The role of Arg165 towards base flipping, base stabilization and catalysis in M.HhaI

Arg165 forms part of a previously identified base flipping motif in the bacterial DNA cytosine methyltransferase, M.HhaI. Replacement of Arg165 with Ala has no detectable effect on either DNA or AdoMet affinity, yet causes the base flipping and restacking transitions to be decreased approximately 16 and 190-fold respectively, thus confirming the importance of this motif. However, these kinetic changes cannot account for the mutant's observed 10(5)-fold decreased catalytic rate. The mutant enzyme/cognate DNA cocrystal structure (2.79 A resolution) shows the target cytosine to be positioned approximately 30 degrees into the major groove, which is consistent with a major groove pathway for nucleotide flipping. The pyrimidine-sugar chi angle is rotated to approximately +171 degrees, from a range of -95 degrees to -120 degrees in B DNA, and -77 degrees in the WT M.HhaI complex. Thus, Arg165 is important for maintaining the cytosine positioned for nucleophilic attack by Cys81. The cytosine sugar pucker is in the C2'-endo-C3'-exo (South conformation), in contrast to the previously reported C3'-endo (North conformation) described for the original 2.70 A resolution cocrystal structure of the WT M.HhaI/DNA complex. We determined a high resolution structure of the WT M.HhaI/DNA complex (1.96 A) to better determine the sugar pucker. This new structure is similar to the original, lower resolution WT M.HhaI complex, but shows that the sugar pucker is O4'-endo (East conformation), intermediate between the South and North conformers. In summary, Arg165 plays significant roles in base flipping, cytosine positioning, and catalysis. Furthermore, the previously proposed M.HhaI-mediated changes in sugar pucker may not be an important contributor to the base flipping mechanism. These results provide insights into the base flipping and catalytic mechanisms for bacterial and eukaryotic DNA methyltransferases.     

Engineered extrahelical base destabilization enhances sequence discrimination of DNA methyltransferase M.HhaI

Improved sequence specificity of the DNA cytosine methyltransferase HhaI was achieved by disrupting interactions at a hydrophobic interface between the active site of the enzyme and a highly conserved flexible loop. Transient fluorescence experiments show that mutations disrupting this interface destabilize the positioning of the extrahelical, "flipped" cytosine base within the active site. The ternary crystal structure of the F124A M.HhaI bound to cognate DNA and the cofactor analogue S-adenosyl-l-homocysteine shows an increase in cavity volume between the flexible loop and the core of the enzyme. This cavity disrupts the interface between the loop and the active site, thereby destabilizing the extrahelical target base. The favored partitioning of the base-flipped enzyme-DNA complex back to the base-stacked intermediate results in the mutant enzyme discriminating better than the wild-type enzyme against non-cognate sites. Building upon the concepts of kinetic proofreading and our understanding of M.HhaI, we describe how a 16-fold specificity enhancement achieved with a double mutation at the loop/active site interface is acquired through destabilization of intermediates prior to methyltransfer rather than disruption of direct interactions between the enzyme and the substrate for M.HhaI.     

Reversing Evolution: Re-establishing Obligate Metal Ion Dependence in a Metal-independent KDO8P Synthase

Two distinct groups of 3-deoxy-d-manno-octulosonate 8-phosphate synthase (KDO8PS), a key enzyme of cell-wall biosynthesis, differ by their requirement for a divalent metal ion for enzymatic activity. The unique difference between these groups is the replacement of the metal-binding Cys by Asn. Substitution of just this Asn for a Cys in metal-independent KDO8PS does not create the obligate metal-ion dependency of natural metal-dependent enzymes. We describe how three or four mutations of the metal-independent KDO8PS from Neisseria meningitidis produce a fully functional, obligately metal-dependent KDO8PS. For the substitutions Asn23Cys, Asp247Glu (this Asp binds to the metal ion in all metal-dependent KDO8PS) and Pro249Ala, and for double and triple combinations, mutant enzymes that contained Cys in place of Asn showed an increase in activity in the presence of divalent metal ions. However, combining these mutations with substitution by Ser of the Cys residue in the conserved ²⁴⁶CysAspGlyPro²⁴⁹ motif of metal-independent KDO8PS created enzymes with obligate metal dependency. The quadruple mutant (Asn23Cys/Cys246Ser/Asp247Glu/Pro249Ala) showed comparable activity to wild-type enzymes only in the presence of metal ions, with maximum activity with Cd²⁺, the metal ion that is strongly inhibitory at micromolar concentrations for the wild-type enzyme. In the absence of metal ions, activity was barely detectable for this quadruple mutant or for triple mutants bearing both Cys246Ser and Asn23Cys mutations. The structures of NmeKDO8PS and its Asn23Cys/Asp247Glu/Pro249Ala and quadruple mutants at pH 4.6 were characterized at resolutions better than 1.85 Å. Aged crystals of the Asn23Cys/Asp247Glu/Pro249Ala mutant featured a Cys23-Cys246 disulfide linkage, explaining the spectral bleaching observed when this mutant was incubated with Cu²⁺. Such bleaching was not observed for the quadruple mutant. Reverse evolution to a fully functional obligately metal-dependent KDO8PS has been achieved with just three directed mutations for enzymes that have, at best, 47% identity between metal-dependent and metal-independent pairs.     

