Crystal structures of Aspergillus oryzae Rib2 deaminase: the functional mechanism involved in riboflavin biosynthesis

Crystal structures of Rib2 deaminase reveal its substrate specificity and catalytic mechanism.


Introduction
The FAD/FMN coenzymes derived from riboflavin are essential for a wide variety of physiological processes and hence occur in all organisms (Macheroux et al., 2011). Their precursor riboflavin is biosynthesized in most prokaryotes, fungi and plants (García-Angulo, 2017;Haase et al., 2014;Roje, 2007). In contrast, animals lack this biosynthetic pathway and hence must obtain this vitamin from nutritional sources. Some pathogenic microorganisms are unable to take up riboflavin from the environment and can only obtain this vitamin by biosynthesis. Therefore, the enzymes of riboflavin biosynthesis have potential as antimicrobial drug targets, particularly for the development of new chemotherapeutic agents against antibiotic-resistant pathogens (Haase et al., 2014;Kaiser et al., 2007;Long et al., 2010;Meir & Osherov, 2018).
Intriguingly, the biosynthesis pathway for riboflavin in archaea and fungi is slightly different from that in plants and eubacteria. In this pathway, DAROPP is reduced to 2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5 0 -phosphate (DARIPP) by Rib7 and subsequently deaminated to ARIPP by Rib2 [ Fig. 1(a)] (Graupner et al., 2002;Behm-Ansmant et al., 2004). Therefore, the deaminase domains of RibG and Rib2 possess distinct substrate specificities. The deaminase domain of fungal Rib2 enzymes also belongs to the CDA superfamily and contains conserved HXE and PCXXC signatures; nevertheless, these enzymes vary widely in length and show obvious distinct variations in sequence and in the structural domains that are present. For example, yeast Rib2 usually comprises an N-terminal pseudouridine synthase domain and a C-terminal deaminase domain (Behm-Ansmant et al., 2004). However, mold Rib2 only contains the deaminase domain [ Fig. 1(b)]. In other words, only the deaminase domain is involved in the riboflavin-biosynthesis pathway.
To investigate the deaminase in the biosynthetic pathway specific to archaea and fungi, we solved the crystal structure of Methanosarcina mazei Rib7 (MmRib7; Chen et al., 2018). Because structural information regarding fungal Rib2 remains unavailable, a three-dimensional structural study of Rib2 would further expand our understanding of the structural differences between the fungal and eubacterial pyrimidine deaminases involved in riboflavin biosynthesis. Nevertheless, all attempts to obtain crystal structures of the Rib2s from Pichia pastoris and Saccharomyces cerevisiae (ScRib2) have been unsuccessful.   residues (LEHHHHHH) at the C-terminus. The cell pellets were resuspended in lysis buffer consisting of 20 mM sodium phosphate pH 7.5, 500 mM NaCl, 1 mM phenylmethanesulfonyl fluoride and lysed by sonication. After the removal of cellular debris by centrifugation at 20 000g at 277 K for 30 min, the crude extract was applied onto a 6 ml nickelnitrilotriacetic acid column. After washing with 20, 40 and 60 mM imidazole, the protein was eluted with 250 mM imidazole. Fractions containing AoRib2 were dialysed and then pooled for additional purification using a Q Sepharose column (GE Healthcare). The protein eluted at 10 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM dithiothreitol. AoRib2 was pooled and concentrated to 14 mg ml À1 for crystallization screening. In addition, AoRib2 188 and AoRib2 177 mutations were introduced into pET-21b. Expression and purification of the deletion mutants were performed as described above.

