866.]) and were autobuilt in Buccaneer (Cowtan, 2006[Cowtan, K. (2006). Acta Cryst. D62, 1002-1011.]). The final model was obtained using alternating manual rebuilding in Coot and maximum-likelihood refinement in PHENIX (Afonine et al., 2012[Afonine, P. V., Grosse-Kunstleve, R. W., Echols, N., Headd, J. J., Moriarty, N. W., Mustyakimov, M., Terwilliger, T. C., Urzhumtsev, A., Zwart, P. H. & Adams, P. D. (2012). Acta Cryst. D68, 352-367.]). The refinement statistics are given in Table 1[link].

The atomic coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 5kzm (FtTrpAB), 5kin (SpTrpAB), 5k9x (LpPhTrpA) and 5kmy (LpPaTrpA).

2.8. Preparation of material for kinetic assays

Prior to kinetic and/or biophysical characterization, MtTrpAB was dialyzed for 2–4 h in TrpAB buffer (20 mM HEPES pH 8.0, 100 mM KCl, 1 mM TCEP, 40 µM PLP) to remove glycerol. After dialysis for 2–4 h, the buffer was exchanged with fresh buffer and dialysis continued overnight. The three other orthologs, however, were stored in 20 mM HEPES pH 8.0, 200 mM NaCl, 2 mM DTT buffer containing no glycerol after purification and did not require dialysis before use.

The compounds F9, F6 and IPP were custom-synthesized by GVK Bio (Cambridge, Massachusetts, USA). The MtTrpAB inhibitor BRD4592 was synthesized internally at the Broad Institute as described previously (Wellington et al., 2017[Wellington, S., Nag, P. P., Michalska, K., Johnston, S. E., Jedrzejczak, R. P., Kaushik, V. K., Clatworthy, A. E., Siddiqi, N., McCarren, P., Bajrami, B., Maltseva, N. I., Combs, S., Fisher, S. L., Joachimiak, A., Schreiber, S. L. & Hung, D. T. (2017). Nature Chem. Biol. 13, 943-950.]).

2.9. Measurement of enzyme kinetics by UV absorption

Enzyme kinetics for each ortholog were determined over 30 min under saturating substrate conditions (200 µM indole and 60 mM L-Ser) in 1 ml TrpAB buffer. An Agilent Technologies Cary 400 Series UV–Vis spectrophotometer set to 290 nm was used for UV absorption measurements. A baseline reading with no enzyme was established, after which enzyme was added every 2 min to give a final concentration range from 50 nM to 2.4 µM. Product progress curves were determined at appropriate enzyme concentrations over a 10 min period in which product generation was linear to determine the Km and kcat parameters. A value of Δ = 1890 M−1 cm−1 was used for the indole to L-Trp conversion. In all cases, these enzymes were studied at room temperature (22°C). These experiments were performed on triplicate test occasions with triplicate replicates in each case.

2.10. LC-MS assay

For the liquid chromatography-mass spectrometry (LC-MS) assay, all reagents were prepared in a 96-well plate with a final reaction volume of 50 µl. Compound IC50 reactions were run at substrate Km conditions (10 µM indole, 20 mM L-serine). Compound concentrations ranged from 0 to 200 µM. 10× Km substrate solutions were prepared, with 5 µl additions of both indole and serine solutions to the wells. The final concentrations of each protein were as follows: 100 nM SpTrpAB, 5 nM FtTrpAB, 600 nM LpPhTrpAB and 100 nM MtTrpAB prepared in TrpAB buffer.

Standard curves for L-Trp and indole were included with each mass-spectrometry experiment for quantification purposes only. An L-Ser standard curve was also included as a biological check for each ortholog. Final L-Ser standard curve concentrations included 48, 24, 12, 6, 3, 1.5, 0.75 and 0 mM at saturating (500 µM) indole (5× solution at 2.5 mM indole with 10 µl additions). After all compound, substrate and standard curve solutions had been prepared, 30 µl of a 1.67× protein solution was added to each well to start the reaction.

After mixing and allowing 10 min incubation at room temperature, the reactions were quenched using 150 µl 0.1% formic acid in methanol followed by storage at 4°C for at least 2 h. The sample plates were then centrifuged for 15 min at 3900 rev min−1 (∼3061g) and an aliquot of the supernatant was diluted 1:10 with water. 3.75 µl of this final solution was injected and analyzed. L-Trp and indole were detected by UPLC-MS (Waters, Milford, Massachusetts, USA). Compounds were quantified by selected ion recording (SIR) on an SQ mass spectrometer by negative electrospray ionization. The SIR method was set for L-Trp at 203.4 m/z and for indole at 116.3 m/z. Mobile phase A consisted of 0.1% ammonium hydroxide in water, while mobile phase B consisted of 0.1% ammonium hydroxide in acetonitrile. The gradient ran from 2% to 95% mobile phase B over 2.65 min at 0.9 ml min−1. An Acquity BEH C18, 1.7 µm, 2.1 × 50 mm column was used with the column temperature maintained at 65°C.

2.11. Data analysis

Kinetic experiments were run in triplicate and the reported values represent the average of at least three independent experiments. Km, kcat and IC50 data were plotted using GraphPad Prism 7.0 and Origin 8.0.

3. Results

3.1. Protein preparation

The recombinant tryptophan synthases from the pathogenic bacteria F. tularensis, S. pneumoniae and L. pneumophila Philadelphia have been produced for detailed characterization and comparison with the previously studied enzymes from S. typhimurium, E. coli and M. tuberculosis (Wellington et al., 2017[Wellington, S., Nag, P. P., Michalska, K., Johnston, S. E., Jedrzejczak, R. P., Kaushik, V. K., Clatworthy, A. E., Siddiqi, N., McCarren, P., Bajrami, B., Maltseva, N. I., Combs, S., Fisher, S. L., Joachimiak, A., Schreiber, S. L. & Hung, D. T. (2017). Nature Chem. Biol. 13, 943-950.]). The level of pairwise sequence identity between the TrpBs from these organisms ranges from 51% to 59%, with the exception of the FtTrpB/StTrpB pair, which show 81% conserved residues. The TrpAs are more variable, with only 25–33% sequence identity for most pairs and 50% for the FtTrpA/StTrpA pair (Table 2[link]), suggesting that there are different evolutionary pressures on the two subunits.