Structure determinants and substrate recognition of serine carboxypeptidase-like acyltransferases from plant secondary metabolism

Structures of the serine carboxypeptidase-like enzymes 1-O-sinapoyl-beta-glucose:L-malate sinapoyltransferase (SMT) and 1-O-sinapoyl-beta-glucose:choline sinapoyltransferase (SCT) were modeled to gain insight into determinants of specificity and substrate recognition. The structures reveal the alpha/beta-hydrolase fold as scaffold for the catalytic triad Ser-His-Asp. The recombinant mutants of SMT Ser173Ala and His411Ala were inactive, whereas Asp358Ala displayed residual activity of 20%. 1-O-sinapoyl-beta-glucose recognition is mediated by a network of hydrogen bonds. The glucose moiety is recognized by a hydrogen bond network including Trp71, Asn73, Glu87 and Asp172. The conserved Asp172 at the sequence position preceding the catalytic serine meets sterical requirements for the glucose moiety. The mutant Asn73Ala with a residual activity of 13% underscores the importance of the intact hydrogen bond network. Arg322 is of key importance by hydrogen bonding of 1-O-sinapoyl-beta-glucose and L-malate. By conformational change, Arg322 transfers L-malate to a position favoring its activation by His411. Accordingly, the mutant Arg322Glu showed 1% residual activity. Glu215 and Arg219 establish hydrogen bonds with the sinapoyl moiety. The backbone amide hydrogens of Gly75 and Tyr174 were shown to form the oxyanion hole, stabilizing the transition state. SCT reveals also the catalytic triad and a hydrogen bond network for 1-O-sinapoyl-beta-glucose recognition, but Glu274, Glu447, Thr445 and Cys281 are crucial for positioning of choline.     

Helix Dynamics in LacY: Helices II and IV

Biochemical and biophysical studies based upon crystal structures of both a mutant and wild-type lactose permease from Escherichia coli (LacY) in an inward-facing conformation have led to a model for the symport mechanism in which both sugar and H⁺ binding sites are alternatively accessible to both sides of the membrane. Previous findings indicate that the face of helix II with Asp68 is important for the conformational changes that occur during turnover. As shown here, replacement of Asp68 at the cytoplasmic end of helix II, particularly with Glu, abolishes active transport but the mutants retain the ability to bind galactopyranoside. In the x-ray structure, Asp68 and Lys131 (helix IV) lie within ∼ 4.2 Å of each other. Although a double mutant with Cys replacements at both position 68 and position 131 cross-links efficiently, single replacements for Lys131 exhibit very significant transport activity. Site-directed alkylation studies show that sugar binding by the Asp68 mutants causes closure of the cytoplasmic cavity, similar to wild-type LacY; however, strikingly, the probability of opening the periplasmic pathway upon sugar binding is markedly reduced. Taken together with results from previous mutagenesis and cross-linking studies, these findings lead to a model in which replacement of Asp68 blocks a conformational transition involving helices II and IV that is important for opening the periplasmic cavity. Evidence suggesting that movements of helices II and IV are coupled functionally with movements in the pseudo-symmetrically paired helices VIII and X is also presented.     

