The identification of the acid-base catalyst of alpha-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycoside hydrolase

The alpha-L-arabinofuranosidase from Geobacillus stearothermophilus T-6 (AbfA T-6) belongs to the retaining family 51 glycoside hydrolases. The conserved Glu175 was proposed to be the acid-base catalytic residue. AbfA T-6 exhibits residual activity towards aryl beta-D-xylopyranosides. This phenomenon was used to examine the catalytic properties of the putative acid-base mutant E175A. Data from kinetic experiments, pH profiles, azide rescue, and the identification of the xylopyranosyl azide product provide firm support to the assignment of Glu175 as the acid-base catalyst of AbfA T-6.     

Investigation of the functional relevance of the catalytically important Glu(28) in family 51 arabinosidases

The alpha-L-arabinofuranosidase (AbfD3) from Thermobacillus xylanilyticus is a family 51 glycosyl hydrolase. According to classification hierarchy, family 51 belongs to clan GH-A. While the major GH-A motifs, the catalytic acid-base and nucleophile, are conserved in AbfD3, a third catalytically important residue (Glu(28)) does not appear to be analogous to any known GH-A motif. To evaluate the importance of Glu(28), bioinformatics analyses and site-saturation mutagenesis were performed. The results indicate that Glu(28) forms part of a family 51 arabinosidase motif which might be functionally homologous to a conserved N-terminal motif found in exo-acting enzymes from families 1 and 5. Importantly, the data reveal that Glu(28) is a key determinant of substrate recognition in the -1 subsite, where it may also play an important role in water-mediated deglycosylation of the glycosyl-enzyme covalent intermediate.     

Mechanism of the family 1 beta-glucosidase from Streptomyces sp: catalytic residues and kinetic studies

The Streptomyces sp. beta-glucosidase (Bgl3) is a retaining glycosidase that belongs to family 1 glycosyl hydrolases. Steady-state kinetics with p-nitrophenyl beta-D-glycosides revealed that the highest k(cat)/K(M) values are obtained with glucoside (with strong substrate inhibition) and fucoside (with no substrate inhibition) substrates and that Bgl3 has 10-fold glucosidase over galactosidase activity. Reactivity studies by means of a Hammett analysis using a series of substituted aryl beta-glucosides gave a biphasic plot log k(cat) vs pK(a) of the phenol aglycon: a linear region with a slope of beta(lg) = -0.8 for the less reactive substrates (pK(a) > 8) and no significant dependence for activated substrates (pK(a) < 8). Thus, according to the two-step mechanism of retaining glycosidases, formation of the glycosyl-enzyme intermediate is rate limiting for the former substrates, while hydrolysis of the intermediate is for the latter. To identify key catalytic residues and on the basis of sequence similarity to other family 1 beta-glucosidases, glutamic acids 178 and 383 were changed to glutamine and alanine by site-directed mutagenesis. Mutation of Glu178 to Gln and Ala yielded enzymes with 250- and 3500-fold reduction in their catalytic efficiencies, whereas larger reduction (10(5)-10(6)-fold) were obtained for mutants at Glu383. The functional role of both residues was probed by a chemical rescue methodology based on activation of the inactive Ala mutants by azide as exogenous nucleophile. The E178A mutant yielded the beta-glucosyl azide adduct (by (1)H NMR) with a 200-fold increase on k(cat) for the 2,4-dinitrophenyl glucoside but constant k(cat)/K(M) on azide concentration. On the other hand, the E383A mutant with the same substrate gave the alpha-glucosyl azide product and a 100-fold increase in k(cat) at 1 M azide. In conclusion, Glu178 is the general acid/base catalyst and Glu383 the catalytic nucleophile. The results presented here indicate that Bgl3 beta-glucosidase displays kinetic and mechanistic properties similar to other family 1 enzymes analyzed so far. Subtle differences in behavior would lie in the fine and specific architecture of their respective active sites.     