Table 2
Primary structure identity and structural similarity between orthologous TrpA and TrpB

The first number corresponds to the percentage sequence identity (calculated in EMBOSS Needle; Rice et al., 2000[Rice, P., Longden, I. & Bleasby, A. (2000). Trends Genet. 16, 276-277.]), followed by r.m.s.d. (in Å) for Cα-atom superposition for the number of pairs given in parentheses (calculated in CCP4; Winn et al., 2011[Winn, M. D., Ballard, C. C., Cowtan, K. D., Dodson, E. J., Emsley, P., Evans, P. R., Keegan, R. M., Krissinel, E. B., Leslie, A. G. W., McCoy, A., McNicholas, S. J., Murshudov, G. N., Pannu, N. S., Potterton, E. A., Powell, H. R., Read, R. J., Vagin, A. & Wilson, K. S. (2011). Acta Cryst. D67, 235-242.], Krissinel & Henrick, 2004[Krissinel, E. & Henrick, K. (2004). Acta Cryst. D60, 2256-2268.]).

  SpTrpB FtTrpB LpPhTrpB MtTrpB StTrpB
SpTrpA   53, 0.87 (385) 57 54, 0.85 (389) 53, 1.02 (377)
FtTrpA 29, 1.89 (233)   53 51, 0.82 (381) 81, 0.65 (388)
LpPhTrpA 32, 1.71 (230) 32, 1.38 (244)   59 53
MtTrpA 31, 1.81 (241) 25, 1.37 (244) 33, 1.64 (240)   51, 1.01 (381)
StTrpA 29, 1.74 (225) 58, 0.84 (253) 31, 1.36 (240) 26, 1.56 (244)  

To obtain sufficient amounts of protein-complex samples, TrpA and TrpB were coexpressed from individual vectors in E. coli. In all cases, either the TrpA (FtTrpAB and LpPhTrpAB) or TrpB (SpTrpAB) subunits were equipped with an N-terminal His6 tag, which was subsequently removed by treatment with TEV protease. The resulting proteins carry an additional three N-terminal residues SNA on the tagged subunit. In addition to TrpABs, TrpAs from the L. pneumophila strains Paris and Philadelphia (LpPaTrpA and LpPhTrpA, respectively; 99% identical) have been produced for crystallographic studies, also with a removable N-terminal His6 tag. FtTrpAB and LpPhTrpA were produced as SeMet-labeled derivatives, while all other proteins were expressed in the native form. The purified proteins were at least 90% pure as judged by PAGE.

3.2. Structure determination

The SpTrpAB protein was crystallized in space group P21 with the entire αββα heterotetramer present in the asymmetric unit (Fig. 1[link], Table 1[link]). The structure, which was determined at 2.45 Å resolution, was solved by molecular replacement. In chains A and C, corresponding to TrpA (amino-acid residues 1–258), residues 1, 180–189 and 182–187, respectively, were not modeled owing to a lack of interpretable electron density. Similarly, in TrpB (amino-acid residues 4–407) the N-terminal SNA sequence and the C-terminal end (residues 403–407) are not present in the respective chains B and D. The other ortholog, FtTrpAB, crystallized in space group C2221 and the asymmetric unit contains only one αβ module. This structure was solved by experimental SAD phasing and was refined to 2.80 Å resolution. In FtTrpAB, TrpA (chain A; residues 1–269) lacks the N-terminal SNA sequence and residues 183–191, while in TrpB (chain B; residues 1–396) the C-terminal residue is not present. For L. pneumophila only the TrpA subunit could be crystallized. The LpPaTrpA and LpPhTrpA structures were determined by experimental SAD phasing at 1.91 and 2.02 Å resolution, respectively. The LpPhTrpA protein crystallized in the orthorhombic space group P212121. The asymmetric unit contains one molecule of TrpA and the model lacks the N-terminal SNA residues, residues 57–59, residues 180–186 and the C-terminal residue 272. LpPaTrpA also crystallized in space group P212121 with one chain in the asymmetric unit. The N-terminal SN residues and residues 180–187 and 270–273 are missing from the final model.

3.3. Kinetic characterization

Simultaneously with structural characterization, we performed kinetic analyses of the three new orthologs (FtTrpAB, SpTrpAB and LpPhTrpAB) and compared them with the MtTrpAB reference. A UV-based assay was used to measure the production of L-Trp from indole and L-Ser. Firstly, the enzyme concentration versus catalytic rate relationship was determined to identify the linear rate dependencies. Both the SpTrpAB and FtTrpAB enzymes displayed specific activities that were comparable to (SpTrpAB, 1.4 M L-Trp s−1M−1 enzyme) or higher (FtTrpAB, 26 M L-Trp s−1M−1 enzyme) than that of MtTrpAB (2.0 M L-Trp s−1M−1 enzyme), with the rate being linearly dependent on enzyme concentration over the entire tested range. The LpPhTrpAB enzyme, however, was less active than the MtTrpAB enzyme, displaying a biphasic dependency with both components appearing to be linear. The specific activity at low enzyme concentrations (50–800 nM) was much lower (0.17 M L-Trp s−1M−1 enzyme), while the higher concentration range (1000–2400 nM) displayed an improved but still significantly lower specific activity (0.38 M L-Trp s−1M−1 enzyme) (Fig. 4[link]). The source of this higher order effect is not obvious, but could be explained by the equilibrium between α subunits and ββ dimers and αββα tetramers, with higher protein concentrations favoring the more active αββα oligomeric state. We have observed such an equilibrium for the MtTrpAB enzyme (Wellington et al., 2017[Wellington, S., Nag, P. P., Michalska, K., Johnston, S. E., Jedrzejczak, R. P., Kaushik, V. K., Clatworthy, A. E., Siddiqi, N., McCarren, P., Bajrami, B., Maltseva, N. I., Combs, S., Fisher, S. L., Joachimiak, A., Schreiber, S. L. & Hung, D. T. (2017). Nature Chem. Biol. 13, 943-950.]). The specific activity order is as follows: FtTrpAB >> MtTrpAB, SpTrpAB >> LpPhTrpAB.

