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

Journal logoSTRUCTURAL
BIOLOGY
ISSN: 2059-7983

The structure of the AliC GH13 α-amylase from Ali­cyclobacillus sp. reveals the accommodation of starch branching points in the α-amylase family

aYork Structural Biology Laboratory, Department of Chemistry, The University of York, York YO10 5DD, England, bDepartment of Chemistry, Technical University of Denmark, 2800 Lyngby, Denmark, and cNovozymes A/S, Krogshoejvej 36, 2880 Bagsvaerd, Denmark
*Correspondence e-mail: keith.wilson@york.ac.uk, gideon.davies@york.ac.uk

Edited by Z. S. Derewenda, University of Virginia, USA (Received 2 July 2018; accepted 21 October 2018; online 4 January 2019)

α-Amylases are glycoside hydrolases that break the α-1,4 bonds in starch and related glycans. The degradation of starch is rendered difficult by the presence of varying degrees of α-1,6 branch points and their possible accommodation within the active centre of α-amylase enzymes. Given the myriad industrial uses for starch and thus also for α-amylase-catalysed starch degradation and modification, there is considerable interest in how different α-amylases might accommodate these branches, thus impacting on the potential processing of highly branched post-hydrolysis remnants (known as limit dextrins) and societal applications. Here, it was sought to probe the branch-point accommodation of the Alicyclobacillus sp. CAZy family GH13 α-amylase AliC, prompted by the observation of a molecule of glucose in a position that may represent a branch point in an acarbose complex solved at 2.1 Å resolution. Limit digest analysis by two-dimensional NMR using both pullulan (a regular linear polysaccharide of α-1,4, α-1,4, α-1,6 repeating trisaccharides) and amylopectin starch showed how the Alicyclo­bacillus sp. enzyme could accept α-1,6 branches in at least the −2, +1 and +2 subsites, consistent with the three-dimensional structures with glucosyl moieties in the +1 and +2 subsites and the solvent-exposure of the −2 subsite 6-hydroxyl group. Together, the work provides a rare insight into branch-point acceptance in these industrial catalysts.

1. Introduction

The enzymatic hydrolysis of starch is not merely central to human health and nutrition, but also to a vast and diverse array of industries. Starch degradation is central to the production of high-fructose corn syrups, modern detergents and starch-derived biofuels, in brewing and fermentation, and in the adhesive, textile and paper sectors. The estimated value of the starch market in 2018 has been proposed to be around $77 billion, growing at a rate of greater than 7% per year (http://www.prweb.com/pdfdownload/10923341.pdf; de Souza & de Oliveira e Magalhães, 2010[Souza, P. M. de & de Oliveira e Magalhães, P. (2010). Braz. J. Microbiol. 41, 850-861.]). There is thus a massive interest in the enzymatic degradation and modification of starch from both academic and industrial perspectives (van der Maarel et al., 2002[Maarel, M. J. E. C. van der, van der Veen, B., Uitdehaag, J. C., Leemhuis, H. & Dijkhuizen, L. (2002). J. Biotechnol. 94, 137-155.]; Liu & Xu, 2008[Liu, X. D. & Xu, Y. (2008). Bioresour. Technol. 99, 4315-4320.]). Indeed, the engineering of starch-degrading enzymes, informed by their three-dimensional structure, has been important for their application (reviewed, for example, in Shaw et al., 1999[Shaw, A., Bott, R. & Day, A. G. (1999). Curr. Opin. Biotechnol. 10, 349-352.]; Nielsen & Borchert, 2000[Nielsen, J. E. & Borchert, T. V. (2000). Biochim. Biophys. Acta, 1543, 253-274.]). Starch degradation requires a consortium of enzymes, notably endo-acting α-amylases (EC 3.2.1.1) and chain-end-acting glucoamylases (EC 3.2.1.3) in microbes. In recent times these two players have been accompanied by copper-dependent lytic polysaccharide monooxygenases that break down starch, including highly recalcitrant forms, through an oxidative mechanism (Vu et al., 2014[Vu, V. V., Beeson, W. T., Span, E. A., Farquhar, E. R. & Marletta, M. A. (2014). Proc. Natl Acad. Sci. USA, 111, 13822-13827.]; Lo Leggio et al., 2015[Lo Leggio, L., Simmons, T. J., Poulsen, J. C., Frandsen, K. E., Hemsworth, G. R., Stringer, M. A., von Freiesleben, P., Tovborg, M., Johansen, K. S., De Maria, L., Harris, P. V., Soong, C. L., Dupree, P., Tryfona, T., Lenfant, N., Henrissat, B., Davies, G. J. & Walton, P. H. (2015). Nature Commun. 6, 5961.]).