Identification of active site residues in E. coli ketopantoate reductase by mutagenesis and chemical rescue

Ketopantoate reductase (EC 1.1.1.169) catalyzes the NADPH-dependent reduction of alpha-ketopantoate to D-(-)-pantoate in the biosynthesis of pantothenate. The pH dependence of V and V/K for the E. coli enzyme suggests the involvement of a general acid/base in the catalytic mechanism. To identify residues involved in catalysis and substrate binding, we mutated the following six strictly conserved residues to Ala: Lys72, Lys176, Glu210, Glu240, Asp248, and Glu256. Of these, the K176A and E256A mutant enzymes showed 233- and 42-fold decreases in V(max), and 336- and 63-fold increases in the K(m) value of ketopantoate, respectively, while the other mutants exhibited WT kinetic properties. The V(max) for the K176A and E256A mutant enzymes was markedly increased, up to 25% and 75% of the wild-type level, by exogenously added primary amines and formate, respectively. The rescue efficiencies for the K176A and E256A mutant enzymes were dependent on the molecular volume of rescue agents, as anticipated for a finite active site volume. The protonated form of the amine is responsible for recovery of activity, suggesting that Lys176 functions as a general acid in catalysis of ketopantoate reduction. The rescue efficiencies for the K176A mutant by primary amines were independent of the pK(a) value of the rescue agents (Bronsted coefficient, alpha = -0.004 +/-0.008). Insensitivity to acid strength suggests that the chemical reaction is not rate-limiting, consistent with (a) the catalytic efficiency of the wild-type enzyme (k(cat)/K(m) = 2x10(6) M(-1) s(-1) and (b) the small primary deuterium kinetic isotope effects, (D)V = 1.3 and (D)V/K = 1.5, observed for the wild-type enzyme. Larger primary deuterium isotope effects on V and V/K were observed for the K176A mutant ((D)V = 3.0, (D)V/K = 3.7) but decreased nearly to WT values as the concentration of ethylamine was increased. The nearly WT activity of the E256A mutant in the presence of formate argues for an important role for this residue in substrate binding. The double mutant (K176A/E256A) has no detectable ketopantoate reductase activity. These results indicate that Lys176 and Glu256 of the E. coli ketopantoate reductase are active site residues, and we propose specific roles for each in binding ketopantoate and catalysis.     

Genetic and biochemical characterization of a highly thermostable alpha-L-arabinofuranosidase from Thermobacillus xylanilyticus

The gene encoding an alpha-L-arabinofuranosidase from Thermobacillus xylanilyticus D3, AbfD3, was isolated. Characterization of the purified recombinant alpha-L-arabinofuranosidase produced in Escherichia coli revealed that it is highly stable with respect to both temperature (up to 90 degrees C) and pH (stable in the pH range 4 to 12). On the basis of amino acid sequence similarities, this 56, 071-Da enzyme could be assigned to family 51 of the glycosyl hydrolase classification system. However, substrate specificity analysis revealed that AbfD3, unlike the majority of F51 members, displays high activity in the presence of polysaccharides.     

Fast kinetic analysis of conformational changes in mutants of the Ca(2+)-ATPase of sarcoplasmic reticulum

Rapid quench experiments at 25 degrees C were carried out on selected mutants of the sarco(endo)plasmic reticulum Ca(2+)-ATPase to assess the kinetics of the conformational changes of the dephosphoenzyme associated with ATP binding/phosphoryl transfer and the binding and dissociation of Ca(2+) at the cytoplasmically facing transport sites. The mutants Gly(233) --> Glu, Gly(233) --> Val, Pro(312) --> Ala, Leu(319) --> Arg, and Lys(684) --> Arg differed conspicuously with respect to the behavior of the dephosphoenzyme, although they were previously shown to display a common block of the transformation of the phosphoenzyme from an ADP-sensitive to an ADP-insensitive form. The maximum rate of the ATP binding/phosphoryl transfer reaction was reduced 3.6-fold in mutant Gly(233) --> Glu and more than 50-fold in mutant Lys(684) --> Arg, relative to wild type. In mutant Leu(319) --> Arg, the rate of the Ca(2+)-binding transition was reduced as much as 10-30-fold depending on the presence of ATP. In mutants Gly(233) --> Glu, Gly(233) --> Val, and Pro(312) --> Ala, the rate of the Ca(2+)-binding transition was increased at least 2-3-fold at acid pH but not significantly at neutral pH, suggesting a destabilization of the protonated form. The rate of Ca(2+) dissociation was reduced 12-fold in mutant Pro(312) --> Ala and 3.5-fold in Leu(319) --> Arg, and increased at least 4-fold in a mutant in which the putative Ca(2+) liganding residue Glu(309) was replaced by aspartate. The data support a model in which Pro(312) and Leu(319) are closely associated with the cation binding pocket, Gly(233) is part of a long-range signal transmission pathway between the ion-binding sites and the catalytic site, and Lys(684) is an essential catalytic residue that may function in the same way as its counterpart in the soluble hydrolases belonging to the haloacid dehalogenase superfamily.     