[Figure 4]
Figure 4
Enzyme versus reaction rate dependency for TrpAB enzymes from (a) F. tularensis (26 M L-Trp s−1M−1 enzyme), (b) S. pneumoniae (1.4 M L-­Trp s−1M−1 enzyme), (c) M. tuberculosis (2.0 M L-Trp s−1M−1 enzyme), (d) L. pneumophila (all concentrations), (e) L. pneumophila (low concentrations) (0.17 M L-Trp s−1M−1 enzyme) and (f) L. pneumophila (high concentrations) (0.38 M L-­Trp s−1M−1 enzyme).

These data were used to set the appropriate enzyme concentrations (5 nM FtTrpAB, 100 nM MtTrpAB, 100 nM SpTrpAB and 600 nM LpPhTrpAB), resulting in linear L-Trp production progress curves over a 10 min reaction period, to determine the apparent Km and kcat parameters using the LC-MS assay. The apparent Km values are similar across all of the species for both substrates tested (indole and L-Ser). The kcat values were reproducible across experiment replicates and substrates, suggesting that saturation was achieved for the independent substrate in each case. The absolute kcat values were consistent with the specific activities described above, following the activity order FtTrpAB >> MtTrpAB, SpTrpAB >> LpPhTrpAB (Fig. 5[link]).

[Figure 5]
Figure 5
Kinetics of TrpAB orthologs. (a) FtTrpAB at 5 nM, (b) LpPhTrpAB at 600 nM, (c) SpTrpAB at 100 nM, (d) MtTrpAB at 100 nM. The left panels show reaction rates versus indole concentration in the presence of 48 mM L-Ser; the right panels show reaction rates versus L-Ser concentration in the presence of 0.5 mM indole.

3.4. Inhibition studies

In addition, the three TrpAB orthologs were profiled against the reported commercially available inhibitors F9 [N-(4′-trifluoromethoxybenzenesulfonyl)-2-aminoethyl phosphate; CID identifier 16122526], F6 [N-(4′-trifluoromethoxy­benzoyl)-2-aminoethyl phosphate; CID identifier 16122525] and IPP (indole­propanol phosphate; CID identifier 3713), as well as the recently discovered MtTrpAB inhibitor BRD4592 (CID identifier 54650477; Wellington et al., 2017[Wellington, S., Nag, P. P., Michalska, K., Johnston, S. E., Jedrzejczak, R. P., Kaushik, V. K., Clatworthy, A. E., Siddiqi, N., McCarren, P., Bajrami, B., Maltseva, N. I., Combs, S., Fisher, S. L., Joachimiak, A., Schreiber, S. L. & Hung, D. T. (2017). Nature Chem. Biol. 13, 943-950.]) (Fig. 6[link]). The LC-MS-based assays examined inhibition of the β reaction with indole and L-Ser as substrates. F9 was found to be a potent inhibitor (ICβ50 = 114 nM) of FtTrpAB under substrate Km conditions (10 µM indole, 20 mM L-Ser), while only slightly inhibiting LpPhTrpAB. Interestingly, F9 appears to be an activator of SpTrpAB (Fig. 6[link]). A similar profile is again exhibited with F6 and IPP, whereby inhibition was only observed for the FtTrpAB enzyme, with ICβ50 = 1.46 µM for F6 and ICβ50 = 0.08 µM for IPP. A different profile was seen when using the MtTrpAB inhibitor BRD4592. All three orthologs are slightly inhibited; however, a measureable IC50 was only obtained for the SpTrpAB ortholog (ICβ50 = 21 µM) (Fig. 6[link]).

[Figure 6]
Figure 6
Inhibition of TrpAB orthologs with (a) IPP, (b) F6, (c) F9 and (d) BRD4592. Enzyme concentrations for all experiments are shown in (a).

4. Discussion

4.1. Structural comparison with other TrpAB orthologs

We have determined the structures of the FtTrpAB and SpTrpAB αββα heterotetramers and of the α subunits LpPaTrpA and LpPhTrpA. The overall structures of the complexes, along with the α subunits, are essentially identical to those of the orthologs characterized previously, with the heterotetramer representing the complete functional unit (Fig. 1[link]). Despite the rather low sequence identity of the TrpAs, the three polypeptides superpose with r.m.s.d.s of 1.4–1.9 Å amongst themselves and with the orthologs MtTrpA or StTrpA (Table 2[link], Fig. 7[link]). The enzyme from F. tularensis, which is the most closely related to StTrpAB, shows even better agreement, with an r.m.s.d. of 0.8 Å for corresponding StTrpA Cα atoms. A similar pattern is observed for the TrpBs, which overlap with r.m.s.d.s of 0.7–1.0 Å.

[Figure 7]
Figure 7
Comparison of TrpAB orthologs. (a) Superposition of SpTrpB (yellow, TrpA, chain C; coral/cyan, TrpB, chain D) with FtTrpB (blue), MtTrpB (purple; chains A and B; PDB entry 5tcf; Wellington et al., 2017[Wellington, S., Nag, P. P., Michalska, K., Johnston, S. E., Jedrzejczak, R. P., Kaushik, V. K., Clatworthy, A. E., Siddiqi, N., McCarren, P., Bajrami, B., Maltseva, N. I., Combs, S., Fisher, S. L., Joachimiak, A., Schreiber, S. L. & Hung, D. T. (2017). Nature Chem. Biol. 13, 943-950.]) and StTrpB (gray; PDB entry 1bks; Rhee et al., 1996[Rhee, S., Parris, K. D., Ahmed, S. A., Miles, E. W. & Davies, D. R. (1996). Biochemistry, 35, 4211-4221.]). PLP from SpTrpAB is shown in a sphere representation. TrpA is shown to indicate the mutual orientation of the subunits. (b) Superposition of TrpA extracted from the TrpAB heterodimers. (c) Stereoview of the TrpA active-site superposition of SpTrpA (yellow), FtTrpA (blue) and StTrpA in complex with IPP (gray; PDB entry 1qop; Weyand & Schlichting, 1999[Weyand, M. & Schlichting, I. (1999). Biochemistry, 38, 16469-16480.]).