The majority of endo-acting α-amylases in industrial starch-degradation processes are CAZy (http://www.cazy.org; see Lombard et al., 2014[Lombard, V., Golaconda Ramulu, H., Drula, E., Coutinho, P. M. & Henrissat, B. (2014). Nucleic Acids Res. 42, D490-D495.]) family GH13 enzymes. GH13 is one of the most well studied glycoside hydrolase families (reviewed in CAZypedia at http://www.cazypedia.org/index.php/Glycoside_Hydrolase_Family_13; The CAZypedia Consortium, 2018[The CAZypedia Consortium (2018). Glycobiology, 28, 3-8.]). Over 111 different three-dimensional structures of GH13 enzymes are now known (see http://www.cazy.org/GH13_structure.html). One particularly important subset of GH13 enzymes are the `Termamyl'-like α-amylases, historically named after an enzyme from Bacillus licheniformis. These enzymes typically feature a three-domain `A, B, C' arrangement with a C-terminal β-sheet domain and with domain B being a protrusion from the (β/α)8 fold of domain A. The catalytic centre is placed in domain A, whilst the A–B interface forms the substrate-binding cleft. Many three-dimensional structures of `Termamyl'-like α-amylases are known. Some notable members include that from B. licheniformis (Machius et al., 1995[Machius, M., Wiegand, G. & Huber, R. (1995). J. Mol. Biol. 246, 545-559.]), a chimeric B. licheniformis/B. amyloliquefaciens enzyme (Brzozowski et al., 2000[Brzozowski, A. M., Lawson, D. M., Turkenburg, J. P., Bisgaard-Frantzen, H., Svendsen, A., Borchert, T. V., Dauter, Z., Wilson, K. S. & Davies, G. J. (2000). Biochemistry, 39, 9099-9107.]), an enzyme from Geobacillus stearothermophilus (Suvd et al., 2001[Suvd, D., Fujimoto, Z., Takase, K., Matsumura, M. & Mizuno, H. (2001). J. Biochem. 129, 461-468.]) and an enzyme from B. halmapalus (Davies et al., 2005[Davies, G. J., Brzozowski, A. M., Dauter, Z., Rasmussen, M. D., Borchert, T. V. & Wilson, K. S. (2005). Acta Cryst. D61, 190-193.]). Notably, as well as having stabilizing Ca2+ ions in various domains, a characteristic Ca2+–Na+–Ca2+ triad is observed at the A/B-domain interface (for the historical context, see Machius et al., 1998[Machius, M., Declerck, N., Huber, R. & Wiegand, G. (1998). Structure, 6, 281-292.]; Brzozowski et al., 2000[Brzozowski, A. M., Lawson, D. M., Turkenburg, J. P., Bisgaard-Frantzen, H., Svendsen, A., Borchert, T. V., Dauter, Z., Wilson, K. S. & Davies, G. J. (2000). Biochemistry, 39, 9099-9107.]).

Currently, the CAZy classification lists over 100 different three-dimensional structures of α-amylases from family GH13. Remarkably, to our knowledge only one of these, the Bacteroides thetaiotaomicron SusG protein, contains a branched substrate within its active centre. In this case, following elegant work by the Koropatkin and Brumer groups (Arnal et al., 2018[Arnal, G., Cockburn, D. W., Brumer, H. & Koropatkin, N. M. (2018). Protein Sci. 27, 1093-1101.]), an α-1,6 branch was observed in the +1 subsite. The GlgE protein from Streptomyces coelicolor (PDB entry 5lgw; Syson et al., 2016[Syson, K., Stevenson, C. E., Miah, F., Barclay, J. E., Tang, M., Gorelik, A., Rashid, A. M., Lawson, D. M. & Bornemann, S. (2016). J. Biol. Chem. 291, 21531-21540.]) also contains a branched oligosaccharide, but this ligand is bound far from the active centre and is instead located on a distal starch-binding domain. Here, we report the three-dimensional structure of a `Termamyl'-like α-amylase, the AliC α-amylase from Alicyclobacillus sp. 18711. An initial ligand-bound structure with a transglycosyl­ated acarbose-derived oligosaccharide at a resolution of 2.1 Å revealed a noncovalently linked glucose moiety, hinting at a putative branch-accommodation site around the +2/+3 subsites. A subsequent lower resolution (approximately 3 Å) analysis revealed the binding of a branched ligand in the +1/+2 subsites with an α-1,6-linked glucose branch bound to the +1 subsite sugar. Motivated by these observations, two-dimensional NMR was used to map the subsite branch preferences on the basis of the structures of the observed limit dextrin products, highlighting how the AliC α-amylase can accommodate amylopectin and pullulan substrates.

2. Methods

2.1. Crystallization

Alicyclobacillus sp. 18711 α-amylase (GenBank MH533021) was a kind gift from Novozymes A/S (Bagsvaerd, Denmark), where it had been cloned in a strain variant of B. subtilis PL1801 from Alicyclobacillus sp. 18711 isolated from a Danish forest floor. A two-amino-acid deletion (T182*G183*) was introduced by SOE PCR (Higuchi et al., 1988[Higuchi, R., Krummel, B. & Saiki, R. K. (1988). Nucleic Acids Res. 16, 7351-7367.]) using synthetic oligonucleotides purchased from Invitrogen, and the α-amylase variant was expressed by fermenting at 37°C for four days in a soy- and starch-based broth.

The fermentation supernatant was filtrated through a 0.45 µm filter followed by filtration through a 0.2 µm filter. After the addition of 1 M ammonium sulfate and adjustment of the pH to pH 8, the supernatant was applied onto a 69 ml Butyl TOYOPEARL column. Prior to loading, the column had been equilibrated in three column volumes (CV) of 25 mM borate pH 8, 2 mM CaCl2, 1 M ammonium sulfate. In order to remove unbound material, the column was washed with 3 CV of 25 mM borate pH 8, 2 mM CaCl2, 1 M ammonium sulfate. Elution of the target protein was obtained with a decreasing salt gradient from 1 to 0 M ammonium sulfate in 25 mM borate pH 8, 2 mM CaCl2, followed by 3 CV of 100% 25 mM borate pH 8, 2 mM CaCl2. The flow rate was 10 ml min−1. Relevant fractions were selected and pooled based on the chromatogram and on SDS–PAGE analysis. The amylase activity of the purified enzymes was confirmed using the AMYL liquid amylase assay (Roche/Hitachi system).

