Molecular genetic analysis of the catalytic site of Streptococcus mutans glucosyltransferases.

In the present communication molecular genetic approaches have been utilized to confirm the nature of the catalytic site of Streptococcus mutans glucosyltransferases (GTF)s. Site-directed mutagenesis was used to convert the putative sucrose binding Asp-451 of the GTF-I enzyme from S. mutans GS5 to Glu, Asn, and Thr. All three of the resulting mutated enzymes displayed no detectable sucrase or GTF activities. By contrast, mutation of nearby Asp residues did not markedly reduce enzymatic activity. The inactive enzymes also appear to bind acceptor dextrans as well as the parental enzyme. These results confirm the essential role of Asp-451 of the GTF-I from strain GS5 and analogous Asp residues in other related GTFs in enzymatic activity.     

Identification of amino acid residues in Streptococcus mutans glucosyltransferases influencing the structure of the glucan product.

The glucosyltransferases (GTFs) of mutans streptococci are important virulence factors in the sucrose-dependent colonization of tooth surfaces by these organisms. To investigate the structure-function relationship of the GTFs, an approach was initiated to identify amino acid residues of the GTFs which affect the incorporation of glucose residues into the glucan polymer. Conserved amino acid residues were identified in the GTF-S and GTF-I enzymes of the mutans streptococci and were selected for site-directed mutagenesis in the corresponding enzymes from Streptococcus mutans GS5. Conversion of six amino acid residues of the GTF-I enzyme to those present at the corresponding positions in GTF-S, either singly or in multiple combinations, resulted in enzymes synthesizing increased levels of soluble glucans. The enzyme containing six alterations synthesized 73% water-soluble glucan in the absence of acceptor dextran T10, while parental enzyme GTF-I synthesized no such glucan product. Conversely, when residue 589 of the GTF-S enzyme was converted from Thr to either Asp or Glu, the resulting enzyme synthesized primarily water-insoluble glucan in the absence of the acceptor. Therefore, this approach has identified several amino acid positions which influence the nature of the glucan product synthesized by GTFs.     

Why are mammalian alkaline phosphatases much more active than bacterial alkaline phosphatases?

Mammalian alkaline phosphatases are 20-30-fold more active than the corresponding bacterial enzymes even though their amino acid sequences are 25–30% absolutely conserved. In the active-site region there are two noticeable differences between the sequences of the bacterial and mammalian enzymes, in the Escherichia coli enzyme positions 153 and 328 are Asp and Lys, respectively, but in the mammalian enzymes His is observed at both of these positions. Site-specific mutagenesis, genetic and X-ray crystallographic data, which will be summarized here, suggest that the His substitutions at positions 153 and 328 are primarily responsible for the differences in properties between the bacterial and mammalian alkaline phosphatases.     

Functional consequences of single amino acid substitutions in calmodulin-activated adenylate cyclase of Bordetella pertussis.

Calmodulin-activated adenylate cyclase of Bordetella pertussis and Bacillus anthracis are two cognate bacterial toxins. Three short regions of 13-24 amino acid residues in these proteins exhibit between 66 and 80% identity. Site-directed mutagenesis of four residues in B. pertussis adenylate cyclase situated in the second (Asp188, Asp190) and third (His298, Glu301) segments of identity were accompanied by important decrease, or total loss, of enzyme activity. The calmodulin-binding properties of mutated proteins showed no important differences when compared to the wild-type enzyme. Apart from the loss of enzymatic activity, the most important change accompanying replacement of Asp188 by other amino acids was a dramatic decrease in binding of 3'-anthraniloyl-2'-deoxyadenosine 5'-triphosphate, a fluorescent analogue of ATP. From these results we concluded that the two neighbouring aspartic acid residues in B. pertussis adenylate cyclase, conserved in many other ATP-utilizing enzymes, are essential for binding the Mg(2+)-nucleotide complex, and for subsequent catalysis. Replacement of His298 and Glu301 by other amino acid residues affected the nucleotide-binding properties of adenylate cyclase to a lesser degree suggesting that they might be important in the mechanism of enzyme activation by calmodulin, rather than being involved directly in catalysis.     