Crystallization and data collection
The initial crystallization screening was performed with Hampton Research Crystal Screens using the hanging-drop vapor-diffusion method at 288 K. Crystals of AoRib2 and AoRib2 188 were grown using a combination of 2 ml reservoir solution and 2 ml protein solution (14 mg ml À1 ). Crystals appeared and reached final dimensions of $0.1 Â 0.1 Â 0.3 mm within 10-14 days at 288 K. X-ray diffraction data were collected at 110 K with the addition of 20% glycerol as a cryoprotectant. Data were collected at the National Synchrotron Radiation Research Center, Taiwan. An AoRib2 crystal was used for single-wavelength anomalous dispersion (Zn-SAD) data collection at the peak wavelength on the TPS-05A beamline ( = 1.2824 Å and 50% beam attenuation). The DARIPP derivative was prepared by soaking crystals for 20 min in reservoir solution containing 50-100 mM DARIPP. Diffraction data were processed using HKL-2000(Otwinowski & Minor, 1997. The resolution cutoff of our diffraction data was at an average I/(I) of >2 in the highest resolution shell. The data statistics are summarized in Table 1.

Structure determination
The initial phase was obtained by the Zn-SAD method using Phaser (McCoy et al., 2007) and an ab initio model was built using ARP/wARP (Carolan & Lamzin, 2014). One zinc site was identified and the initial phases calculated from the site were further improved by density modification. The resulting electron-density map was used to build most of the AoRib2 structure. The structures of the apo form and binary complex of AoRib2 188 were solved using Phaser  i jI i ðhklÞ À hIðhklÞij= P hkl P i I i ðhklÞ. ‡ R work = P hkl jF obs j À jF calc j = P hkl jF obs j. R free was calculated using 5% of the data that were excluded from refinement.

Biophysical characterization
The molecular mass in solution was estimated using a Beckman-Coulter XL-A Analytical Ultracentrifuge with an An60Ti rotor. Prior to the experiments, the samples were diluted to a protein concentration of 0.3 mg ml À1 with 20 mM phosphate-buffered saline (PBS) pH 7.5. Sedimentationvelocity analysis was performed at 293 K and 42 000 rev min À1 with standard double-sector centerpieces. The UV absorption of the cells was scanned every 5 min for 6 h and the data were analyzed using SEDFIT (Schuck, 2000).
CD spectra were recorded at 5 C and a protein concentration of 0.5 mg ml À1 in 20 mM sodium phosphate pH 7.5 with a cuvette of 0.1 cm path length. Thermal unfolding profiles between 25 and 95 C were recorded by increasing the temperature at a ramping rate of 1 C min À1 while monitoring the CD signal at 222 nm. The midpoints of thermal unfolding were calculated using SigmaPlot.

Deaminase activity assay
DAROPP was prepared by the addition of 0.5 M GTP to a solution of E. coli GTP cyclohydrolase II (5 mg ml À1 ) to a final concentration of 10-30 mM. The reaction was complete in about 10-15 min; P. pastoris Rib7 (1 mg) and 25 mM NADPH were added for 20 min to produce DARIPP. The activities of wild-type and mutant AoRib2 were assayed using the changes in fluorescence intensity after diacetyl modification. The reaction mixtures were incubated for 30 min at 310 K. Diacetyl was added to a final concentration of 1%(v/v) and the mixture was incubated at 368 K for 10 min. The fluorescence spectra were recorded using a Perkin Elmer LS-50B luminescence spectrometer.