Crystal structures of D-tagatose 3-epimerase from Pseudomonas cichorii and its complexes with D-tagatose and D-fructose

Pseudomonas cichoriiid-tagatose 3-epimerase (P. cichoriid-TE) can efficiently catalyze the epimerization of not only d-tagatose to d-sorbose, but also d-fructose to d-psicose, and is used for the production of d-psicose from d-fructose. The crystal structures of P. cichoriid-TE alone and in complexes with d-tagatose and d-fructose were determined at resolutions of 1.79, 2.28, and 2.06 Å, respectively. A subunit of P. cichoriid-TE adopts a (β/α)₈ barrel structure, and a metal ion (Mn²⁺) found in the active site is coordinated by Glu152, Asp185, His211, and Glu246 at the end of the β-barrel. P. cichoriid-TE forms a stable dimer to give a favorable accessible surface for substrate binding on the front side of the dimer. The simulated omit map indicates that O2 and O3 of d-tagatose and/or d-fructose coordinate Mn²⁺, and that C3-O3 is located between carboxyl groups of Glu152 and Glu246, supporting the previously proposed mechanism of deprotonation/protonation at C3 by two Glu residues. Although the electron density is poor at the 4-, 5-, and 6-positions of the substrates, substrate-enzyme interactions can be deduced from the significant electron density at O6. The O6 possibly interacts with Cys66 via hydrogen bonding, whereas O4 and O5 in d-tagatose and O4 in d-fructose do not undergo hydrogen bonding to the enzyme and are in a hydrophobic environment created by Phe7, Trp15, Trp113, and Phe248. Due to the lack of specific interactions between the enzyme and its substrates at the 4- and 5-positions, P. cichoriid-TE loosely recognizes substrates in this region, allowing it to efficiently catalyze the epimerization of d-tagatose and d-fructose (C4 epimer of d-tagatose) as well. Furthermore, a C3-O3 proton-exchange mechanism for P. cichoriid-TE is suggested by X-ray structural analysis, providing a clear explanation for the regulation of the ionization state of Glu152 and Glu246.     

The effects of multiple ancestral residues on the Thermus thermophilus 3-isopropylmalate dehydrogenase

Previously, we showed that mutants of Thermus thermophilus 3-isopropylmalate dehydrogenase (IPMDH) each containing a residue (ancestral residue) that had been predicted to exist in a postulated common ancestor protein often have greater thermal stabilities than does the contemporary wild-type enzyme. In this study, the combined effects of multiple ancestral residues were analyzed. Two mutants, containing multiple mutations, Sup3mut (Val181Thr/Pro324Thr/Ala335Glu) and Sup4mut (Leu134Asn/Val181Thr/Pro324Thr/Ala335Glu) were constructed and show greater thermal stabilities than the wild-type and single-point mutant IPMDHs do. Most of the mutants have similar or improved catalytic efficiencies at 70 degrees C when compared with the wild-type IPMDH.     

Structure-guided engineering of the coenzyme specificity of Pseudomonas fluorescens mannitol 2-dehydrogenase to enable efficient utilization of NAD(H) and NADP(H)

The structure of Pseudomonas fluorescens mannitol 2-dehydrogenase with bound NAD+ leads to the suggestion that the carboxylate group of Asp(69) forms a bifurcated hydrogen bond with the 2' and 3' hydroxyl groups of the adenosine of NAD+ and contributes to the 400-fold preference of the enzyme for NAD+ as compared to NADP+. Accordingly, the enzyme with the Asp(69)-->Ala substitution was found to use NADP(H) almost as well as wild-type enzyme uses NAD(H). The Glu(68)-->Lys substitution was expected to enhance the electrostatic interaction of the enzyme with the 2'-phosphate of NADP+. The Glu(68)-->Lys:Asp(69)-->Ala doubly mutated enzyme showed about a 10-fold preference for NADP(H) over NAD(H), accompanied by a small decrease in catalytic efficiency for NAD(H)-dependent reactions as compared to wild-type enzyme.     

Three-dimensional structures of apo- and holo-L-alanine dehydrogenase from Mycobacterium tuberculosis reveal conformational changes upon coenzyme binding