Glutamic acid 160 is the acid-base catalyst of beta-xylosidase from Bacillus stearothermophilus T-6: a family 39 glycoside hydrolase

A beta-xylosidase from Bacillus stearothermophilus T-6 was cloned, overexpressed in Escherichia coli and purified to homogeneity. Based on sequence alignment, the enzyme belongs to family 39 glycoside hydrolases, which itself forms part of the wider GH-A clan. The conserved Glu160 was proposed as the acid-base catalyst. An E160A mutant was constructed and subjected to steady state and pre-steady state kinetic analysis together with azide rescue and pH activity profiles. The observed results support the assignment of Glu160 as the acid-base catalytic residue.     

Mutational analysis of Asp51 of Anabaena azollae glutamine synthetase. D51E mutation confers resistance to the active site inhibitors L-methionine-DL-sulfoximine and phosphinothricin.

The role of Asp51 in the catalytic activity of glutamine synthetase from the cyanobacterium Anabaena azollae has been analyzed. Five mutant enzymes, D51S, D51A, D51E, D51N and D51R, were constructed by site-directed mutagenesis and characterized. Asp51 appears to participate in the binding of ammonium ion, as affinity for this substrate was affected in all cases, although it varied according to the charge and/or size of the amino-acid residue, decreasing in the order Glu > Asn > Ser > Ala. The replacement of Asp51 by Glu (D51E) conferred besides a high resistance to the herbicides L-methionine-DL-sulfoximine and phosphinothricin, as a result of a decreased phosphorylation ability.     

1.2 A crystal structure of the serine carboxyl proteinase pro-kumamolisin; structure of an intact pro-subtilase

Kumamolisin, an extracellular proteinase derived from an acido/thermophilic Bacillus, belongs to the sedolisin family of endopeptidases characterized by a subtilisin-like fold and a Ser-Glu-Asp catalytic triad. In kumamolisin, the Asp82 carboxylate hydrogen bonds to Glu32-Trp129, which might act as a proton sink stabilizing the catalytic residues. The 1.2/1.3 A crystal structures of the Glu32-->Ala and Trp129-->Ala mutants show that both mutations affect the active-site conformation, causing a 95% activity decrease. In addition, the 1.2 A crystal structure of the Ser278-->Ala mutant of pro-kumamolisin was determined. The prodomain exhibits a half-beta sandwich core docking to the catalytic domain similarly as the equivalent subtilisin prodomains in their catalytic-domain complexes. This pro-kumamolisin structure displays, for the first time, the uncleaved linker segment running across the active site and connecting the prodomain with the properly folded catalytic domain. The structure strongly points to an initial intramolecular activation cleavage in subtilases, as presumed for pro-subtilisin and pro-furin.     

Catalytic mechanism of phosphorylase kinase probed by mutational studies.