Overall structure of AoRib2
AoRib2 is composed of a deaminase domain (residues 1-188) and a flexible C-terminal region (residues 189-222) rich in charged residues. Both the full length and the deaminase domain (AoRib2 188 ) of AoRib2 were bacterially produced and crystallized. AoRib2 crystals in space group P4 1 2 1 2 were grown in 1.5 M ammonium sulfate, 0.1 M sodium acetate pH 4.6 (AoRib2 pH4.6 ), while crystals of AoRib2 188 were grown in two different space groups. Crystals of AoRib2 188 with space group P4 1 2 1 2 were obtained by equilibration against a reservoir solution consisting of 2 M sodium chloride, 0.1 M sodium acetate pH 4.6 (AoRib2 188 pH4.6 ) and crystals of AoRib2 188 with space group P2 1 2 1 2 were obtained using a reservoir solution consisting of 0.1 M MES pH 6.5, 0.2 M ammonium sulfate, 30% PEG 5000 MME (AoRib2 188 pH6.5 ). There is one molecule in the asymmetric unit for all three crystals. Interestingly, the atomic model of AoRib2 pH4.6 contains only residues 3-188 due to unclear electron density for the C-terminal region after residue 188, indicating relatively high thermal motion and a disordered conformation. The structure of the deaminase domain is composed of a central five-stranded -sheet (1-5) sandwiched by three -helices (A, E and F) on one side and three -helices (B-D) on the other side [ Fig. 2(a)]. AoRib2 has a conserved core architecture akin to that of CDA superfamily members.
Compared with deaminases of known structure involved in riboflavin biosynthesis, which present an oligomeric form, analytical ultracentrifugation (AUC) experiments clearly demonstrated that AoRib2 exists as a monomer in solution [ Fig. 2(b)]. A previous study reported that the N-terminal deaminase domain (residues 1-147) of BsRibG was sufficient for enzyme activity, while the truncated protein could not be purified because of poor stability (Richter et al., 1997). Unlike the deaminase domain of BsRibG (BsRibG-D), the deaminase domain of AoRib2 could be purified and stabilized in solution. Comparing the molecular size, the deaminase domain of AoRib2 is larger than BsRibG-D. The additional residues of the AoRib2 deaminase domain are located in the L 1-2 , L B-3 and L C-4 loops. These three loops are ordered and participate in hydrogen bonds and hydrophobic interactions, which may make a significant contribution to stabilizing the core structure of AoRib2. Moreover, -strands (1 and 3) and helix B of AoRib2 are also involved in the interaction of these three loops. The additional interactions lead to an increase in the number of hydrophobic patches in the core structure, which may result in the structural stability of AoRib2 being greater than that of the truncated BsRibG protein.

Structural comparison with other CDA deaminases
Based on a structural alignment search using the DALI server, AoRib2 shares high structural similarity with other CDA superfamily members, including BsRibG (PDB entry 4g3m; Z-score 16.9; 25% identity; Chen et al., 2013), yCD (PDB entry 1uaq; Z-score 14.5; 23% identity; Ko et al., 2003) and EcTadA (PDB entry 1z3a; Z-score 14.0; 24% identity; Kim et al., 2006). The -sheet (1-5) and three -helices (A-C) of AoRib2 are virtually superimposable on the structures of these CDA members, but the C-terminal segment differs significantly [ Fig. 2(c)]. The C-terminal F helix of yCD forms a cap narrowing the opening of the active-site cavity for the nucleobase, whereas the C-terminal helices in EcTadA, AoRib2 and BsRibG swing the active-site cavity away to accommodate their larger substrates (Kuratani et al., 2005;Losey et al., 2006). The C-terminal helix of EcTadA is involved in tRNA interaction. Nonetheless, the C-terminal helices of AoRib2 and BsRibG do not participate in substrate binding, thus contributing to maintaining structural integrity.