l-Alanine dehydrogenase from Mycobacterium tuberculosis catalyzes the NADH-dependent reversible conversion of pyruvate and ammonia to l-alanine. Expression of the gene coding for this enzyme is up-regulated in the persistent phase of the organism, and alanine dehydrogenase is therefore a potential target for pathogen control by antibacterial compounds. We have determined the crystal structures of the apo- and holo-forms of the enzyme to 2.3 and 2.0 Å resolution, respectively. The enzyme forms a hexamer of identical subunits, with the NAD-binding domains building up the core of the molecule and the substrate-binding domains located at the apical positions of the hexamer. Coenzyme binding stabilizes a closed conformation where the substrate-binding domains are rotated by about 16° toward the dinucleotide-binding domains, compared to the open structure of the apo-enzyme. In the structure of the abortive ternary complex with NAD+ and pyruvate, the substrates are suitably positioned for hydride transfer between the nicotinamide ring and the C2 carbon atom of the substrate. The approach of the nucleophiles water and ammonia to pyruvate or the reaction intermediate iminopyruvate, respectively, is, however, only possible through conformational changes that make the substrate binding site more accessible. The crystal structures identified the conserved active-site residues His96 and Asp270 as potential acid/base catalysts in the reaction. Amino acid replacements of these residues by site-directed mutagenesis led to inactive mutants, further emphasizing their essential roles in the enzymatic reaction mechanism.     

The Crystal Structure of the Novobiocin Biosynthetic Enzyme NovP: The First Representative Structure for the TylF O-Methyltransferase Superfamily

NovP is an S-adenosyl-l-methionine-dependent O-methyltransferase that catalyzes the penultimate step in the biosynthesis of the aminocoumarin antibiotic novobiocin. Specifically, it methylates at 4-OH of the noviose moiety, and the resultant methoxy group is important for the potency of the mature antibiotic: previous crystallographic studies have shown that this group interacts directly with the target enzyme DNA gyrase, which is a validated drug target. We have determined the high-resolution crystal structure of NovP from Streptomyces spheroides as a binary complex with its desmethylated cosubstrate S-adenosyl-l-homocysteine. The structure displays a typical class I methyltransferase fold, in addition to motifs that are consistent with a divalent-metal-dependent mechanism. This is the first representative structure of a methyltransferase from the TylF superfamily, which includes a number of enzymes implicated in the biosynthesis of antibiotics and other therapeutics. The NovP structure reveals a number of distinctive structural features that, based on sequence conservation, are likely to be characteristic of the superfamily. These include a helical ‘lid’ region that gates access to the cosubstrate binding pocket and an active center that contains a 3-Asp putative metal binding site. A further conserved Asp likely acts as the general base that initiates the reaction by deprotonating the 4-OH group of the noviose unit. Using in silico docking, we have generated models of the enzyme-substrate complex that are consistent with the proposed mechanism. Furthermore, these models suggest that NovP is unlikely to tolerate significant modifications at the noviose moiety, but could show increasing substrate promiscuity as a function of the distance of the modification from the methylation site. These observations could inform future attempts to utilize NovP for methylating a range of glycosylated compounds.     

Thermodynamic determination of the binding constants of angiotensin-converting enzyme inhibitors by a displacement method

Somatic angiotensin I-converting enzyme (s-ACE) plays a central role in blood pressure regulation and has been the target of most antihypertensive drugs. A displacement isothermal titration calorimetry method has been used to accurately determine the binding constant of three strong s-ACE inhibitors. Under the experimental conditions studied in this work, the relative potency of the inhibitors was determined to be enalaprilat>lisinopril>captopril. We analyze the thermodynamic behaviour of the binding process using the new structural information provided by the ACE structures, as well as the conformational changes that occur upon binding.     

The coupling of tight DNA binding and base flipping: identification of a conserved structural motif in base flipping enzymes

Val(121) is positioned immediately above the extrahelical cytosine in HhaI DNA C(5)-cytosine methyltransferase, and replacement with alanine dramatically interferes with base flipping and catalysis. DNA binding and k(cat) are decreased 10(5)-fold for the Val(121) --> Ala mutant that has a normal circular dichroism spectrum and AdoMet affinity. The magnitude of this loss of function is comparable with removal of the essential catalytic Cys(81). Surprisingly, DNA binding is completely recovered (increase of 10(5)-fold) with a DNA substrate lacking the target cytosine base (abasic). Thus, interfering with the base flipping transition results in a dramatic loss of binding energy. Our data support an induced fit mechanism in which tight DNA binding is coupled to both base flipping and protein loop rearrangement. The importance of the proximal protein segment (His(127)-Thr(132)) in maintaining this critical interaction between Val(121) and the flipped cytosine was probed with single site alanine substitutions. None of these mutants are significantly altered in secondary structure, AdoMet or DNA affinity, k(methylation), k(inactivation), or k(cat). Although Val(121) plays a critical role in both extrahelical base stabilization and catalysis, its position and mobility are not influenced by individual residues in the adjacent peptide region. Structural comparisons with other DNA methyltransferases and DNA repair enzymes that stabilize extrahelical nucleotides reveal a motif that includes a positively charged or polar side chain and a hydrophobic residue positioned adjacent to the target DNA base and either the 5'- or 3'-phosphate.     