Catalytic mechanism of xylose (glucose) isomerase from Clostridium thermosulfurogenes. Characterization of the structural gene and function of active site histidine

The gene coding for thermophilic xylose (glucose) isomerase of Clostridium thermosulfurogenes was isolated and its complete nucleotide sequence was determined. The structural gene (xylA) for xylose isomerase encodes a polypeptide of 439 amino acids with an estimated molecular weight of 50,474. The deduced amino acid sequence of thermophilic C. thermosulfurogenes xylose isomerase displayed higher homology with those of thermolabile xylose isomerases from Bacillus subtilis (70%) and Escherichia coli (50%) than with those of thermostable xylose isomerases from Ampullariella (22%), Arthrobacter (23%), and Streptomyces violaceoniger (24%). Several discrete regions were highly conserved throughout the amino acid sequences of all these enzymes. To identify the histidine residue of the active site and to elucidate its function during enzymatic xylose or glucose isomerization, histidine residues at four different positions in the C. thermosulfurogenes enzyme were individually modified by site-directed mutagenesis. Substitution of His101 by phenylalanine completely abolished enzyme activity whereas substitution of other histidine residues by phenylalanine had no effect on enzyme activity. When His101 was changed to glutamine, glutamic acid, asparagine, or aspartic acid, approximately 10-16% of wild-type enzyme activity was retained by the mutant enzymes. The Gln101 mutant enzyme was resistant to diethylpyrocarbonate inhibition which completely inactivated the wild-type enzyme, indicating that His101 is the only essential histidine residue involved directly in enzyme catalysis. The constant Vmax values of the Gln101, Glu101, Asn101, and Asp101 mutant enzymes over the pH range of 5.0-8.5 indicate that protonation of His101 is responsible for the reduced Vmax values of the wild-type enzyme at pH below 6.5. Deuterium isotope effects by D-[2-2H]glucose on the rate of glucose isomerization indicated that hydrogen transfer and not substrate ring opening is the rate-determining step for both the wild-type and Gln101 mutant enzymes. These results suggest that the enzymatic sugar isomerization does not involve a histidine-catalyzed proton transfer mechanism. Rather, essential histidine functions to stabilize the transition state by hydrogen bonding to the C5 hydroxyl group of the substrate and this enables a metal-catalyzed hydride shift from C2 to C1.     

Identification of the histidine and aspartic acid residues essential for enzymatic activity of a family I.3 lipase by site-directed mutagenesis

A lipase from Pseudomonas sp. MIS38 (PML) is a member of the lipase family I.3. We analyzed the roles of the five histidine residues (His(30), His(274), His(291), His(313), and His(365)) and five acidic amino acid residues (Glu(253), Asp(255), Asp(262), Asp(275), and Asp(290)), which are fully conserved in the amino acid sequences of family I.3 lipases, by site-directed mutagenesis. We showed that the mutation of His(313) or Asp(255) to Ala almost fully inactivated the enzyme, whereas the mutations of other residues to Ala did not seriously affect the enzymatic activity. Measurement of the far- and near-UV circular dichroism spectra suggests that inactivation by the mutation of His(313) or Asp(255) is not due to marked changes in the tertiary structure. We propose that His(313) and Asp(255), together with Ser(207), form a catalytic triad in PML.     

Altering of the metal specificity of Escherichia coli alkaline phosphatase

Analysis of sequence alignments of alkaline phosphatases revealed a correlation between metal specificity and certain amino acid side chains in the active site that are metal-binding ligands. The Zn(2+)-requiring Escherichia coli alkaline phosphatase has an Asp at position 153 and a Lys at position 328. Co(2+)-requiring alkaline phosphatases from Thermotoga maritima and Bacillus subtilis have a His and a Trp at these positions, respectively. The mutations D153H, K328W, and D153H/K328W were induced in E. coli alkaline phosphatase to determine whether these residues dictate the metal dependence of the enzyme. The wild-type and D153H enzymes showed very little activity in the presence of Co(2+), but the K328W and especially the D153H/K328W enzymes effectively use Co(2+) for catalysis. Isothermal titration calorimetry experiments showed that in all cases except for the D153H/K328W enzyme, a possible conformation change occurs upon binding Co(2+). These data together indicate that the active site of the D153H/K328W enzyme has been altered significantly enough to allow the enzyme to utilize Co(2+) for catalysis. These studies suggest that the active site residues His and Trp at the E. coli enzyme positions 153 and 328, respectively, at least partially dictate the metal specificity of alkaline phosphatase.     