2.1.1. Acarbose complex

For the acarbose complex, co-crystallization screening was carried out using sitting-drop vapour diffusion with drops set up using a Mosquito Crystal liquid-handling robot (TTP LabTech, UK) with 150 nl protein solution plus 150 nl reservoir solution in 96-well format plates (MRC 2-well crystallization microplates; Swissci, Switzerland) equilibrated against 54 µl reservoir solution. Initial crystallization experiments were carried out at room temperature using a number of commercial screens. Diffraction-quality crystals were obtained in PACT screen condition G11 [0.2 M sodium citrate, 0.1 M bis-tris propane (BTP) pH 6.5, 20% PEG 3350]. The crystals were tested in-house prior to being sent to the synchrotron. Crystallization conditions are given in Table 1[link].

Table 1
Crystallization

  Acarbose complex Branched-ligand complex
Method Vapour diffusion, sitting drop Vapour diffusion, hanging drop
Plate type Swissci 96-well Linbro 24-well
Temperature (K) 293 293
Protein concentration (mg ml−1) 20 20
Buffer composition of protein solution 25 mM borate, 2 mM CaCl2 pH 8 + 40 mM acarbose 25 mM borate, 2 mM CaCl2 pH 8 + 20 mM GMT
Composition of reservoir solution 0.2 M sodium citrate, 0.1 M BTP pH 6.5, 20% PEG 3350 (PACT G11) 20%(w/v) PEG 3350, 0.1 M BTP pH 8.5, 0.2%(w/v) sodium sulfate
Volume and ratio of drop 300 nl total, 1:1 ratio 1 µl total, 1:1 ratio
Volume of reservoir (µl) 54 1000
2.1.2. Branched-ligand complex

Crystals of the complex with 20 mM 63-α-D-glucosyl-maltotriose (GMT; a branched ligand) were obtained by manual optimization in a 24-well Linbro tray (hanging drops) in 20% PEG 3350, 0.1 M BTP pH 8.5, 0.2% sodium sulfate with seeding. The initial seeding stock was prepared by crushing crystals of the acarbose complex, adding 50 µl mother liquor and vortexing the mixture for 1 min using a Seed Bead (Hampton Research), based on the protocol described in D'Arcy et al. (2014[D'Arcy, A., Bergfors, T., Cowan-Jacob, S. W. & Marsh, M. (2014). Acta Cryst. F70, 1117-1126.]). Different seed dilutions were screened; the final crystals grew using a 1:1000 seed dilution.

Details of the crystallization experiments are given in Table 1[link].

2.2. Data collection and processing, structure solution and refinement

Computations were carried out using programs from the CCP4 suite (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.]) unless otherwise stated. For the structure of the acarbose complex, data were collected to 2.1 Å resolution on beamline I04 at Diamond Light Source (DLS). The crystal belonged to space group P41212, with unit-cell parameters a = b = 180.90, c = 77.85 Å. The data were processed with xia2 (Winter et al., 2013[Winter, G., Lobley, C. M. C. & Prince, S. M. (2013). Acta Cryst. D69, 1260-1273.]). The structure was solved using MOLREP (Vagin & Teplyakov, 2010[Vagin, A. & Teplyakov, A. (2010). Acta Cryst. D66, 22-25.]) with the maltohexaose-producing amylase from alkalophilic Bacillus sp. 707 as a search model (PDB entry 1wp6; Kanai et al., 2004[Kanai, R., Haga, K., Akiba, T., Yamane, K. & Harata, K. (2004). Biochemistry, 43, 14047-14056.]).

For the branched-ligand complex, data were collected to 2.95 Å resolution on beamline I04 at DLS. The crystals belonged to space group P61, with unit-cell parameters a = b = 212.18, c = 172.22 Å. The data were processed with xia2 (Winter et al., 2013[Winter, G., Lobley, C. M. C. & Prince, S. M. (2013). Acta Cryst. D69, 1260-1273.]). The structure was solved by MOLREP using the acarbose complex (minus all ligands) as a search model. Data-collection statistics are given in Table 2[link].

Table 2
Data collection and processing

Values in parentheses are for the outer shell.