The contributions to catalysis of the conserved catalytic aspartate (Asp149) in the phosphorylase kinase catalytic subunit (PhK; residues 1-298) have been studied by kinetic and crystallographic methods. Kinetic studies in solvents of different viscosity show that PhK, like cyclic AMP dependent protein kinase, exhibits a mechanism in which the chemical step of phosphoryl transfer is fast and the rate-limiting step is release of the products, ADP and phosphoprotein, and possibly viscosity-dependent conformational changes. Site-directed mutagenesis of Asp149 to Ala and Asn resulted in enzymes with a small increase in K(m) for glycogen phosphorylase b (GPb) and ATP substrates and dramatic decreases in k(cat) (1.3 x 10(4) for Asp149Ala and 4.7 x 10(3) for Asp149Asn mutants, respectively). Viscosometric kinetic measurements with the Asp149Asn mutant showed a reduction in the rate-limiting step for release of products by 4.5 x 10(3) and a significant decrease (possibly as great as 2.2 x 10(3)) in the rate constant characterizing the chemical step. The date combined with the crystallographic evidence for the ternary PhK-AMPPNP-peptide complex [Lowe et al. (1997) EMBO J. 6, 6646-6658] provide powerful support for the role of the carboxyl of Asp149 in binding and orientation of the substrate and in catalysis of phosphoryl transfer. The constitutively active subunit PhK has a glutamate (Glu182) residue in the activation segment, in place of a phosphorylatable serine, threonine, or tyrosine residue in other protein kinases that are activated by phosphorylation. Site-directed mutagenesis of Glu182 and other residues involved in a hydrogen bond network resulted in mutant proteins (Glu182Ser, Arg148Ala, and Tyr206Phe) with decreased catalytic efficiency (approximate average decrease in k(cat)/K(m) by 20-fold). The crystal structure of the mutant Glu182Ser at 2.6 A resolution showed a phosphate dianion about 2.6 A from the position previously occupied by the carboxylate of Glu182. There was no change in tertiary structure from the native protein, but the activation segment in the region C-terminal to residue 182 showed increased disorder, indicating that correct localization of the activation segment is necessary in order to recognize and present the protein substrate for catalysis.     

Probing the catalytically essential residues of the alpha-L-fucosidase from the hyperthermophilic archaeon Sulfolobus solfataricus

Retaining glycosidases promote the hydrolysis of the substrate by following a double-displacement mechanism involving a covalent intermediate. The catalytic residues are a general acid/base catalyst and the nucleophile. Experimental identification of these residues in a specific glycosidase allows for the assigning of the corresponding residues in all of the other enzymes belonging to the same family. By means of sequence alignment, mutagenesis, and detailed kinetic studies of the alpha-fucosidase from Sulfolobus solfataricus (Ssalpha-fuc) (family 29), we show here that the residues, invariant in this family, have the function inferred from the analysis of the 3D structure of the enzyme from Thermotoga maritima (Tmalpha-fuc). These include in Ssalpha-fuc the substrate-binding residues His46 and His123 and the nucleophile of the reaction, previously described. The acid/base catalyst could be assigned less easily. The k(cat) of the Ssalpha-fucGlu292Gly mutant, corresponding to the acid/base catalyst of Tmalpha-fuc, is reduced by 154-fold but could not be chemically rescued. Instead, the Ssalpha-fucGlu58Gly mutant revealed a 4000-fold reduction of k(cat)/K(M) if compared to the wild-type and showed the rescue of the k(cat) by sodium azide at wild-type levels. Thus, our data suggest that a catalytic triad, namely, Glu58, Glu292, and Asp242, is involved in catalysis. Glu58 and Glu292 cooperate in the role of acid/base catalyst, while Asp242 is the nucleophile of the reaction. Our data suggest that in glycosidase family 29 alpha-fucosidases promoting the retaining mechanism with slightly different catalytic machineries coexist.     

Dissection of the energetic coupling across the Src SH2 domain-tyrosyl phosphopeptide interface