Characterization of the active site and thermostability regions of endoxylanase from Thermoanaerobacterium saccharolyticum B6A-RI.

Deletion mutants were constructed from pZEP12, which contained the intact Thermoanaerobacterium saccharolyticum endoxylanase gene (xynA). Deletion of 1.75 kb from the N-terminal end of xynA resulted in a mutant enzyme that retained activity but lost thermostability. Deletion of 1.05 kb from the C terminus did not alter thermostability or activity. The deduced amino acid sequence of T. saccharolyticum B6A-RI endoxylanase XynA was aligned with five other family F beta-glycanases by using the PILEUP program of the Genetics Computer Group package. This multiple alignment of amino acid sequences revealed six highly conserved motifs which included the consensus sequence consisting of a hydrophobic amino acid, Ser or Thr, Glu, a hydrophobic amino acid, Asp, and a hydrophobic amino acid in the catalytic domain. Endoxylanase was inhibited by EDAC [1-(3-dimethylamino propenyl)-3-ethylcarbodiimide hydrochloride], suggesting that Asp and/or Glu was involved in catalysis. Three aspartic acids, two glutamic acids, and one histidine were conserved in all six enzymes aligned. Hydrophobic cluster analysis revealed that two Asp and one Glu occur in the same hydrophobic clusters in T. saccharolyticum B6A-RI endoxylanase and two other enzymes belonging to family F beta-glycanases and suggests their involvement in a catalytic triad. These two Asp and one Glu in XynA from T. saccharolyticum were targeted for analysis by site-specific mutagenesis. Substitution of Asp-537 and Asp-602 by Asn and Glu-600 by Gln completely destroyed endoxylanase activity. These results suggest that these three amino acids form a catalytic triad that functions in a general acid catalysis mechanism.     

Directed mutagenesis of apecific active site residues on Fibrobacter succinogenes 1,3-1,4-beta -D-glucanase significantly affects catalysis and enzyme structural stability

The functional and structural significance of amino acid residues Met(39), Glu(56), Asp(58), Glu(60), and Gly(63) of Fibrobacter succinogenes 1,3-1,4-beta-d-glucanase was explored by the approach of site-directed mutagenesis, initial rate kinetics, fluorescence spectroscopy, and CD spectrometry. Glu(56), Asp(58), Glu(60), and Gly(63) residues are conserved among known primary sequences of the bacterial and fungal enzymes. Kinetic analyses revealed that 240-, 540-, 570-, and 880-fold decreases in k(cat) were observed for the E56D, E60D, D58N, and D58E mutant enzymes, respectively, with a similar substrate affinity relative to the wild type enzyme. In contrast, no detectable enzymatic activity was observed for the E56A, E56Q, D58A, E60A, and E60Q mutants. These results indicated that the carboxyl side chain at positions 56 and 60 is mandatory for enzyme catalysis. M39F, unlike the other mutants, exhibited a 5-fold increase in K(m) value. Lower thermostability was found with the G63A mutant when compared with wild type or other mutant forms of F. succinogenes 1,3-1,4-beta-d-glucanase. Denatured wild type and mutant enzymes were, however, recoverable as active enzymes when 8 m urea was employed as the denaturant. Structural modeling and kinetic studies suggest that Glu(56), Asp(58), and Glu(60) residues apparently play important role(s) in the catalysis of F. succinogenes 1,3-1,4-beta-d-glucanase.     

Site-directed mutagenesis analysis of amino acids critical for activity of the type I signal peptidase of the archaeon Methanococcus voltae

Site-directed mutagenesis studies of the signal peptidase of the methanogenic archaeon Methanococcus voltae identified three conserved residues (Ser52, His122, and Asp148) critical for activity. The requirement for one conserved aspartic acid residue distinguishes the archaeal enzyme from both the Escherichia coli and yeast Sec11 enzymes.     