  Acarbose complex Branched-ligand complex
Diffraction source I04, DLS I04, DLS
Wavelength (Å) 0.98 0.98
Temperature (K) 100 100
Detector ADSC Quantum 315 PILATUS 6M Prosport+
Crystal-to-detector distance (mm) 254.8 475.8
Rotation range per image (°) 0.5 0.2
Total rotation range (°) 180 180
Exposure time per image (s) 0.5 0.2
Space group P41212 P61
a, b, c (Å) 180.9, 180.9, 77.85 212.18, 212.18, 172.22
α, β, γ (°) 90, 90, 90 90, 90, 120
Mosaicity (°) 0.20 0.13
Resolution range (Å) 57.21–2.07 (2.12–2.07) 66.9–2.95 (3.03–2.95)
Total No. of reflections 1147294 (85223) 921282 (71482)
No. of unique reflections 78951 (5774) 92496 (6812)
Completeness (%) 99.6 (100) 100 (100)
Multiplicity 14.5 (14.8) 10.0 (10.5)
CC1/2 1.0 (0.907) 0.998 (0.914)
I/σ(I)〉 16.7 (4.5) 13.4 (2.7)
Rmerge (%) 17.3 (74.5) 12.6 (74.8)
Rr.i.m. (%) 18.4 (79.9) 14.1 (83.1)
Overall B factor from Wilson plot (Å2) 15.4 50.9
†CC1/2 values for Imean are calculated by splitting the data randomly into half data sets.
‡Estimated Rr.i.m. = Rmerge [N/(N − 1)]1/2, where N is the data multiplicity and Rmerge is defined as [\textstyle \sum_{hkl}\sum_{i}|I_{i}(hkl)- \langle I(hkl)\rangle|/][\textstyle \sum_{hkl}\sum_{i}I_{i}(hkl)], where I(hkl) is the intensity of the reflection.

Both structures were refined by REFMAC (Murshudov et al., 2011[Murshudov, G. N., Skubák, P., Lebedev, A. A., Pannu, N. S., Steiner, R. A., Nicholls, R. A., Winn, M. D., Long, F. & Vagin, A. A. (2011). Acta Cryst. D67, 355-367.]) iterated with manual model correction using Coot (Emsley et al., 2010[Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. (2010). Acta Cryst. D66, 486-501.]). Those monosaccharides that were expected to be in their minimal energy conformation (4C1 for D-glucopyranose) were additionally restrained to adopt torsional values consistent with such a conformation. This was performed using a dictionary containing unimodal dihedral restraints produced by Privateer (Agirre et al., 2015[Agirre, J., Iglesias-Fernández, J., Rovira, C., Davies, G. J., Wilson, K. S. & Cowtan, K. D. (2015). Nature Struct. Mol. Biol. 22, 833-834.]). Including these restraints in the refinement caused the Rfree values to decrease for both structures. The final R and Rfree are 0.138 and 0.176 for the acarbose ligand complex and 0.156 and 0.183 for the branched-ligand complex, respectively. Validation was performed using 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.]), EDSTATS (Tickle, 2012[Tickle, I. J. (2012). Acta Cryst. D68, 454-467.]) and Privateer (Agirre et al., 2015[Agirre, J., Iglesias-Fernández, J., Rovira, C., Davies, G. J., Wilson, K. S. & Cowtan, K. D. (2015). Nature Struct. Mol. Biol. 22, 833-834.]) through the use of the CCP4i2 interface (Potterton et al., 2018[Potterton, L., Agirre, J., Ballard, C., Cowtan, K., Dodson, E., Evans, P. R., Jenkins, H. T., Keegan, R., Krissinel, E., Stevenson, K., Lebedev, A., McNicholas, S. J., Nicholls, R. A., Noble, M., Pannu, N. S., Roth, C., Sheldrick, G., Skubak, P., Turkenburg, J., Uski, V., von Delft, F., Waterman, D., Wilson, K., Winn, M. & Wojdyr, M. (2018). Acta Cryst. D74, 68-84.]). During this work, a MolProbity graphical interface for CCP4i2 was developed. Aside from supporting the usual reporting, the functionality of the interface was extended to cover automated 180° rotation of suggested histidine, asparagine and glutamine side chains around the last χ angle, with an additional real-space refinement step, and real-time compression and decompression of the results from PROBE, with a typical ratio of reduction in file size of 8:1. This new interface is available in CCP4 through use of the `Analyse model geometry' task.

Data-processing and refinement statistics for both structures are given in Table 3[link].

Table 3
Structure solution and refinement

  Acarbose complex Branched-ligand complex
PDB code 6gxv 6gya
Resolution range (Å) 57.21–2.07 66.86–2.95
No. of reflections, working set 78885 92448
No. of reflections, test set 3965 4492
Final Rcryst 0.138 0.156
Final Rfree 0.176 0.183
Cruickshank DPI 0.14 0.37
No. of non-H atoms
 Protein 7770 15151
 Ion 8 16
 Ligand 272 312
 Water 1092 224
 Total 9142 15703
R.m.s. deviations
 Bonds (Å) 0.012 0.016
 Angles (°) 1.5 1.8
Average B factors2)
 Protein 18 (6) 61 (13)
 Ion 18 (8) 60 (9)
 Ligand 34 (16) 92 (26)
 Water 33 (13) 48 (10)
Ramachandran plot
 Most favoured (%) 920 (96.03%) 1830 (95.51%)
 Allowed (%) 33 (3.44%) 76 (3.97%)
 Outliers (%) 5 (0.52%) 10 (0.52%)
†Values in parentheses are standard deviations.

2.3. Degradation of pullulan and amylopectin by the purified Alicyclobacillus α-amylase

The enzymatic specificity of the Alicyclobacillus α-amylase was experimentally determined to complement the search for potential branch-point accommodation in the active site in the crystal structures. To this end, pullulan and amylopectin were subjected to degradation prior to NMR analysis of the fragments formed. Pullulan (Sigma–Aldrich, St Louis, Missouri, USA) samples were degraded by purified Alicyclobacillus α-amylase at room temperature and samples were withdrawn, inactivated at 90°C for 10 min, condensed by lyophilization and redissolved in 600 µl D2O (99.9%; Cambridge Isotope Laboratories, Andover, Massachusetts, USA) to obtain partially degraded and fully degraded samples. The samples were transferred to 5 mm NMR sample tubes for analysis. Amylopectin (from potato starch; Sigma–Aldrich) was incubated at 30°C overnight with the purified Alicyclobacillus α-amylase, inactivated at 90°C for 10 min, condensed by lyophilization and redissolved in 600 µl D2O for NMR analysis.