As expected in the absence of any TrpA ligand, the α subunit adopts an open conformation with a disordered loop αL6, regardless of whether the subunit is complexed with TrpB or alone. In isolated LpPhTrpA parts of loop αL2 could not be modeled, indicating its high flexibility. The TrpA binding pocket and these critical loops are generally well conserved in terms of composition, including the catalytic residues, one of which is provided by loop αL2. One important feature, although only noted at the sequence level owing to disorder, is the lack of conservation in the N-terminal region of loop αL6. In the Salmonella enzyme this section carries αArg179, which has been shown to provide loop stabilization via hydrogen bonds between the guanidinium group and the main-chain atoms (Schneider et al., 1998[Schneider, T. R., Gerhardt, E., Lee, M., Liang, P. H., Anderson, K. S. & Schlichting, I. (1998). Biochemistry, 37, 5394-5406.]). With the exception of FtTrpA, this residue is replaced by much smaller and in some cases hydrophobic residues, Ile in SpTrpA, Leu in LpTrpA and Thr in MtTrpA, and cannot form interactions equivalent to those of αArg179. It has previously been shown that an αArg179Leu mutation reduces the affinity of the substrate IGP for StTrpA and slows the TrpAB reaction (Brzović et al., 1993[Brzović, P. S., Hyde, C. C., Miles, E. W. & Dunn, M. F. (1993). Biochemistry, 32, 10404-10413.]). It is not clear that this is a valid assumption for the other orthologs; however, MtTrpAB indeed has a higher Km for IGP than StTrpA. In addition, it is also consistent with the relative rank order of specific activities observed across this panel of TrpAB orthologs, although only in the context of the β reaction.

Within the ordered fragments of the TrpA pocket, some sequence variability is observed at the positions of αPro129Sp (the equivalent residues are αPro135Mt, αAla130Ft, αAla129St and αVal129Lp), αMet100Sp (αMet100Lp and αMet106Mt but αLeu101Ft and αLeu100St) and αTyr23Sp (replaced by Phe in FtTrpA, LpTrpA and StTrpA). Notably, though, despite the good superposition of the main-chain atoms throughout most of the subunit, the side chains adopt slightly different conformations (Fig. 7[link]). The most pronounced discrepancy is observed for αPhe212Sp, a residue that T-stacks against the aromatic ring of indole in the ligand-bound StTrpA state (Weyand & Schlichting, 1999[Weyand, M. & Schlichting, I. (1999). Biochemistry, 38, 16469-16480.]). The position of this residue is affected by the mobile αL6 loop in the substrate-bound closed state that reinforces the proper placement of the Phe side chain with respect to the substrate moiety. Without such constraints, in SpTrpA, as well as in LpTrpA, it points somewhat outside of the binding pocket towards the helical layer of the protein. In FtTrpA it is oriented more towards the cavity, but its position is still halfway from the state achieved in the substrate-bound complex (Fig. 7[link]). Interestingly, this residue is replaced by αLeu218 in the MtTrpA ortholog, where it also swings outside the binding pocket. The catalytic αGlu52Sp and its equivalents in other orthologs also display some conformational diversity; in some cases, such as FtTrpA or StTrpA, it points towards the protein core, while in others (SpTrpA and MtTrpA) it faces the binding pocket. There are no apparent structural differences between TrpA in the TrpAB complex versus TrpA alone. The only exception is a slight movement of loop αL2 towards the active site of TrpA in the αβ heterodimer unit.

In our FtTrpAB and StTrpAB structures the β subunits exist in the open conformation, or more precisely in the expanded open conformation βeO reported previously for several StTrpAB structures [PDB entries 2j9z (Blumenstein et al., 2007[Blumenstein, L., Domratcheva, T., Niks, D., Ngo, H., Seidel, R., Dunn, M. F. & Schlichting, I. (2007). Biochemistry, 46, 14100-14116.]), 1qoq (Weyand & Schlichting, 1999[Weyand, M. & Schlichting, I. (1999). Biochemistry, 38, 16469-16480.]) and 1kfb (Kulik et al., 2002[Kulik, V., Weyand, M., Seidel, R., Niks, D., Arac, D., Dunn, M. F. & Schlichting, I. (2002). J. Mol. Biol. 324, 677-690.])], the P. furiosus ortholog [PDB entries 5e0k (Buller et al., 2015[Buller, A. R., Brinkmann-Chen, S., Romney, D. K., Herger, M., Murciano-Calles, J. & Arnold, F. H. (2015). Proc. Natl Acad. Sci. USA, 112, 14599-14604.]) and 1wdw (Lee et al., 2005[Lee, S. J., Ogasahara, K., Ma, J., Nishio, K., Ishida, M., Yamagata, Y., Tsukihara, T. & Yutani, K. (2005). Biochemistry, 44, 11417-11427.])] and MtTrpAB (PDB entry 5tcf; Wellington et al., 2017[Wellington, S., Nag, P. P., Michalska, K., Johnston, S. E., Jedrzejczak, R. P., Kaushik, V. K., Clatworthy, A. E., Siddiqi, N., McCarren, P., Bajrami, B., Maltseva, N. I., Combs, S., Fisher, S. L., Joachimiak, A., Schreiber, S. L. & Hung, D. T. (2017). Nature Chem. Biol. 13, 943-950.]), suggesting that this state may be more common than previously indicated. The active site carries a PLP moiety covalently attached to βLys91Sp (βLys86Ft, βLys101Mt). The β active site is very conserved both in terms of sequence and the conformation of the PLP cofactor and side chains, with a few exceptions. FtTrpB and SpTrpB share an Ala with StTrpB (βAla84, βAla89 and βAla85, respectively), but MtTrpB has an equivalent βSer99 that makes a direct hydrogen bond to PLP. This interaction is missing in the other three orthologs. βThr87 is present in SpTrpB (and βThr97 in MtTrpB), which is replaced by glycine in FtTrpB and StTrpB. There is no obvious role for this substitution. Two important catalytic residues, threonine (βThr114Sp, βThr109Ft, βThr124Mt and βThr110St) and aspartic acid (βAsp310Sp, βAsp304Ft, βAsp319Mt and βAsp305St), show a very different conformational behavior in the open state of β-subunit orthologs. The threonine, which is involved in coordination of the substrate/product carboxylate, shows nearly the same conformation in all four orthologs, while the conformations of the aspartic acid, which is involved in interaction with the amino group of the reagents, are very different. Larger conformational diversity is also observed for βGln118, a residue that is conserved in all four enzymes. However, only in MtTrpB does this residue form a direct hydrogen bond to O3 of the PLP cofactor. The side chains of a few other residues (βGln89, βSer234 and βLys381 in FtTrpB) also show somewhat different conformations, but these are much less pronounced. The phosphate group of PLP is anchored by interaction with the N-terminal dipole of helix βH9, direct hydrogen bonds to several main-chain amino groups (helix βH9 and a short loop between βS7 and βH9) and three conserved side chains (βHis85, βSer234 and βAsn235 in FtTrpB and βHis90, βSer240 and βAsn241 in SpTrpB). These small changes in sequence and conformational propensity may explain the differences in substrate affinities and reaction rates.