Asparagine362 is essential for zinc binding and catalysis in the peptidase reaction of Saccharomyces cerevisiae leukotriene A4 hydrolase

Zinc metallopeptidases are ubiquitous enzymes with diverse cellular functions that can be found in most organisms. Leukotriene A₄ hydrolase (LTA4H; E.C. 3.3.2.6) is an unusual zinc metallopeptidase of the M1 family that also possesses an epoxide hydrolase activity; however, the role of its peptidase activity remains unknown. To further characterize the peptidase activity of LTA4H and other closely related metallopeptidases, a multiple sequence alignment and predicted structure were used to target three amino acid residues of yeast LTA4H for mutagenesis: Asn362, Trp365, and Asp399. Although mutating Trp365 and Asp399 had little effect on catalysis, altering Asn362 had varying effects on catalysis, depending on the replacement residue. Mutation of Asn362 to glutamine (N362Q) caused minor catalytic defects, while mutation to leucine (N362L) or glutamate (N362E) caused large reductions in activity. Both N362L and N362E also exhibited an altered pH dependence of catalysis, reduced chloride activation, and reduced zinc affinity and content, indicating that Asn362 may interact with the nearby zinc coordinating residue His344, and possibly with Glu363 as well, to polarize and/or orient these residues.     

Specification of amino acid residues essential for the catalytic reaction of cold-active protein-tyrosine phosphatase of a psychrophile, Shewanella sp

Protein-tyrosine phosphatase [EC 3.1.3.48] from a psychrophile, Shewanella sp. shows high activity at low temperatures and has the conserved amino acid sequence of protein-Ser/Thr-phosphatases. Site-directed mutagenesis with the conserved amino acid residues indicated that His148 could be important as a general acid catalyst and Asp115 assists the protonation with His148 of the leaving group of a substrate, and that Asp76 and Asp112 were involved in binding to magnesium ions.     

A mutagenic study of beta-1,4-galactosyltransferases from Neisseria meningitidis

N-terminal His-tagged recombinant beta-1,4-galactosyltransferase from Neisseria meningitidis was expressed and purified to homogeneity by column chromatography using Ni-NTA resin. Mutations were introduced to investigate the roles of, Ser68, His69, Glu88, Asp90, and Tyr156, which are components of a highly conserved region in recombinant beta-1,4 galactosyltransferase. Also, the functions of three other cysteine residues, Cys65, Cys139, and Cys205, were investigated using site-directed mutagenesis to determine the location of the disulfide bond and the role of the sulfhydryl groups. Purified mutant galactosyltransferases, His69Phe, Glu88Gln and Asp90Asn completely shut down wild-type galactosyltransferase activity (1-3 %). Also, Ser68Ala showed much lower activity than wild-type galactosyltransferase (19 %). However, only the substitution of Tyr156Phe resulted in a slight reduction in galactosyltransferase activity (90 %). The enzyme was found to remain active when the cysteine residues at positions 139 and 205 were replaced separately with serine. However, enzyme reactivity was found to be markedly reduced when Cys65 was replaced with serine (27 %). These results indicate that conserved amino acids such as Cys65, Ser68, His69, Glu88, and Asp90 may be involved in the binding of substrates or in the catalysis of the galactosyltransferase reaction.     

Function of conserved aromatic residues in the Gal/GalNAc-glycosyltransferase motif of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 1

UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc transferases), which initiate mucin-type O-glycan biosynthesis, have broad acceptor substrate specificities, and it is still unclear how they recognize peptides with different sequences. To increase our understanding of the catalytic mechanism of GalNAc-T1, one of the most ubiquitous isozymes, we studied the effect of substituting six conserved aromatic residues in the highly conserved Gal/GalNAc-glycosyltransferase motif with leucine on the catalytic properties of the enzyme. Our results indicate that substitutions of Trp302 and Phe325 have little impact on enzyme function and that substitutions of Phe303 and Tyr309 could be made with only limited impact on the interaction(s) with donor and/or acceptor substrates. By contrast, Trp328 and Trp316 are essential residues for enzyme functions, as substitution with leucine, at either site, led to complete inactivation of the enzymes. The roles of these tryptophan residues were further analyzed by evaluating the impact of substitutions with additional amino acids. All evaluated substitutions at Trp328 resulted in enzymes that were completely inactive, suggesting that the invariant Trp328 is essential for enzymatic activity. Trp316 mutant enzymes with nonaromatic replacements were again completely inactive, whereas two mutant enzymes containing a different aromatic amino acid, at position 316, showed low catalytic activity. Somewhat surprisingly, a kinetic analysis revealed that these two amino acid substitutions had a moderate impact on the enzyme's affinity for the donor substrate. By contrast, the drastically reduced affinity of the Trp316 mutant enzymes for the acceptor substrates suggests that Trp316 is important for this interaction.     