Sugar binding in lactose permease: anomeric state of a disaccharide influences binding structure

Lactose permease in Escherichia coli (LacY) transports both anomeric states of disaccharides but has greater affinity for α-sugars. Molecular dynamics (MD) simulations are used to probe the protein-sugar interactions, binding structures, and global protein motions in response to sugar binding by investigating LacY (the experimental mutant and wild-type) embedded in a fully hydrated lipid bilayer. A total of 12 MD simulations of 20-25 ns each with β(α)-d-galactopyranosyl-(1,1)-β-d-galactopyranoside (ββ-(Galp)₂) and αβ-(Galp)₂ result in binding conformational families that depend on the anomeric state of the sugar. Both sugars strongly interact with Glu126 and αβ-(Galp)₂ has a greater affinity to this residue. Binding conformations are also seen that involve protein residues not observed in the crystal structure, as well as those involved in the proton translocation (Phe118, Asn119, Asn240, His322, Glu325, and Tyr350). Common to nearly all protein-sugar structures, water acts as a hydrogen bond bridge between the disaccharide and protein. The average binding energy is more attractive for αβ-(Galp)₂ than ββ-(Galp)₂, i.e. −10.7(±0.7) and −3.1(±1.0) kcal/mol, respectively. Of the 12 helices in LacY, helix-IV is the least stable with ββ-(Galp)₂ binding resulting in larger distortion than αβ-(Galp)₂.     

Critical role of N-terminal N-glycosylation for proper folding of the human formyl peptide receptor

The human formyl peptide receptor (FPR) is N-glycosylated and activates phagocytes via G(i)-proteins. The FPR expressed with G(i)alpha(2)beta(1)gamma(2) in Sf9 insect cells exhibits high constitutive activity as assessed by strong inhibitory effects of an inverse agonist and Na(+) on basal guanosine 5(')-O-(3-thiotriphosphate) (GTPgammaS) binding. The aim of our study was to analyze the role of N-glycosylation in FPR function. Site-directed mutagenesis of extracellular Asn residues prevented FPR glycosylation but not FPR expression in Sf9 membranes. However, in terms of high-affinity agonist binding, kinetics of GTPgammaS binding, number of G(i)-proteins activated, and constitutive activity, non-glycosylated FPR was much less active than native FPR. FPR-Asn4Gln/Asn10Gln/Asn179Gln and FPR-Asn4Gln/Asn10/Gln exhibited similar defects. Our data indicate that N-glycosylation of N-terminal Asn4 and Asn10 but not of Asn179 in the second extracellular loop is essential for proper folding and, hence, function of FPR. FPR deglycosylation by bacterial glycosidases could be a mechanism by which bacteria compromise host defense.     

Mechanism of DNA Methylation: The Double Role of DNA as a Substrate and as a Cofactor

Methylation of cytosine residues in the DNA is one of the most important epigenetic marks central to the control of differential expression of genes. We perform quantum mechanical calculations to investigate the catalytic mechanism of the bacterial HhaI DNA methyltransferase. We find that the enzyme nucleophile, Cys81, can attack C6 of cytosine only after it is deprotonated by the DNA phosphate group, a reaction facilitated by a bridging water molecule. This finding, which indicates that the DNA acts as both the substrate and the cofactor, can explain the total loss of activity observed in an analogous enzyme, thymidylate synthase, when the phosphate group of the substrate was removed. Furthermore, our results displaying the inability of the phosphate group to deprotonate the side chain of serine is in agreement with the total, or the large extent of, inactivity observed for the C81S mutant. In contrast to results from previous calculations, we find that the active site conserved residues, Glu119, Arg163, and Arg165, are crucial for catalysis. In addition, the enzyme-DNA adduct formation and the methyl transfer from the cofactor S-adenosyl-l-methionine are not concerted but proceed via stepwise mechanism. In many of the different steps of this methylation reaction, the transfer of a proton is found to be necessary. To render these processes possible, we find that several water molecules, found in the crystal structure, play an important role, acting as a bridge between the donating and accepting proton groups.     