2.4. NMR spectroscopy

All NMR spectra were recorded on an 800 MHz Avance II spectrometer (Bruker, Fällanden, Switzerland) equipped with a TCI Z-gradient CryoProbe and an 18.7 T magnet (Oxford Magnet Technology, Oxford, England). Highly resolved 1H–13C HSQC spectra employing a sweep width of 10 p.p.m. centred near the 13C chemical shift of the α-anomeric signals were recorded as data matrices of 1024 × 256 complex data points sampling acquisition times of 143 and 127 ms in the 1H and 13C dimensions, respectively. High-precision signal measurements in the two-dimensional spectra were thus used to enumerate the number of signals in the resultant reaction products and for the identification of the products by comparison with authentic standards including glucose, malto­oligosaccharides, panose and limit dextrins (Petersen et al., 2014[Petersen, B. O., Hindsgaul, O. & Meier, S. (2014). Analyst, 139, 401-406.], 2015[Petersen, B. O., Motawie, M. S., Møller, B. L., Hindsgaul, O. & Meier, S. (2015). Carbohydr. Res. 403, 149-156.]).

All spectra were processed with extensive zero filling in both dimensions using a shifted sine-bell apodization function and were analysed with TopSpin 2.1 pl 5 (Bruker).

3. Results and discussion

3.1. Three-dimensional structure of AliC α-amylase and its acarbose-derived complex

The complex of AliC with acarbose was solved by molecular replacement, with two molecules of AliC in the asymmetric unit, at a resolution of 2.1 Å. The fold, as expected, is a canonical three-domain arrangement with the A, B and C domains defined approximately as A, residues 4–104 and 210–397; B, residues 105–209; and C, residues 398–484. A classical Ca2+–Na+–Ca2+ triad (Machius et al., 1998[Machius, M., Declerck, N., Huber, R. & Wiegand, G. (1998). Structure, 6, 281-292.]; Brzozowski et al., 2000[Brzozowski, A. M., Lawson, D. M., Turkenburg, J. P., Bisgaard-Frantzen, H., Svendsen, A., Borchert, T. V., Dauter, Z., Wilson, K. S. & Davies, G. J. (2000). Biochemistry, 39, 9099-9107.]) is found at the A/B-domain interface. At the time of writing, structural similarity searches using PDBeFold (Krissinel & Henrick, 2004[Krissinel, E. & Henrick, K. (2004). Acta Cryst. D60, 2256-2268.]) showed that the closest three-dimensional match to AliC is the B. halmapalus α-amylase (Davies et al., 2005[Davies, G. J., Brzozowski, A. M., Dauter, Z., Rasmussen, M. D., Borchert, T. V. & Wilson, K. S. (2005). Acta Cryst. D61, 190-193.]), with 67% sequence identity and with 479 aligned Cα atoms overlapping with an r.m.s.d. of 0.49 Å (PDBeFold Q score 0.95, Z-score 27.8). Other close structural homologs are the malto­hexaose-producing amylase from Bacillus sp. 707 (Kanai et al., 2004[Kanai, R., Haga, K., Akiba, T., Yamane, K. & Harata, K. (2004). Biochemistry, 43, 14047-14056.]) and the calcium-free amylase AmyK38 from Bacillus sp. strain KSM-K38 (Nonaka et al., 2003[Nonaka, T., Fujihashi, M., Kita, A., Hagihara, H., Ozaki, K., Ito, S. & Miki, K. (2003). J. Biol. Chem. 278, 24818-24824.]).

The structure of AliC was determined in the presence of the inhibitor acarbose. As with many (retaining) α-amylase complexes [some examples from the author's laboratory include those reported in Brzozowski et al. (2000[Brzozowski, A. M., Lawson, D. M., Turkenburg, J. P., Bisgaard-Frantzen, H., Svendsen, A., Borchert, T. V., Dauter, Z., Wilson, K. S. & Davies, G. J. (2000). Biochemistry, 39, 9099-9107.]), Davies et al. (2005[Davies, G. J., Brzozowski, A. M., Dauter, Z., Rasmussen, M. D., Borchert, T. V. & Wilson, K. S. (2005). Acta Cryst. D61, 190-193.]), Brzozowski & Davies (1997[Brzozowski, A. M. & Davies, G. J. (1997). Biochemistry, 36, 10837-10845.]), Dauter et al. (1999[Dauter, Z., Dauter, M., Brzozowski, A. M., Christensen, S., Borchert, T. V., Beier, L., Wilson, K. S. & Davies, G. J. (1999). Biochemistry, 38, 8385-8392.]) and Offen et al. (2015[Offen, W. A., Viksoe-Nielsen, A., Borchert, T. V., Wilson, K. S. & Davies, G. J. (2015). Acta Cryst. F71, 66-70.])], the acarbose is observed as a transglycosylated species, here a hexasaccharide which contains two of the acarviosin disaccharide motifs. The complex defines six subsites, −4 to +2, with the expected catalytic GH13 signature triad of Asp234 (nucleophile), Glu265 (acid/base) and Asp332 (interacting with O2/O3 of the −1 subsite sugar) all disposed for catalysis, here around the 2H3 half-chair of the unsaturated cyclohexitol moiety.