Secretion of mono- and diacylglycerol lipase from Penicillium camembertii U-150 by Saccharomyces cerevisiae and site-directed mutagenesis of the putative catalytic sites of the lipase.

Yeast cells carrying intronless mono- and diacylglycerol lipase (MDGL) genes, constructed by recombination of the genomic gene and cDNA, secreted MDGL into the culture supernatant. Most of the yeast MDGL were extensively glycosylated while they had a similar glyceride specificity to that of native MDGL. Site-directed mutagenesis was used to directly confirm the involvements in enzyme activity of the presumptive amino acid residues to form the catalytic center of MDGL. These residues were conserved in the primary structure alignment of a lipase family from filamentous fungi. Mutant lipase proteins in which Ser83, Ser145, or His259 was replaced with glycine were secreted by yeast transformants as inactive proteins. Mutant proteins replacing Asp199 with glycine or asparagine were not detected in the culture supernatant. Replacing other two highly conserved aspartic acids (at positions 232 and 243) with glycine did not render the enzyme inactive. These results indicate that Ser83, Ser145, and His259 in MDGL, are essential to enzyme activity. Asp199 is also likely to be involved.     

Putative reaction mechanism of heterologously expressed octopine dehydrogenase from the great scallop, Pecten maximus (L)

cDNA for octopine dehydrogenase (ODH) from the adductor muscle of the great scallop, Pecten maximus, was cloned using 5'- and 3'-RACE. The cDNA comprises an ORF of 1197 nucleotides and the deduced amino acid sequence encodes a protein of 399 amino acids. ODH was heterologously expressed in Escherichia coli with a C-terminal penta His-tag. ODH-5His was purified to homogeneity using metal-chelate affinity chromatography and Sephadex G-100 gel filtration. Recombinant ODH had kinetic properties similar to those of wild-type ODH isolated from the scallop's adductor muscle. Site-directed mutagenesis was used to elucidate the involvement of several amino acid residues for the reaction catalyzed by ODH. Cys148, which is conserved in all opine dehydrogenases known to date, was converted to serine or alanine, showing that this residue is not intrinsically important for catalysis. His212, Arg324 and Asp329, which are also conserved in all known opine dehydrogenase sequences, were subjected to site-directed mutagenesis. Modification of these residues revealed their importance for the catalytic activity of the enzyme. Conversion of each of these residues to alanine resulted in strong increases in K(m) and decreases in k(cat) values for pyruvate and L-arginine, but had little effect on the K(m) and k(cat) values for NADH. Assuming a similar structure for ODH compared with the only available structure of a bacterial opine dehydrogenase, these three amino acids may function as a catalytic triad in ODH similar to that found in lactate dehydrogenase or malate dehydrogenase. The carboxyl group of pyruvate is then stabilized by Arg324. In addition to orienting the substrate, His212 will act as an acid-base catalyst by donating a proton to the carbonyl group of pyruvate. The acidity of this histidine is further increased by the proximity of Asp329.     

Insights into the catalytic properties of bamboo vacuolar invertase through mutational analysis of active site residues

Plant acid invertases, which are either associated with the cell wall or present in vacuoles, belong to family 32 of glycoside hydrolases (GH32). Homology modeling of bamboo vacuolar invertase Boβfruct3 using Arabidopsis cell-wall invertase AtcwINV1 as a template showed that its overall structure is similar to GH32 enzymes, and that the three highly conserved motifs, NDPNG, RDP and EC, are located in the active site. This study also used site-directed mutagenesis to examine the roles of the conserved amino acid residues in these three motifs, which include Asp135, Arg259, Asp260, Glu316 and Cys317, and a conserved Trp residue (Trp159) that resides between the NDPNG and RDP motifs. The mutants W159F, W159L, E316Q and C317A retained acid invertase activity, but no invertase activity was observed for the mutant E316A or mutants with changes at Asp135, Arg259, or Asp260. The apparent K_m values of the four mutants with invertase activity were all higher than that of the wild-type enzyme. The mutants W159L and E316Q exhibited lower k_cat values than the wild-type enzyme, but an increase in the k_cat value was observed for the mutants W159F and C317A. The results of this study demonstrate that these residues have individual functions in catalyzing sucrose hydrolysis.     