DARIPP recognition by AoRib2
AoRib2 is a member of the CDA superfamily, with conserved HXE and PCX 8-9 C tetrahedral zinc-binding motifs (Chen et al., 2006). The Zn 2+ ion is coordinated by His61 N 1 (2.0 Å ), Cys102 S (2.2 Å ) and Cys112 S (2.3 Å ), and a fourth research papers coordination is provided by a water molecule. The zinc-bound water molecule interacts with Glu63 O "2 (2.6 Å ). The zinc ion and Glu63 are proposed to activate the zinc-bound water required for the deamination activity (Betts et al., 1994). In the absence of a substrate, the structure of ligand-free AoRib2 reveals that the active site of the enzyme is occupied by water and a sulfate ion, which occupies the phosphate-binding site. Hence, we reduced the concentration of ammonium sulfate and performed ligand-soaking experiments. As the result of ligand soaking, DARIPP could be built nicely into strong electron density and is firmly embedded in the active site of the AoRib2 crystal structure [ Fig. 3(a)]. Analysis of the interaction reveals that the pyrimidine N 3 H group of DARIPP interacts with the side-chain O "1 atom of Glu63 (3.1 Å ), the O 4 atom of DARIPP interacts with the backbone N atom of Ala62 (2.9 Å ) and the side-chain O 1 atom of Thr53 (2.6 Å ), the N 5 H 2 group of DARIPP interacts with the backbone carbonyl group of Phe26 (3.2 Å ), and the N 2 H 2 group of DARIPP interacts with the side-chain O "2 atom of Glu63 (3.2 Å ) and the backbone carbonyl group of Glu100 (3.5 Å ). In addition, the O 2 0 H, O 3 0 H and O 4 0 H groups of the ribitol make close contacts with the side-chain O "2 atom of Glu136 (2.7 Å ), its phosphate group (2.6 Å ) and the backbone N atom of Leu106 (3.0 Å ), respectively. The phosphate moiety of DARIPP forms hydrogen bonds to the side-chain atoms N 2 of Asn59 (3.0 Å ), N 2 of His61 (2.8 Å ), N 1 of Arg105 (3.0 Å ) and O of Ser107 (2.6 Å ) [ Fig. 3(b)]. Of note, an intramolecular hydrogen bond is formed in the ligand between the O 3 0 H group and the phosphate moiety. Moreover, the side chain of His61 stacks against the pyrimidine ring with an interplanar distance of about 3.5 Å . The substrate-interacting residues are all conserved in the fungal Rib2.
In eubacteria and fungi, the deamination and reduction steps in riboflavin biosynthesis proceed in the opposite order [ Fig. 1(a)]. Therefore, the substrates of RibG and Rib2 are distinct: DAROPP with a cyclic ribose versus DARIPP with a linear ribitol. Structural superimposition of DARIPP-bound AoRib2 and AROPP-bound BsRibG-D revealed that the conformations of their active-site loops are quite different [ Fig. 4(a)]. The residues of the two structures involved in the recognition of the pyrimidine moiety of DARIPP and AROPP occupy an equivalent spatial position [ Fig. 4(b)], while the residues of AoRib2 involved in the recognition of the ribitol (or ribose for BsRibG) and phosphate moieties are distinct [ Fig. 4(c)]. In addition, among the phosphate-interacting residues in AoRib2, Arg105 seems to be particularly important because it forms a salt bridge with the phosphate group of DARIPP. Lys79 in BsRibG plays a similar role to Arg105 in AoRib2 [ Fig. 4  L 3-C and L 4-D loops, which move towards AROPP. DARIPP does not induce a significant conformation in the L 3-C and L 4-D loops of AoRib2, whereas the L A-1 loop moves towards DARIPP. Therefore, AoRib2 possesses a different ligandinduced conformational change mechanism compared with BsRibG.