The structures of the FtTrpAB and SpTrpAB αββα heterotetramers provide a new set of high-quality models and enable comparison of the intermolecular tunnel connecting the TrpA and TrpB catalytic pockets. In contrast to the active sites, the composition of the tunnel, which is mostly encompassed by TrpB, varies between the orthologs (Fig. 8[link]), although generally SpTrpAB shares some features with MtTrpAB while FtTrpAB is similar to StTrpAB. This is consistent with the relative specific activities and the conservation of local primary sequence. The cross-comparisons indicate a number of differences. For example, one side of the SpTrpB tunnel contains βTyr311, βHis285 and the neighboring βLeu284, with the tyrosine rotated towards the active site of TrpB, where it could potentially interfere with the β reaction. The opposite side contributes βVal174, βLeu178 and βLeu192. In FtTrpB all of the former residues are replaced by phenyl­alanines (βPhe305, βPhe279 and βPhe278, respectively), while the leucines are conserved and βVal174Sp is replaced by βCys169Ft. A similar scenario is present in StTrpB (βPhe306 and βPhe280), with the exception of βTyr279St, which substitutes for βPhe278Ft. In MtTrpB the equivalent residues are βTyr320, βHis294 and βPhe293, resembling the SpTrpB composition, but in this case the tyrosine ring points in a different direction, making a hydrogen bond to βHis294. Such an arrangement would be more constrained in SpTrpB owing to the proximity of βLeu196, a residue that is substituted by a much smaller Ala in the other enzymes. MtTrpB also contains phenylalanines (βPhe188 and βPhe202) instead of the leucines that are conserved in the three other TrpBs, and βIle184Mt takes the place of βVal174Sp. Previous data for the StTrpB ortholog showed that large side chains, such as Phe or Trp, in this position hamper indole channeling (Anderson et al., 1995[Anderson, K. S., Kim, A. Y., Quillen, J. M., Sayers, E., Yang, X.-J. & Miles, E. W. (1995). J. Biol. Chem. 270, 29936-29944.]; Schlichting et al., 1994[Schlichting, I., Yang, X.-J., Miles, E. W., Kim, A. Y. & Anderson, K. S. (1994). J. Biol. Chem. 269, 26591-26593.]; Weyand & Schlichting, 2000[Weyand, M. & Schlichting, I. (2000). J. Biol. Chem. 275, 41058-41063.]). Therefore, it appears that these variations in the residues composing the tunnel may have a direct impact on the rate of indole transfer and influence the kinetic activities of these enzymes. This may represent a fine-tuning of the enzyme activity without directly involving the residues in the catalytic sites.

[Figure 8]
Figure 8
Comparison of the intersubunit tunnel. (a) Superposition of SpTrpAB (yellow, TrpA, chain C; coral/cyan, TrpB, chain D) with ligand-free MtTrpAB (purple; chains A and B; PDB entry 5tcf; Wellington et al., 2017[Wellington, S., Nag, P. P., Michalska, K., Johnston, S. E., Jedrzejczak, R. P., Kaushik, V. K., Clatworthy, A. E., Siddiqi, N., McCarren, P., Bajrami, B., Maltseva, N. I., Combs, S., Fisher, S. L., Joachimiak, A., Schreiber, S. L. & Hung, D. T. (2017). Nature Chem. Biol. 13, 943-950.]). (b) Superposition of ligand-free MtTrpAB (purple; chains A and B; PDB entry 5tcf) with MtTrpAB in complex with BRD4592 (pink; chains C and D; PDB entry 5tci; Wellington et al., 2017[Wellington, S., Nag, P. P., Michalska, K., Johnston, S. E., Jedrzejczak, R. P., Kaushik, V. K., Clatworthy, A. E., Siddiqi, N., McCarren, P., Bajrami, B., Maltseva, N. I., Combs, S., Fisher, S. L., Joachimiak, A., Schreiber, S. L. & Hung, D. T. (2017). Nature Chem. Biol. 13, 943-950.]). Note that in the shown chain D TrpB adopts the βO state. Chain B exists as a mixture of the βO and βeO states. (c) Superposition of FtTrpAB (green, TrpA; blue/navy, TrpB) with StTrpAB (gray; PDB entry 1bks; Rhee et al., 1996[Rhee, S., Parris, K. D., Ahmed, S. A., Miles, E. W. & Davies, D. R. (1996). Biochemistry, 35, 4211-4221.]). (d) StTrpAB (gray; PDB entry 1bks) with StTrpAB in complex with F6 (black; PDB entry 4wx2; Hilario et al., 2016[Hilario, E., Caulkins, B. G., Huang, Y.-M. M., You, W., Chang, C.-E. A., Mueller, L. J., Dunn, M. F. & Fan, L. (2016). Biochim. Biophys. Acta, 1864, 268-279.]). Key residues are shown in stick representation; inhibitors are shown in ball-and-stick representation.