3.2. Limit digest analysis of the action of AliC on pullulan and amylopectin

Of particular interest to us was the observation of a `lone' ordered glucose moiety that was not covalently linked to the acarbose-derived oligosaccharide in a position that could be indicative of the accommodation of branch points at either the +2 or +3 positions of AliC. An additional isolated glucose molecule, modelled in both α and β anomeric forms (occupancy set at 0.5 for each and in different orientations; omitted from Fig. 1[link] for clarity), was observed near a potential +3 site. We speculated whether these isolated glucose moieties could provide insight into branch-point recognition. The accommodation of branch points was therefore investigated by an analysis of limit digestion products (the characteristic oligosaccharides remaining after enzymatic digestion) on both pullulan (a regular linear polysaccharide of α-1,4, α-1,4, α-1,6 repeating trisaccharides) and amylopectin, an α-1,6-branched starch structure. The action of AliC on pullulan results in the production of the trisaccharide panose, glucose α-1,6-glucose α-1,4-glucose (Fig. 2[link]), demonstrating that the enzyme must be able to accommodate α-1,6 linkages to glucose moieties in both the +1 and −2 subsites.

[Figure 1]
Figure 1
Three-dimensional structure of the Alicyclobacillus α-amylase AliC. (a) Three-dimensional protein cartoon, coloured by domain, with metal ions shown as shaded spheres and the acarbose and the +2′ glucose shown as van der Waals spheres. (b) Electron density for the transglycosylated acarbose in subsites −4 to +2 (and +2′ Glc) binding; density for an isolated `+3' glucose is not shown. (c) Electron density for the binding of the branched oligosaccharide in subsites +1, +1 and +1′. Electron-density maps are REFMAC maximum-likelihood-weighted 2FoFc syntheses contoured at 1σ. This figure was drawn with CCP4mg (McNicholas et al., 2011[McNicholas, S., Potterton, E., Wilson, K. S. & Noble, M. E. M. (2011). Acta Cryst. D67, 386-394.]).
[Figure 2]
Figure 2
HSQC spectra of pullulan degradation by purified Alicyclobacillus α-amylase recorded with extensive sampling of the 13C dimension. Only the α-anomeric spectral region is shown. Three different glucopyranosyl units occur in pullulan (left). Signals of α-1,6 anomeric glucopyranosyl units at the nonreducing end emerge (see the inset in the middle spectrum) owing to cleavage at the indicated position (middle top). Pullulan is degraded to panose as the final product (right), as demonstrated by comparison with an authentic standard (grey). These experiments identify the pullulan-cleaving activity of Alicyclobacillus α-amylase as that of a panose-forming neopullulanase (EC 3.2.1.135).

Action on amylopectin produced the limit dextrin 62-α-maltosyl maltotriose (for NMR assignments, see Petersen et al., 2015[Petersen, B. O., Motawie, M. S., Møller, B. L., Hindsgaul, O. & Meier, S. (2015). Carbohydr. Res. 403, 149-156.]; Jodelet et al., 1998[Jodelet, A., Rigby, N. M. & Colquhoun, I. J. (1998). Carbohydr. Res. 312, 139-151.]) (Fig. 3[link]) demonstrating that AliC must also be able to accommodate starches with α-1,6 branches in both the −2 and +2 subsites. Taken together, the action on pullulan and amylopectin shows that AliC is able to accommodate 1,6 linkages in the −2, +1 and +2 subsites (Fig. 4[link]).

[Figure 3]
Figure 3
1H–13C HSQC NMR spectrum of degradation products from potato amylopectin using purified Alicyclobacillus α-amylase. Glucose, maltose, maltotriose and 62-α-maltosylmaltotriose are the main products. The asterisk denotes the overlapping reducing-end α-glucopyranosyl signal of oligosaccharides. Several signals are detected for each molecule, as the individual glucopyranosyl units in oligosaccharides give separate signals.
[Figure 4]
Figure 4
Interpretation of the limit digest patterns in terms of protein structure. Black and red arrows indicate the two/three places at which bonds must be cut to accommodate the limit dextrins observed. To generate panose from pullulan, both the +1 and −2 subsites must accommodate α-1,6 branches. The branched-ligand complex shows how subsite +1 can accommodate a branch (Fig. 1c), and in subsite −2 the O6 of acarbose was solvent-exposed. To generate the branched limit dextrin from amylopectin (Fig. 3[link]), AliC must also be able to accommodate branching in the +2 subsite, which is consistent with the glucose moiety seen adjacent to O6 of the +2 sugar (Fig. 1[link]b).

3.3. Branched-ligand complex of AliC

These branch patterns are consistent with the initial three-dimensional structure of AliC with the acarbose-derived oligosaccharide in which the −2 subsite O6 points into solvent and in which we observed a glucosyl moiety approximately where an O6 branch in either the +2 or +3 site might lie. In order to try and access a branched complex with a branch in the +1 subsite (the position of which is harder to model from the 6-deoxy sugars present in acarbose alone) we sought to obtain a complex by co-crystallizing AliC with 63-α-D-glucosyl maltotriose (Megazyme, Wicklow, Ireland) and observing what was obtained with this active enzyme.