Conformational changes of AoRib2
In the crystal structures of fulllength AoRib2 and AoRib2 188 pH4.6 , the L 4-D loop projects into the active site and sterically occludes the binding site for substrate entry. Due to the fact that the two crystal forms were crystallized under acidic conditions, AoRib2 tends to be in an occluded form at low pH. The structure of AoRib2 188 pH4.6 is virtually identical to that of AoRib2, with a rootmean-squared deviation (r.m.s.d.) of 0.25 Å for 186 C atoms. In AoRib2 188 pH6.5 , the L 4-D loop becomes disordered (no electron density is visible for residues 139-143). The volume of the active site of the structure can accommodate its substrate. Therefore, the neutral pH structure can be considered as an open form. As DARIPP binds to AoRib2 188 , the L A-1 loop moves towards the active site in order to be sterically complementary to the substrate using van der Waals interactions and water-mediated hydrogen bonds. Most of the substrateinteracting residues do not change significantly, except Glu136, which rotates to interact with the O 2 0 H group of the ribitol. The phosphate-binding site (L 2-B and L 3-C loops) adopts a similar conformation whether or not DARIPP is bound. This may indicate that the phosphatebinding site is structurally well organized in the absence of bound substrate. On the basis of these structures, we were able to    Fig. 5(a)]. These are likely to represent different functional states that would be controlled by the pH and the substrate.
Structural analysis of the occluded form reveals that the protonated O "1 group of Glu100 forms a hydrogen bond to the backbone carbonyl group of Gly143 (2.7 Å ). This interaction may contribute to the rigidity of the L 4-D loop. In the open form, a deprotonated Glu100 could not make close contacts with the L 4-D loop. Part of the L 4-D loop becomes disordered and allows substrate entry. Glu100 is highly conserved among fungal Rib2 deaminases [ Fig. 5(b)], which leads to the possibility that the pH-triggered conformational switching mechanism could also apply to other Rib2s.

Importance of the C-terminal helix
Sequence analysis of fungal Rib2s shows that a stretch of additional residues are found at the C-terminus of AoRib2 that form a flexible C-terminal tail and helix F. In order to understand the function of the flexible C-terminal tail and helix F, two constructs with C-terminal deletions were generated (AoRib2 188 and AoRib2 177 ). The two deletion mutants were purified using the purification protocol used for AoRib2, but AoRib2 177 had a slight tendency to aggregate. AoRib2 188 and AoRib2 177 were both found to be monomeric by analytical ultracentrifugation [ Fig. 6(a)].
The T m values of AoRib2 and the deletion mutants were measured by equilibrium thermal denaturation using circulardichroism (CD) spectroscopy. AoRib2 188 and the full-length protein have similar T m values, but the T m value of AoRib2 177 is 5.4 C lower than that of the full-length protein [Figs. 6(b), 6(c) and 6(d)]. This can be explained by the structures: the flexible C-terminal tail is not involved in the structural core and does not influence the stability of the domain. The packing of helix F against helix E seems to increase the structural stability. In the deaminase assay, when the AoRib2 protein concentration increases the fluorescence intensity shows a two-state-like curve. The protein concentration at the midpoint of the transition is $2.5 mg ml À1 for the wild type and AoRib2 188 , while it is $6.6 mg ml À1 for AoRib2 177 [Fig. 6(e)]. The AoRib2 177 mutant lacking helix F shows a reduced deaminase activity, which may be due to a decrease in the stability of the C-terminal structure. Helix F plays an indispensable role in deaminase function. However, sequence analysis shows that the C-terminal helix F of ScRib2 is shorter than that of AoRib2 [ Fig. 5(b)]. This may indicate that ScRib2 has an alternative method of enhancing its structural  stability. During evolution, nature has utilized distinct strategies to improve the structural stability of proteins, for instance through the formation of higher oligomers, more disulfide bonds, domain fusions, cofactor binding, salt bridges and hydrophobic patches. AoRib2 has longer loops and helix F to increase the number of hydrophobic patches, whereas ScRib2 may use a domain-fusion strategy to compensate for the short C-terminal helix F in structural stability.