Src Homology (SH2) domains play critical roles in signaling pathways by binding to phosphotyrosine (pTyr)-containing sequences, thereby recruiting SH2 domain-containing proteins to tyrosine-phosphorylated sites on receptor molecules. Investigations of the peptide binding specificity of the SH2 domain of the Src kinase (Src SH2 domain) have defined the EEI motif C-terminal to the phosphotyrosine as the preferential binding sequence. A subsequent study that probed the importance of eight specificity-determining residues of the Src SH2 domain found two residues which when mutated to Ala had significant effects on binding: Tyr beta D5 and Lys beta D3. The mutation of Lys beta D3 to Ala was particularly intriguing, since a Glu to Ala mutation at the first (+1) position of the EEI motif (the residue interacting with Lys beta D3) did not significantly affect binding. Hence, the interaction between Lys beta D3 and +1 Glu is energetically coupled. This study is focused on the dissection of the energetic coupling observed across the SH2 domain-phosphopeptide interface at and around the +1 position of the peptide. It was found that three residues of the SH2 domain, Lys beta D3, Asp beta C8 and AspCD2 (altogether forming the so-called +1 binding region) contribute to the selection of Glu at the +1 position of the ligand. A double (Asp beta C8Ala, AspCD2Ala) mutant does not exhibit energetic coupling between Lys beta D3 and +1 Glu, and binds to the pYEEI sequence 0.3 kcal/mol tighter than the wild-type Src SH2 domain. These results suggest that Lys beta D3 in the double mutant is now free to interact with the +1 Glu and that the role of Lys beta D3 in the wild-type is to neutralize the acidic patch formed by Asp beta C8 and AspCD2 rather than specifically select for a Glu at the +1 position as it had been hypothesized previously. A triple mutant (Lys beta D3Ala, Asp beta C8Ala, AspCD2Ala) has reduced binding affinity compared to the double (Asp beta C8Ala, AspCD2Ala) mutant, yet binds the pYEEI peptide as well as the wild-type Src SH2 domain. The structural basis for such high affinity interaction was investigated crystallographically by determining the structure of the triple (Lys beta D3Ala, Asp beta C8Ala, AspCD2Ala) mutant bound to the octapeptide PQpYEEIPI (where pY indicates a phosphotyrosine). This structure reveals for the first time contacts between the SH2 domain and the -1 and -2 positions of the peptide (i.e. the two residues N-terminal to pY). Thus, unexpectedly, mutations in the +1 binding region affect binding of other regions of the peptide. Such additional contacts may account for the high affinity interaction of the triple mutant for the pYEEI-containing peptide.     

Relaxing the nicotinamide cofactor specificity of phosphite dehydrogenase by rational design

Homology modeling was used to identify two particular residues, Glu175 and Ala176, in Pseudomonas stutzeri phosphite dehydrogenase (PTDH) as the principal determinants of nicotinamide cofactor (NAD(+) and NADP(+)) specificity. Replacement of these two residues by site-directed mutagenesis with Ala175 and Arg176 both separately and in combination resulted in PTDH mutants with relaxed cofactor specificity. All three mutants exhibited significantly better catalytic efficiency for both cofactors, with the best kinetic parameters displayed by the double mutant, which had a 3.6-fold higher catalytic efficiency for NAD(+) and a 1000-fold higher efficiency for NADP(+). The cofactor specificity was changed from 100-fold in favor of NAD(+) for the wild-type enzyme to 3-fold in favor of NADP(+) for the double mutant. Isoelectric focusing of the proteins in a nondenaturing gel showed that the replacement with more basic residues indeed changed the effective pI of the protein. HPLC analysis of the enzymatic products of the double mutant verified that the reaction proceeded to completion using either substrate and produced only the corresponding reduced cofactor and phosphate. Thermal inactivation studies showed that the double mutant was protected from thermal inactivation by both cofactors, while the wild-type enzyme was protected by only NAD(+). The combined results provide clear evidence that Glu175 and Ala176 are both critical for nicotinamide cofactor specificity. The rationally designed double mutant might be useful for the development of an efficient in vitro NAD(P)H regeneration system for reductive biocatalysis.     

Acid-base catalysis in Leuconostoc mesenteroides sucrose phosphorylase probed by site-directed mutagenesis and detailed kinetic comparison of wild-type and Glu237-->Gln mutant enzymes