A `branched-ligand' AliC complex was obtained through co-crystallization, with crystals forming in a new space group. This form diffracted poorly and data could only be obtained to 2.95 Å resolution. Weak density in the −1 subsite, largely diffuse but greater than would be expected for discrete solvent, remained unmodelled. Density was clearer for a panose tri­saccharide with an α-1,4-linked disaccharide in subsites +1 and +2 and, crucially, clear density for an α-1,6 branch accommodated in the +1 subsite (Fig. 1[link]c), providing a structural context for the limit digest analysis of action on amylopectin starch (Fig. 4). Notably, this +1′ sugar overlaps in position with the very recently reported SusG amylase branched-ligand complex (Arnal et al., 2018[Arnal, G., Cockburn, D. W., Brumer, H. & Koropatkin, N. M. (2018). Protein Sci. 27, 1093-1101.]).

4. Conclusions

The accommodation of branch points in industrial enzymes is a key factor when considering their utility. How close to branch points an α-1,4 cleaving α-amylase will cleave defines what the ultimate limit dextrin product will be. The product profile impacts both on the cocktail of enzymes that are required for complete hydrolysis to glucose and on the physical properties of the limit dextrin itself (which are important in food and brewing processes, including the `mouthfeel' of beer), such that insight into branch-point accommodation can provide powerful insight to aid protein-engineering campaigns. Yet, surprisingly, there has been very little structural insight into possible branch-point accommodation in α-amylases. Here, we have shown how serendipitous observation of a `lone' glucosyl moiety close to the O6 position of an oligosaccharide complex inspired analysis of limit dextrins on substrates containing α-1,6 linkages, both linear and branched. Such combined X-ray and product-analysis NMR approaches should prove valuable in the future for interrogating, defining and ultimately exploiting the branch-point accommodation in this massively widespread family of starch-degrading catalysts.

Supporting information


Acknowledgements

The authors would like to thank Diamond Light Source for beamtime (proposals mx-7864 and mx-9948) and the staff of beamline I04 for assistance with crystal testing and data collection. NMR spectra were acquired using the instruments of the NMR center, Technical University of Denmark (DTU).

Funding information

JA is a Royal Society University Research Fellow (award reference UF160039). GJD is a Royal Society Ken Murray Research Professor. The authors would like to thank Novozymes A/S for partially funding this work.