Discussion
Prior to this report, details of the three-dimensional structure of Rib2 and the residues involved in substrate binding were not known. Sequence and structural analysis demonstrated that Rib2 is a representative member of the CDA superfamily.  (a) Sedimentation-velocity analysis of AoRib2 deletion mutants. Top, AoRib2 188 ; bottom, AoRib2 177 . The two deletion mutants were found to exist in a monomeric form. To compare protein stability, equilibrium thermal denaturation CD measurements of (b) AoRib2, (c) AoRib2 188 and (d) AoRib2 177 were conducted in 20 mM sodium phosphate pH 7.5. (e) A plot of the average fluorescence emission intensity against protein concentration for AoRib2 (black circles), AoRib2 188 (red inverted triangles) and AoRib2 177 (green squares). change in Rib2 and trigger changes in the protein dissimilar to those in RibG-AROPP. DARIPP triggers a conformational change in the L A-1 loop of AoRib2, while AROPP induces a change in the L 3-C and L 4D loops of RibG. This finding shows that the fungal Rib2 and the eubacterial RibG use different strategies to ensure substrate specificity.
Previous studies of CDA superfamily members have demonstrated that they share a conserved hydrolytic deamination mechanism (Liaw et al., 2004;Betts et al., 1994;Ko et al., 2003;Kim et al., 2006). Despite Rib2 and RibG acting on different substrates, they possess similar substrateinteracting networks with pyrimidine moieties and an identical catalytic center containing a zinc ion and a glutamate general base. It could be expected that Rib2 has a similar deamination mechanism to RibG (Chen et al., 2013). Based on the AoRib2-DARIPP complex and previous studies of other CDA members, the deamination mechanism of Rib2 has been proposed (Fig. 7). The substrate DARIPP binds to the active site with its amino group stabilized by Glu63 O "2 and Glu100 O. Glu63 is proposed to activate the nucleophile water molecule and facilitate attack on the C 2 atom of the pyrimidine ring of DARIPP. The L A-1 loop moves towards DARIPP. Glu51 facilitates stabilization of the intermediate and proton transfer, producing ARIPP and an ammonium ion. Similar to the BsRibG-AROPP complex, the carbonyl group of ARIPP may bend out of the pyrimidine ring and ligate to the zinc ion. Glu63 O "2 and Glu100 O create unfavorable contacts with the carbonyl group of ARIPP to promote product release.
To the best of our knowledge, most mononucleotide CDA members are oligomers. Yeast cytosine deaminase (yCD) and B. subtilis guanine deaminase (BsGD) are dimeric (Ko et al., 2003;Liaw et al., 2004). BsRibG, cytidine deaminase (CDA) and blasticidin-S deaminase (BSD) are tetrameric (Chen et al., 2006;Sá nchez-Quitian et al., 2011;Kumasaka et al., 2007). dCMP deaminase (dCMPD) is organized as hexamer (Hou et al., 2008). Unexpectedly, AoRib2 is the only known monomeric mononucleotide deaminase in the CDA superfamily. Structural analysis of AoRib2 indicates that three loops (L 1-2 , L B-3 and L C-4 ) and longer secondary-structural elements (1, 3 and B) enlarge the structural core to maintain structural stability. These unique structural features may maintain a single deaminase domain of AoRib2 in solution. A structural comparison between AoRib2 and three members of the CDA superfamily shows the expected similarities in the three-layer // structure containing five -strands (1-5) and three helices (A-C). A mutational analysis of AoRib2 shows that the C-terminal F helix also contributes to structural stability and deaminase activity. Therefore, the diverse C-terminal tails in the CDA superfamily provide different functions.
Here, we present crystal structures of AoRib2 in open and occluded conformations. Structural comparisons of the conformations delineated the L 4-D loop involved in pH change-induced structural switching. In addition, we also determined the structure of AoRib2 in complex with DARIPP. A structural overlay of the open and complex structures revealed a slight conformational change induced by the substrate. The AoRib2-DARIPP complex explains how the fungal pyrimidine deaminase ensures its unique substrate specificity. AoRib2 shares structural similarity with the core of the deaminase domain of BsRibG, but shows divergence in the loops that provides its distinct substrate specificity. Additionally, AoRib2 possesses unique structural features, including a longer C-terminal helix F, compared with ScRib2. The helix F is crucial for maintaining structural stability and enzyme activity. These structural and functional studies of AoRib2 contribute to our further understanding of the biochemical functions of pyrimidine deaminases involved in riboflavin biosynthesis. Crystal structures of AoRib2 providing detailed stereochemistry of the active site and the binding of the DARIPP substrate should stimulate the design of antifungal inhibitors.