The role of acid-base catalysis in the two-step enzymatic mechanism of alpha-retaining glucosyl transfer by Leuconostoc mesenteroides sucrose phosphorylase has been examined through site-directed replacement of the putative catalytic Glu237 and detailed comparison of purified wild-type and Glu237-->Gln mutant enzymes using steady-state kinetics. Reactions with substrates requiring Br?nsted catalytic assistance for glucosylation or deglucosylation were selectively slowed at the respective step, about 10(5)-fold, in E237Q. Azide, acetate and formate but not halides restored catalytic activity up to 300-fold in E237Q under conditions in which the deglucosylation step was rate-determining, and promoted production of the corresponding alpha-glucosides. In situ proton NMR studies of the chemical rescue of E237Q by acetate and formate revealed that enzymatically formed alpha-glucose 1-esters decomposed spontaneously via acyl group migration and hydrolysis. Using pH profiles of kcat/K(m), the pH dependences of kinetically isolated glucosylation and deglucosylation steps were analysed for wild-type and E237Q. Glucosylation of the wild-type proceeded optimally above and below apparent pK(a) values of about 5.6 and 7.2 respectively whereas deglucosylation was dependent on the apparent single ionization of a group of pK(a) approximately 5.8 that must be deprotonated for reaction. Glucosylation of E237Q was slowed below apparent pK(a) approximately 6.0 but had lost the high pH dependence of the wild-type. Deglucosylation of E237Q was pH-independent. The results allow unequivocal assignment of Glu237 as the catalytic acid-base of sucrose phosphorylase. They support a mechanism in which the pK(a) of Glu237 cycles between approximately 7.2 in free enzyme and approximately 5.8 in glucosyl enzyme intermediate, ensuring optimal participation of the glutamate residue side chain at each step in catalysis. Enzyme deglucosylation to an anionic nucleophile took place with Glu237 protonated or unprotonated. The results delineate how conserved active-site groups of retaining glycoside hydrolases can accommodate enzymatic function of a phosphorylase.     

Mutational and crystallographic analyses of the active site residues of the Bacillus circulans xylanase.

Using site-directed mutagenesis we have investigated the catalytic residues in a xylanase from Bacillus circulans. Analysis of the mutants E78D and E172D indicated that mutations in these conserved residues do not grossly alter the structure of the enzyme and that these residues participate in the catalytic mechanism. We have now determined the crystal structure of an enzyme-substrate complex to 108 A resolution using a catalytically incompetent mutant (E172C). In addition to the catalytic residues, Glu 78 and Glu 172, we have identified 2 tyrosine residues, Tyr 69 and Tyr 80, which likely function in substrate binding, and an arginine residue, Arg 112, which plays an important role in the active site of this enzyme. On the basis of our work we would propose that Glu 78 is the nucleophile and that Glu 172 is the acid-base catalyst in the reaction.     

Characterization of the First Fungal Glycosyl Hydrolase Family 19 Chitinase (NbchiA) from Nosema bombycis (Nb)

Chitinases (EC 3.2.1.14), as one kind of glycosyl hydrolase, hydrolyze the β‐(1,4) linkages of chitin. According to the sequence similarity, chitinases can be divided into glycoside hydrolase family 18 and family 19. Here, a chitinase from Nosema bombycis (NbchiA) was cloned and purified by metal affinity chromatography and molecular exclusion chromatography. Sequence analysis indicated that NbchiA belongs to glycoside hydrolase family 19 class IV chitinase. The optimal pH and temperature of NbchiA are 7.0 and 40 °C, respectively. This purified chitinase showed high activity toward soluble substrates such as ethylene glycol chitin and soluble chitosan. The degradation of chitin oligosaccharides (GlcNAc)_2–5 detected by high‐performance liquid chromatography showed that NbchiA hydrolyzed mainly the second glycosidic linkage from the reducing end of (GlcNAc)_3‐5. On the basis of structure‐based multiple‐sequence alignment, Glu51 and Glu60 are believed to be the key catalytic residues. The site‐directed mutation analysis revealed that the enzymatic activity was decreased upon mutation of Glu60, whereas mutation of Glu51 totally abolished the enzymatic activity. This is the first report of a GH19 chitinase in fungi and in Microsporidia.     

Identification of active-site residues of the pro-metastatic endoglycosidase heparanase