Generally, the tunnel displays some level of flexibility and can adapt to enable indole translocation or to specifically bind certain inhibitors. For instance, we showed previously that in MtTrpAB βPhe188 changes conformation to accommodate BRD4592 (Wellington et al., 2017[Wellington, S., Nag, P. P., Michalska, K., Johnston, S. E., Jedrzejczak, R. P., Kaushik, V. K., Clatworthy, A. E., Siddiqi, N., McCarren, P., Bajrami, B., Maltseva, N. I., Combs, S., Fisher, S. L., Joachimiak, A., Schreiber, S. L. & Hung, D. T. (2017). Nature Chem. Biol. 13, 943-950.]) both in the open and closed states of the β subunit, while in StTrpAB βPhe280 and βTyr279 swing away to provide space for the F6 molecule (Hilario et al., 2016[Hilario, E., Caulkins, B. G., Huang, Y.-M. M., You, W., Chang, C.-E. A., Mueller, L. J., Dunn, M. F. & Fan, L. (2016). Biochim. Biophys. Acta, 1864, 268-279.]) in the open state (Fig. 8[link]). The latter work also proposed that the indole moiety enters TrpB in the vicinity of αLeu21St (conserved as αLeu24Sp, αLeu34Mt and αLeu20Ft), βLeu174St and βPhe280St, which need to move to open up a farther segment of the channel that is lined with residues that do not present major obvious obstacles. In principle, an analogous mechanism can be envisioned for the very similar enzyme from F. tularensis. In the other two orthologs alternative mechanisms are most likely to exist. In the SpTrpB/MtTrpB structures, in which βPhe280St is replaced by a histidine, this residue adopts a conformation that is compatible with an open channel both in the βO (SpTrpB/MtTrpB) and βC (MtTrpB) states. Moreover, in MtTrpB such an architecture is stabilized by a hydrogen bond to βTyr320Mt (in βO and βC) and another to βAsn185Mt (in MtβC), suggesting that it represents the most common conformational state. An analogous interaction with asparagine might be created in SpTrpB upon subunit closure, while His–Tyr bonding would require the concomitant movement of βTyr311Sp and βLeu196Sp. This coordinated movement is potentially a necessary step for the COMM-domain shift and TrpB closure, as otherwise βLeu170Sp would clash with βTyr311Sp. On the other hand, the mycobacterial enzyme may need to undergo a different adjustment on the opposite side of the tunnel. Here, there are two bulkier phenylalanine residues, βPhe188 and βPhe202. In both cases these residues appear to be mobile, as in some structures of MtTrpAB βPhe202 exists in double conformations while βPhe188 has been shown to rotate in the complex with the BRD4592 inhibitor. However, for βPhe188 in this alternative state the access from subunit α is blocked; thus, it is possible that the ligand-free conformation of βPhe188 corresponds to the open-tunnel state with only a minor adjustment required.

4.2. Allosteric contacts

Previous investigations of allosteric communication between the TrpAB subunits recognized a number of key interactions at the αβ interface that transmit activation signals. One of them is the main-chain–main-chain hydrogen bond between βSer178 and αGly181 in StTrpAB (Spyrakis et al., 2006[Spyrakis, F., Raboni, S., Cozzini, P., Bettati, S. & Mozzarelli, A. (2006). Biochim. Biophys. Acta, 1764, 1102-1109.]; Schneider et al., 1998[Schneider, T. R., Gerhardt, E., Lee, M., Liang, P. H., Anderson, K. S. & Schlichting, I. (1998). Biochemistry, 37, 5394-5406.]). The former residue is preserved in FtTrpB; however, the other two orthologs contain valine. On the other hand, the glycine residue (αGly181Sp, αGly182Ft and αGly187Mt) belongs to the highly conserved GVTG motif of the αL6 loop. In the S. typhimurium TrpA αC state the conserved threonine residue from this motif, αThr183, binds through its hydroxyl group to the carboxylate of the catalytic αAsp60 (αAsp61Sp, αAsp63Ft and αAsp68Mt), in addition to the main-chain–main-chain interaction with the αL2 loop. Deletions or point mutations within the αL6 loop, such as αThr183Ala in StTrpA, dramatically reduce the α-subunit activity (Yang & Miles, 1992[Yang, X.-J. & Miles, E. W. (1992). J. Biol. Chem. 267, 7520-7528.]). Similar modifications in the αL2 loop, including changes to αPro57St (αPro60Sp, αPro58Ft and αPro65Mt) and αAsp56St (αAsp59Sp, αAsp57Ft and αAsp64Mt) reduce TrpA activity, although significant effects only occur in the context of the TrpAB complex, i.e. not when the α subunit alone is assayed (Ogasahara et al., 1992[Ogasahara, K., Hiraga, K., Ito, W., Miles, E. W. & Yutani, K. (1992). J. Biol. Chem. 267, 5222-5228.]; Rowlett et al., 1998[Rowlett, R., Yang, L.-H., Ahmed, S. A., McPhie, P., Jhee, K.-H. & Miles, E. W. (1998). Biochemistry, 37, 2961-2968.]).

In the available βO and βC states of the mycobacterial enzyme, the side chain of αAsp64Mt (the main chain of αSer63 in βeO) interacts with βLys181 from the COMM domain, while the carbonyl group of αAsp68 binds to βArg189 in some of the subunits, as seen before in the StTrpA ortholog (Weyand & Schlichting, 1999[Weyand, M. & Schlichting, I. (1999). Biochemistry, 38, 16469-16480.]). In the SpTrpAB βeO state there is also a hydrogen bond between the αSer58 carbonyl group and βLys171, but βArg179 is too distant to interact with the catalytic aspartate. None of these contacts is observed in the reported FtTrpAB structure, either owing to disorder or to longer distances between the relevant atoms.