References

First citationAgirre, J., Iglesias-Fernández, J., Rovira, C., Davies, G. J., Wilson, K. S. & Cowtan, K. D. (2015). Nature Struct. Mol. Biol. 22, 833–834.  Web of Science CrossRef CAS Google Scholar
First citationArnal, G., Cockburn, D. W., Brumer, H. & Koropatkin, N. M. (2018). Protein Sci. 27, 1093–1101.  CrossRef CAS Google Scholar
First citationBrzozowski, A. M. & Davies, G. J. (1997). Biochemistry, 36, 10837–10845.  CrossRef CAS PubMed Web of Science Google Scholar
First citationBrzozowski, A. M., Lawson, D. M., Turkenburg, J. P., Bisgaard-Frantzen, H., Svendsen, A., Borchert, T. V., Dauter, Z., Wilson, K. S. & Davies, G. J. (2000). Biochemistry, 39, 9099–9107.  Web of Science CrossRef PubMed CAS Google Scholar
First citationChen, 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.  Web of Science CrossRef CAS IUCr Journals Google Scholar
First citationD'Arcy, A., Bergfors, T., Cowan-Jacob, S. W. & Marsh, M. (2014). Acta Cryst. F70, 1117–1126.  Web of Science CrossRef IUCr Journals Google Scholar
First citationDauter, Z., Dauter, M., Brzozowski, A. M., Christensen, S., Borchert, T. V., Beier, L., Wilson, K. S. & Davies, G. J. (1999). Biochemistry, 38, 8385–8392.  CrossRef CAS Google Scholar
First citationDavies, G. J., Brzozowski, A. M., Dauter, Z., Rasmussen, M. D., Borchert, T. V. & Wilson, K. S. (2005). Acta Cryst. D61, 190–193.  Web of Science CrossRef CAS IUCr Journals Google Scholar
First citationEmsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. (2010). Acta Cryst. D66, 486–501.  Web of Science CrossRef CAS IUCr Journals Google Scholar
First citationHiguchi, R., Krummel, B. & Saiki, R. K. (1988). Nucleic Acids Res. 16, 7351–7367.  CrossRef CAS PubMed Web of Science Google Scholar
First citationJodelet, A., Rigby, N. M. & Colquhoun, I. J. (1998). Carbohydr. Res. 312, 139–151.  CrossRef CAS Google Scholar
First citationKanai, R., Haga, K., Akiba, T., Yamane, K. & Harata, K. (2004). Biochemistry, 43, 14047–14056.  Web of Science CrossRef PubMed CAS Google Scholar
First citationKrissinel, E. & Henrick, K. (2004). Acta Cryst. D60, 2256–2268.  Web of Science CrossRef CAS IUCr Journals Google Scholar
First citationLiu, X. D. & Xu, Y. (2008). Bioresour. Technol. 99, 4315–4320.  CrossRef CAS Google Scholar
First citationLo Leggio, L., Simmons, T. J., Poulsen, J. C., Frandsen, K. E., Hemsworth, G. R., Stringer, M. A., von Freiesleben, P., Tovborg, M., Johansen, K. S., De Maria, L., Harris, P. V., Soong, C. L., Dupree, P., Tryfona, T., Lenfant, N., Henrissat, B., Davies, G. J. & Walton, P. H. (2015). Nature Commun. 6, 5961.  CrossRef Google Scholar
First citationLombard, V., Golaconda Ramulu, H., Drula, E., Coutinho, P. M. & Henrissat, B. (2014). Nucleic Acids Res. 42, D490–D495.  Web of Science CrossRef CAS PubMed Google Scholar
First citationMaarel, M. J. E. C. van der, van der Veen, B., Uitdehaag, J. C., Leemhuis, H. & Dijkhuizen, L. (2002). J. Biotechnol. 94, 137–155.  Google Scholar
First citationMachius, M., Declerck, N., Huber, R. & Wiegand, G. (1998). Structure, 6, 281–292.  Web of Science CrossRef CAS PubMed Google Scholar
First citationMachius, M., Wiegand, G. & Huber, R. (1995). J. Mol. Biol. 246, 545–559.  CrossRef CAS PubMed Web of Science Google Scholar
First citationMcNicholas, S., Potterton, E., Wilson, K. S. & Noble, M. E. M. (2011). Acta Cryst. D67, 386–394.  Web of Science CrossRef CAS IUCr Journals Google Scholar
First citationMurshudov, G. N., Skubák, P., Lebedev, A. A., Pannu, N. S., Steiner, R. A., Nicholls, R. A., Winn, M. D., Long, F. & Vagin, A. A. (2011). Acta Cryst. D67, 355–367.  Web of Science CrossRef CAS IUCr Journals Google Scholar
First citationNielsen, J. E. & Borchert, T. V. (2000). Biochim. Biophys. Acta, 1543, 253–274.  Web of Science CrossRef PubMed CAS Google Scholar
First citationNonaka, T., Fujihashi, M., Kita, A., Hagihara, H., Ozaki, K., Ito, S. & Miki, K. (2003). J. Biol. Chem. 278, 24818–24824.  CrossRef CAS Google Scholar
First citationOffen, W. A., Viksoe-Nielsen, A., Borchert, T. V., Wilson, K. S. & Davies, G. J. (2015). Acta Cryst. F71, 66–70.  CrossRef IUCr Journals Google Scholar
First citationPetersen, B. O., Hindsgaul, O. & Meier, S. (2014). Analyst, 139, 401–406.  CrossRef CAS Google Scholar
First citationPetersen, B. O., Motawie, M. S., Møller, B. L., Hindsgaul, O. & Meier, S. (2015). Carbohydr. Res. 403, 149–156.  CrossRef CAS Google Scholar
First citationPotterton, L., Agirre, J., Ballard, C., Cowtan, K., Dodson, E., Evans, P. R., Jenkins, H. T., Keegan, R., Krissinel, E., Stevenson, K., Lebedev, A., McNicholas, S. J., Nicholls, R. A., Noble, M., Pannu, N. S., Roth, C., Sheldrick, G., Skubak, P., Turkenburg, J., Uski, V., von Delft, F., Waterman, D., Wilson, K., Winn, M. & Wojdyr, M. (2018). Acta Cryst. D74, 68–84.  Web of Science CrossRef IUCr Journals Google Scholar
First citationShaw, A., Bott, R. & Day, A. G. (1999). Curr. Opin. Biotechnol. 10, 349–352.  CrossRef PubMed CAS Google Scholar
First citationSouza, P. M. de & de Oliveira e Magalhães, P. (2010). Braz. J. Microbiol. 41, 850–861.  Google Scholar
First citationSuvd, D., Fujimoto, Z., Takase, K., Matsumura, M. & Mizuno, H. (2001). J. Biochem. 129, 461–468.  Web of Science CrossRef PubMed CAS Google Scholar
First citationSyson, K., Stevenson, C. E., Miah, F., Barclay, J. E., Tang, M., Gorelik, A., Rashid, A. M., Lawson, D. M. & Bornemann, S. (2016). J. Biol. Chem. 291, 21531–21540.  CrossRef CAS Google Scholar
First citationThe CAZypedia Consortium (2018). Glycobiology, 28, 3–8.  Web of Science CrossRef PubMed Google Scholar
First citationTickle, I. J. (2012). Acta Cryst. D68, 454–467.  Web of Science CrossRef CAS IUCr Journals Google Scholar
First citationVagin, A. & Teplyakov, A. (2010). Acta Cryst. D66, 22–25.  Web of Science CrossRef CAS IUCr Journals Google Scholar
First citationVu, V. V., Beeson, W. T., Span, E. A., Farquhar, E. R. & Marletta, M. A. (2014). Proc. Natl Acad. Sci. USA, 111, 13822–13827.  Web of Science CrossRef CAS PubMed Google Scholar
First citationWinn, 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.  Web of Science CrossRef CAS IUCr Journals Google Scholar
First citationWinter, G., Lobley, C. M. C. & Prince, S. M. (2013). Acta Cryst. D69, 1260–1273.  Web of Science CrossRef CAS IUCr Journals Google Scholar

This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.

Journal logoSTRUCTURAL
BIOLOGY
ISSN: 2059-7983
Follow Acta Cryst. D
Sign up for e-alerts
Follow Acta Cryst. on Twitter
Follow us on facebook
Sign up for RSS feeds