Structural determinant for cold inactivation of rodent L-xylulose reductase

L-Xylulose reductase (XR) is a homotetramer belonging to the short-chain dehydrogenase/reductase family. Human XR is stable at low temperature, whereas the enzymes of mouse, rat, guinea pig, and hamster are rapidly dissociated into their inactive dimeric forms. In order to identify amino acid residues that cause cold inactivation of the rodent XRs, we have here selected Asp238, Leu242, and Thr244 in the C-terminal regions of rodent XRs and performed site-directed mutagenesis of the residues of mouse XR to the corresponding residues (Glu, Trp, and Cys) of the human enzyme. Cold inactivation was prevented partially by the single mutation of L242W and the double mutation of L242W/T244C, and completely by the double mutation of D238E/L242W. The L242W and L242W/T244C mutants existed in both tetrameric and dimeric forms at low temperature and the D238E/L242W mutant retained its tetrameric structure. No preventive effect was exerted by the mutations of D238E and T244C, which were dissociated into their dimeric forms upon cooling. Crystallographic analysis of human XR revealed that Glu238 and Trp242 contribute to proper orientation of the guanidino group of Arg203 of the same subunit to the C-terminal carboxylate group of Cys244 of another subunit through the neighboring residues, Gln137 and Phe241. Thus, the determinants for cold inactivation of rodent XRs are Asp238 and Leu242 with small side chains, which weaken the salt bridges between Arg203 and the C-terminal carboxylate group, and lead to cold inactivation.     

Dissecting differential binding of fructose and phosphate as leaving group/nucleophile of glucosyl transfer catalyzed by sucrose phosphorylase

Site-directed mutagenesis was used to examine the specificity of Leuconostoc mesenteroides sucrose phosphorylase for utilization of fructose and phosphate as leaving group/nucleophile of the reaction. The largest catalytic defect in Arg(137)-->Ala (approximately 60-fold) and Tyr(340)-->Ala (approximately 2500-fold) concerned phosphate dependent half-reactions whereas that in Asp(338)-->Asn (approximately 7000-fold) derived from disruption of steps where fructose departs or attacks. The relative efficiencies for enzyme glucosylation by sucrose compared with alpha-d-glucose-1-phosphate and enzyme deglucosylation by phosphate compared with fructose were 5.5 and 6.2 for wild-type, 19 and 2.0 for Arg(137)-->Ala, 950 and 0.17 for Tyr(340)-->Ala, and 0.05 and 180 for Asp(338)-->Asn, respectively. Asp(338) and Tyr(340) have a key role in differential binding of fructose and phosphate, respectively.     

Catalytic Mechanism of Retaining α-Galactosidase Belonging to Glycoside Hydrolase Family 97

Glycoside hydrolase family 97 (GH 97) is a unique glycoside family that contains inverting and retaining glycosidases. Of these, BtGH97a (SusB) and BtGH97b (UniProtKB/TrEMBL entry Q8A6L0), derived from Bacteroides thetaiotaomicron, have been characterized as an inverting α-glucoside hydrolase and a retaining α-galactosidase, respectively. Previous studies on the three-dimensional structures of BtGH97a and site-directed mutagenesis indicated that Glu532 acts as an acid catalyst and that Glu439 and Glu508 function as the catalytic base in the inverting mechanism. However, BtGH97b lacks base catalysts but possesses a putative catalytic nucleophilic residue, Asp415. Here, we report that Asp415 in BtGH97b is the nucleophilic catalyst based on the results of crystal structure analysis and site-directed mutagenesis study. Structural comparison between BtGH97b and BtGH97a indicated that OD1 of Asp415 in BtGH97b is located at a position spatially identical with the catalytic water molecule of BtGH97a, which attacks on the anomeric carbon from the β-face (i.e., Asp415 is poised for nucleophilic attack on the anomeric carbon). Site-directed mutagenesis of Asp415 leads to inactivation of the enzyme, and the activity is rescued by an external nucleophilic azide ion. That is, Asp415 functions as a nucleophilic catalyst. The multiple amino acid sequence alignment of GH 97 members indicated that almost half of the GH 97 enzymes possess base catalyst residues at the end of β-strands 3 and 5, while the other half of the family show a conserved nucleophilic residue at the end of β-strand 4. The different positions of functional groups on the β-face of the substrate, which seem to be due to “hopping of the functional group” during evolution, have led to divergence of catalytic mechanism within the same family.