Overall, the available data suggest that the geometry and contacts established by loops αL6 and αL2 have a pronounced effect on the enzyme activity. Transition from βO to βC triggers the closure of αL6, which, together with the αL2 and βH6 elements, activates the catalytic aspartate residue. Changes in these elements or in their neighborhood possibly lock αL6 into a low-activity open state (Spyrakis et al., 2006[Spyrakis, F., Raboni, S., Cozzini, P., Bettati, S. & Mozzarelli, A. (2006). Biochim. Biophys. Acta, 1764, 1102-1109.]), thus preventing the proper positioning of the catalytic aspartic acid. Simultaneously with the α-subunit malfunction, destabilization of the αL2–βH6 interactions in mutants reduces the β-subunit activity (Ogasahara et al., 1992[Ogasahara, K., Hiraga, K., Ito, W., Miles, E. W. & Yutani, K. (1992). J. Biol. Chem. 267, 5222-5228.]), with the detrimental effect partly alleviated by cation binding. Monovalent cations have been shown to stabilize the StTrpAB enzyme, with large cations (Cs+ and NH4+) exhibiting the most pronounced effect (Rowlett et al., 1998[Rowlett, R., Yang, L.-H., Ahmed, S. A., McPhie, P., Jhee, K.-H. & Miles, E. W. (1998). Biochemistry, 37, 2961-2968.]). These effects might result from the chain of interactions linking αL2 to βH6 and further, via the monovalent cation-binding site (MVC), to the active site of the β subunit. The MVC is established by a set of residues localized in the proximity of the channel and the active site of TrpB, which interact with the cation through four main-chain carbonyl moieties (in S. typhimurium and M. tuberculosis) and a threonine side chain (only in M. tuberculosis owing to the presence of Pro in the equivalent position in StTrpB). While no monovalent cations have been modeled in the current structures, by analogy to the data collected from the MtTrpAB and StTrpAB systems the MVC must be created by βTyr311Sp, βGly313Sp, βAla273Sp, βGly237Sp and βThr275Sp in SpTrpAB and βPhe305Ft, βSer307Ft, βGly267Ft, βGly231Ft in FtTrpAB, with βPro269Ft replacing the threonine residue. Depending on the size of the cation, either all residues equivalent to those in StTrpAB and MtTrpAB would be involved in cation binding, or only a subset, where the unfilled valencies in the coordination sphere may be completed by water molecules. As mentioned above, the MVC is indirectly connected to the βH6 element of the COMM domain and to TrpA via either a histidine (βHis285Sp and βHis294Mt) or a phenylalanine (βPhe279Ft, βPhe280St), switching between hydrophobic Phe–Phe contacts (FtTrpB and StTrpB) and the well defined His–Tyr hydrogen bond seen in MtTrpB and most likely to be present in the activated form of SpTrpB. It is not clear how this different organization of the MVC and its interactions with other structural elements affect the sensitivity of the protein to different cations or how the signal transduction is affected.

4.3. Enzymatic properties

In the β-elimination reaction of TrpAB, with a kβcat of between 1.7 and 78.6 min−1, all of the investigated enzymes appear to be poorer catalysts of the indole-to-tryptophan conversion than the previously studied MtTrpAB (kβcat = 197 min−1; Wellington et al., 2017[Wellington, S., Nag, P. P., Michalska, K., Johnston, S. E., Jedrzejczak, R. P., Kaushik, V. K., Clatworthy, A. E., Siddiqi, N., McCarren, P., Bajrami, B., Maltseva, N. I., Combs, S., Fisher, S. L., Joachimiak, A., Schreiber, S. L. & Hung, D. T. (2017). Nature Chem. Biol. 13, 943-950.]1), EcTrpAB (348 min−1; Lane & Kirschner, 1983[Lane, A. N. & Kirschner, K. (1983). Eur. J. Biochem. 129, 571-582.]) and StTrpAB (288 min−1; Raboni et al., 2007[Raboni, S., Mozzarelli, A. & Cook, P. F. (2007). Biochemistry, 46, 13223-13234.]), at least under the given experimental conditions: at room temperature (20–22°C) at pH 7.6–8.0 in the presence of potassium ion. Similarly, the Km for serine is at least 35 times higher for the SpTrpAB and FtTrpAB enzymes (18.3–43.2 mM) than for those previously characterized (0.37, 4.4 and 0.58 mM for EcTrpAB, MtTrpAB and StTrpAB, respectively). Interestingly, however, the Km for indole is at least approximately three times lower for all of the currently tested orthologs than those reported for MtTrpAB and StTrpAB and is comparable to that of EcTrpAB.