Properties and active center of the thermostable branching enzyme from Bacillus stearothermophilus.

Although the branching enzyme (EC 2.4.1.18) is a member of the alpha-amylase family, the characteristics are not understood. The thermostable branching enzyme gene from Bacillus stearothermophilus TRBE14 was cloned and expressed in Escherichia coli. The branching enzyme was purified to homogeneity, and various enzymatic properties were analyzed by our improved assay method. About 80% of activity was retained when the enzyme was heated at 60 degrees C for 30 min, and the optimum temperature for activity was around 50 degrees C. The enzyme was stable in the range of pH 7.5 to 9.5, and the optimum pH was 7.5. The nucleotide sequence of the gene was determined, and the active center of the enzyme was analyzed by means of site-directed mutagenesis. The catalytic residues were tentatively identified as two Asp residues and a Glu residue by comparison of the amino acid sequences of various branching enzymes from different sources and enzymes of the alpha-amylase family. When the Asp residues and Glu were replaced by Asn and Gln, respectively, the branching enzyme activities disappeared. The results suggested that these three residues are the catalytic residues and that the catalytic mechanism of the branching enzyme is basically identical to that of alpha-amylase. On the basis of these results, four conserved regions including catalytic residues and most of the substrate-binding residues of various branching enzymes are proposed.     

Tyrosine residue 300 is important for activity and stability of branching enzyme from Escherichia coli

Branching enzyme belongs to the alpha-amylase family, which includes enzymes that catalyze hydrolysis or transglycosylation at alpha-(1,4)- or alpha-(1,6)-glucosidic linkages. In the alpha-amylase family, four highly conserved regions are proposed to make up the active site. From amino acid sequence analysis a tyrosine residue is completely conserved in the alpha-amylase family. In Escherichia coli branching enzyme, this residue (Y300) is located prior to the conserved region 1. Site-directed mutagenesis of the Y300 residue in E. coli branching enzyme was used in order to study its possible function in branching enzymes. Replacement of Y300 with Ala, Asp, Leu, Ser, and Trp resulted in mutant enzymes with less than 1% of wild-type activity. A Y300F substitution retained 25% of wild-type activity. Kinetic analysis of Y300F showed no effect on the Km value. The heat stability of Y300F was analyzed, and this was lowered significantly compared to that of the wild-type enzyme. Y300F also showed lower relative activity at elevated temperatures compared to wild-type. Thus, these results show that Tyr residue 300 in E. coli branching enzyme is important for activity and thermostability of the enzyme.     

Catalytic mechanism of endonuclease v: a catalytic and regulatory two-metal model

The enzyme endonuclease V initiates repair of deaminated DNA bases by making an endonucleolytic incision on the 3' side one nucleotide from a base lesion. In this study, we have used site-directed mutagenesis to characterize the role of the highly conserved residues D43, E89, D110, and H214 in Thermotoga maritima endonuclease V catalysis. DNA cleavage and Mn(2+)-rescue analysis suggest that amino acid substitutions at D43 impede the enzymatic activity severely while mutations at E89 and D110 may be tolerated. Mutations at H214 yield enzyme that maintains significant DNA cleavage activity. The H214D mutant exhibits little change in substrate specificity or DNA cleavage kinetics, suggesting the exchangeability between His and Asp at this site. DNA binding analysis implicates the involvement of the four residues in metal binding. Mn(2+)-mediated cleavage of inosine-containing DNA is stimulated by the addition of Ca(2+), a metal ion that does not support catalysis. The effects of Mn(2+) on Mg(2+)-mediated DNA cleavage show a complexed initial stimulatory and later inhibitory pattern. The data obtained from the dual metal ion analyses lead to the notion that two metal ions are involved in endonuclease V-mediated catalysis. A catalytic and regulatory two-metal model is proposed.