Heparanase is a beta-D-endoglucuronidase that cleaves heparan sulfate (HS) and has been implicated in many important physiological and pathological processes, including tumor cell metastasis, angiogenesis, and leukocyte migration. We report herein the identification of active-site residues of human heparanase. Using PSI-BLAST and PHI-BLAST searches of sequence databases, similarities were identified between heparanase and members of several of the glycosyl hydrolase families (10, 39, and 51) from glycosyl hydrolase clan A (GH-A), including strong local identities to regions containing the critical active-site catalytic proton donor and nucleophile residues that are conserved in this clan of enzymes. Furthermore, secondary structure predictions suggested that heparanase is likely to contain an (alpha/beta)(8) TIM-barrel fold, which is common to the GH-A families. On the basis of sequence alignments with a number of glycosyl hydrolases from GH-A, Glu(225) and Glu(343) of human heparanase were identified as the likely proton donor and nucleophile residues, respectively. The substitution of these residues with alanine and the subsequent expression of the mutant heparanases in COS-7 cells demonstrated that the HS-degrading capacity of both was abolished. In contrast, the alanine substitution of two other glutamic acid residues (Glu(378) and Glu(396)), both predicted to be outside the active site, did not affect heparanase activity. These data suggest that heparanase is a member of the clan A glycosyl hydrolases and has a common catalytic mechanism that involves two conserved acidic residues, a putative proton donor at Glu(225) and a nucleophile at Glu(343).     

Role of Glu318 at the putative distal site in the catalytic function of cytochrome P450d.

Most microsomal P450s have a conserved "threonine cluster" composed of three Thrs (Thr319, Thr321, Thr322 for P450d) at a putative distal site. An ionic amino acid at 318 is also well conserved as Glu or Asp for most P450s. To understand the role of these conserved polar amino acids at the putative distal site in the catalytic function of microsomal P450, we studied how mutations at this site of P450d influence the activation of molecular oxygen in the reconstituted system. Catalytic activity (0.02 min-1) toward 7-ethoxycoumarin of the Glu318Ala mutant of P450d was just 6% of that (0.33 min-1) of the wild type, while those of Glu318Asp, Thr319Ala, and Thr322Ala were comparable to or even higher than that of the wild type. Consumption rates of O2 and formation rates of H2O2 of those mutants varied in accord with the catalytic activities. Especially, the efficiency (0.5%) of incorporated oxygen atom to the substrate versus produced H2O2 for the Glu318Ala mutant was much lower than that (3.7%) of the wild type, while that (58.8%) for the mutant Glu318Asp was 16-fold higher than that of the wild type. In addition, the autoxidation [Fe(II)---- Fe(III)] rate (0.074 s-1) of the Glu318Ala mutant was much lower than those (0.374-0.803 s-1) of the wild type and other mutants. Thus, we strongly suggest that Glu318 plays an important role in the catalytic function toward 7-ethoxycoumarin of microsomal P450d.     

Identification of a catalytic residue of Clostridium paraputrificum N-acetyl-beta-D-glucosaminidase Nag3A by site-directed mutagenesis

Clostridium paraputrificum M-21 beta-N-acetylglucosaminidase 3A (Nag3A) is an enzyme classified in family 3 of the glycoside hydrolases. To identify catalytic residues of this enzyme, mutations were introduced into highly conserved Glu and Asp residues. Replacement of Asp175 with Ala abolished the catalytic activity without change in the circular dichroism spectrum, strongly suggesting that this residue is a catalytic residue, a nucleophile/base or a proton donor. Since the K(m) values of mutant enzymes D119N, D229N, D229A and D274N increased 17 to 41 times as compared with that of wild-type enzyme, Asp119, Asp229, and Asp274 appear to be involved in substrate recognition and binding. Taking previous studies into consideration, we presume that Asp303 is the catalytic nucleophile and Asp175 is the proton donor of C. paraputrificum Nag3A.     

Catalytic properties of a mutant beta-galactosidase from Xanthomonas manihotis engineered to synthesize galactosyl-thio-beta-1,3 and -beta-1,4-glycosides

The identity of the acid/base catalyst of the Family 35 beta-galactosidases from Xanthomonas manihotis (BgaX) has been confirmed as Glu184 by kinetic analysis of mutants modified at that position. The Glu184Ala mutant of BgaX is shown to function as an efficient thioglycoligase, which synthesises thiogalactosides with linkages to the 3 and 4 positions of glucosides and galactosides in high (>80%) yields. Kinetic analysis of the thioglycoligase reveals glycosyl donor K(m) values of 1.5-21 microM and glycosyl acceptor K(m) values from 180 to 500 microM. This mutant should be a valuable catalyst for the synthesis of metabolically stable analogues of this important glycosidic linkage.