4.4. Inhibition

Several inhibitors have been designed to study the mechanistic details of TrpAB. A number of them are competitive indole-3-glycerol phosphate analogs that bind to subunit α, such as IPP and similar indole-3-alkyl 1-phosphates (Kirschner et al., 1975[Kirschner, K., Wiskocil, R. L., Foehn, M. & Rezeau, L. (1975). Eur. J. Biochem. 60, 513-523.]), indole-3-acetyl amino acids (Marabotti et al., 2000[Marabotti, A., Cozzini, P. & Mozzarelli, A. (2000). Biochim. Biophys. Acta, 1476, 287-299.]) or aryl compounds linked via an amide/sulfonamide/thioether/thiourea to a phosphoalkyl moiety (Ngo, Harris et al., 2007[Ngo, H., Harris, R., Kimmich, N., Casino, P., Niks, D., Blumenstein, L., Barends, T. R., Kulik, V., Weyand, M., Schlichting, I. & Dunn, M. F. (2007). Biochemistry, 46, 7713-7727.]; Sachpatzidis et al., 1999[Sachpatzidis, A., Dealwis, C., Lubetsky, J. B., Liang, P.-H., Anderson, K. S. & Lolis, E. (1999). Biochemistry, 38, 12665-12674.]). The IC50 parameters for these inhibitors against TrpAB have not been determined, with the exception of thioether-linked substrate analogs (Sachpatzidis et al., 1999[Sachpatzidis, A., Dealwis, C., Lubetsky, J. B., Liang, P.-H., Anderson, K. S. & Lolis, E. (1999). Biochemistry, 38, 12665-12674.]), which showed nanomolar values for the α reaction of StTrpAB. In addition to competitive inhibition of the α reaction, some of the α-binders, for example indole-3-acetyl-amino acids, IPP and F9, exert allosteric effects on subunit β (Marabotti et al., 2000[Marabotti, A., Cozzini, P. & Mozzarelli, A. (2000). Biochim. Biophys. Acta, 1476, 287-299.]; Ngo, Harris et al., 2007[Ngo, H., Harris, R., Kimmich, N., Casino, P., Niks, D., Blumenstein, L., Barends, T. R., Kulik, V., Weyand, M., Schlichting, I. & Dunn, M. F. (2007). Biochemistry, 46, 7713-7727.]). The more promiscuous ligand F6 has been found to bind not only to the active site of TrpA but also to the intersubunit tunnel, close to the β active site (Hilario et al., 2016[Hilario, E., Caulkins, B. G., Huang, Y.-M. M., You, W., Chang, C.-E. A., Mueller, L. J., Dunn, M. F. & Fan, L. (2016). Biochim. Biophys. Acta, 1864, 268-279.]). The influence of competitive inhibitors of TrpA on the TrpB reaction has been linked to their ability to remodel the α site, with the higher degree of ordered TrpA structure triggering more pronounced changes in TrpB (Ngo, Harris et al., 2007[Ngo, H., Harris, R., Kimmich, N., Casino, P., Niks, D., Blumenstein, L., Barends, T. R., Kulik, V., Weyand, M., Schlichting, I. & Dunn, M. F. (2007). Biochemistry, 46, 7713-7727.]).

Here, we have tested the commercially available compounds IPP, F6 and F9 against the β reaction. Notably, we observed potent inhibition only for FtTrpAB, which is the most similar to the prototypical StTrpAB of all the tested enzymes. It therefore seems that the allosteric effect influencing the activity of TrpB is sensitive to local sequence variations and structural features, and consequently might be unique to a subset of orthologs. Alternatively, it is also possible that the lack of TrpB susceptibility originates directly from the poor affinity of these inhibitors for TrpA, but we have not investigated such a scenario biochemically. From a structural perspective, the TrpA active sites are similar enough to at least bind to the very close substrate mimetic IPP, suggesting that the former argument for the lack of inhibition is more likely. Another explanation of these differences involves long-distance effects within and between subunits. The activation of SpTrpAB by the α-binders is unexpected and surprising. However, allosteric sites serve modulatory purposes and a single binding pocket may exert activatory or inhibitory roles. It is therefore possible that the binding of the same ligands to various TrpAB orthologs may result in opposite kinetic effects because of small sequence variations.

In agreement with our previous work demonstrating that BRD4592 inhibition is limited to orthologs containing a glycine residue in the αL2 loop of TrpAB, such as in the case of the MtTrpAB enzyme, no significant effect was observed for all of the tested synthases. The weak inhibition of SpTrpAB, which carries the smallest side chain among the tested enzymes (αVal61 in place of αGly66 in MtTrpAB, αLeu59 in FtTrpAB and αMet58 in LpPhTrpAB), supports the previous conclusion that any substitution in the loop would drastically reduce the size of the BRD4592 binding pocket, limiting the inhibitor affinity.

5. Conclusions

Tryptophan synthases have been shown to be conditionally essential enzymes in a number of important human pathogens, but the enzymes of the family have remained unexplored beyond a limited number of representatives. To broaden our perspective on TrpABs, we have purified and characterized three enzymes from L. pneumophila, F. tularensis and S. pneumoniae to uncover the potential unique features of TrpABs and to support future drug-discovery efforts. X-ray crystallography and biochemical studies show a remarkable structural conservation of the architecture and the catalytic and allosteric sites of the enzyme, suggesting preservation of the catalytic mechanism and regulation. At the same time, these enzymes display local sequence and structural differences in the catalytic, allosteric and metal-binding sites. These enzymes also exhibit differences in kinetic properties and their response to inhibitors, yet they display some correlations between biochemical properties and sequence/structural conservation. Notably, not all enzymes were inhibited by the tested compounds. In fact, for the S. pneumoniae ortholog the reaction was more efficient in the presence of α-binders. Some of the differences can be explained structurally; however, others may result from the altered conditions in which these enzymes operate in cellulo. Nevertheless, understanding these dissimilarities may provide a basis for the design of new species-specific tryptophan synthase inhibitors against both the α and β active sites as well as the allosteric sites, which show higher conformational and sequence variability. Recognition that the targeting of unique allosteric sites may have species-specific effects may be important for the treatment of coexisting infections.

Supporting information


Footnotes

1Note that in the current experiments the parameters for MtTrpAB are somewhat worse than in the previous study, either owing to slow deterioration of the protein at −80°C or variations in the experimental setup.

Acknowledgements

Conflict of interest: the authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. K. Michalska, C. Chang and B. Nocek performed the X-ray structure determination; G. Joachimiak, C. Hatzos-Skintges and L. Bigelow purified, characterized and crystallized proteins for biochemical and biophysical studies; R. P. Jedrzejczak and M. Endres cloned genes and expressed proteins; P. P. Nag synthesized inhibitors; J. Gale, S. E. Johnston, B. Bajrami and S. Wellington performed biochemical, kinetic and inhibitor-sensitivity experiments; A. Joachimiak, K. Michalska, D. T. Hung and S. L. Fisher designed experiments and analyzed data, and together with J. Gale wrote the paper. The beamlines are supported by the US Department of Energy, Office of Biological and Environmental Research under contract DE-AC02-06CH11357.

Funding information

This research was funded by National Institutes of Health grant GM115586 and contracts HHSN272201200026C and HHSN272201700060C.

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