The relationship between thermal stability and pH optimum studied with wild-type and mutant Trichoderma reesei cellobiohydrolase Cel7A.

The major cellulase secreted by the filamentous fungus Trichoderma reesei is cellobiohydrolase Cel7A. Its three-dimensional structure has been solved and various mutant enzymes produced. In order to study the potential use of T. reesei Cel7A in the alkaline pH range, the thermal stability of Cel7A was studied as a function of pH with the wild-type and two mutant enzymes using different spectroscopic methods. Tryptophan fluorescence and CD measurements of the wild-type enzyme show an optimal thermostability between pH 3.5-5.6 (Tm, 62 +/- 2 degrees C), at which the highest enzymatic activity is also observed, and a gradual decrease in the stability at more alkaline pH values. A soluble substrate, cellotetraose, was shown to stabilize the protein fold both at optimal and alkaline pH. In addition, unfolding of the Cel7A enzyme and the release of the substrate seem to coincide at both acidic and alkaline pH, demonstrated by a change in the fluorescence emission maximum. CD measurements were used to show that the five point mutations (E223S/A224H/L225V/T226A/D262G) that together result in a more alkaline pH optimum [Becker, D., Braet, C., Brumer, H., III, Claeyssens, M., Divne, C., Fagerstr?m, R.B., Harris, M., Jones, T.A., Kleywegt, G.J., Koivula, A., et al. (2001) Biochem. J.356, 19-30], destabilize the protein fold both at acidic and alkaline pH when compared with the wild-type enzyme. In addition, an interesting time-dependent fluorescence change, which was not observed by CD, was detected for the pH mutant. Our data show that in order to engineer more alkaline pH cellulases, a combination of mutations should be found, which both shift the pH optimum and at the same time improve the thermal stability at alkaline pH range.     

Catalytic-regulatory subunit interactions and allosteric effects in aspartate transcarbamylase

The x-ray structure of the unliganded aspartate transcarbamylase reveals that Arg-113 of the catalytic chain is involved in an important set of interactions at the interface between the catalytic and regulatory subunits (Honzatko, R.B., Crawford, J.L., Monaco, H.L., Ladner, J.E., Edwards, B.F.P., Evans, D.R., Warren, S.G., Wiley, D.C., Ladner, R.C., and Lipscomb, W. N. (1982) J. Mol. Biol. 160, 219-263). In order to disturb this interaction, site-directed mutagenesis has been used to replace Arg-113 with glycine. This modification results in a substantial weakening of the interface between the catalytic and regulatory subunits leading to a high tendency for dissociation. The unliganded mutant enzyme exhibits a pH dependence and a sensitivity toward mercurials analogous to that obtained for the relaxed conformation of the wild-type enzyme. Moreover, the presence of saturating concentrations of aspartate is accompanied by only a slight shift in the optimal pH for activity. The bisubstrate analog N-(phosphonacetyl)-L-aspartate induces a 2-fold increase in the sulfhydryl reactivity as compared to the 4-fold increase observed for the wild-type enzyme. Despite this change in the interactions at the interface between the catalytic and regulatory subunits, the mutant enzyme still retains homotropic and heterotropic effects and exhibits a normal affinity for aspartate. Together these data show that a substantial weakening of the catalytic-regulatory interface can occur without altering the allosteric properties of the enzyme. These results also indicate that the intersubunit interactions involving Arg-113, between the polar domain of the catalytic chain and the zinc domain of the regulatory chain, do not participate in the homotropic cooperativity of the enzyme.     

Subunit interface of triosephosphate isomerase: site-directed mutagenesis and characterization of the altered enzyme

We have replaced asparagine residues at the subunit interface of yeast triosephosphate isomerase (TIM) using site-directed mutagenesis in order to elucidate the effects of substitutions on the catalytic activity and conformational stability of the enzyme. The mutant proteins were expressed in a strain of Escherichia coli lacking the bacterial isomerase and purified by ion-exchange and immunoadsorption chromatography. Single replacements of Asn-78 by either Thr or Ile residues had little effect on the enzyme's catalytic efficiency, while the single replacement Asn-78----Asp-78 and the double replacement Asn-14/Asn-78----Thr-14/Ile-78 appreciably lowered kcat for the substrate D-glyceraldehyde 3-phosphate. The isoelectric point of the mutant Asn-78----Asp-78 was equivalent to that of wild-type yeast TIM that had undergone a single, heat-induced deamidation, and this mutant enzyme was less resistant than wild-type TIM to denaturation and inactivation caused by elevated temperature, denaturants, tetrabutylammonium bromide, alkaline pH, and proteases.     

Glutamate 264 modulates the pH dependence of the NAD(+)-dependent D-lactate dehydrogenase.

Recently, we amplified the Lactobacillus bulgaricus NAD(+)-dependent D-lactate dehydrogenase gene by the polymerase chain reaction, cloned and overexpressed it in Escherichia coli (Kochhar, S., Chuard, N., and Hottinger, H. (1992) Biochem. Biophys. Res. Commun. 185, 705-712). Polymerase chain reaction-amplified DNA fragments may contain base changes resulting in mutant gene products. A comparison of specific activities of D-lactate dehydrogenase in the crude extracts of 50 recombinant clones indicated that one of the clones had drastically reduced enzyme activity. Nucleotide sequence analysis of the insert DNA showed an exchange of A to G at position 795 resulting in substitution of Glu264 to Gly in the D-lactate dehydrogenase. The purified mutant D-lactate dehydrogenase showed a shift of 2 units in its optimum pH toward the acidic range. The dependence of kcat/Km on the pH of the mutant enzyme showed that the pKa of the free enzyme was around 4, at least 2 pH units lower than that of the wild-type enzyme. Both the wild-type and the mutant enzyme at their respective optimum pH values showed similar kcat and Km values. The data suggest that the highly conserved Glu264 is not critical for enzyme catalysis, but it must be situated within hydrogen bonding distance to amino acid residue(s) involved in substrate binding as well as in catalysis.     

Xylanase XYL1p from Scytalidium acidophilum: Site-directed mutagenesis and acidophilic adaptation

The role of residues Asp60, Tyr35 and Glu141 in the pH-dependent activity of xylanase XYL1p from Scytalidium acidophilum was investigated by site-directed mutagenesis. These amino acids are highly conserved among the acidophilic family 11 xylanases and located near the catalytic site. XYL1p and its single mutants D60N, Y35W and E141A and three combined mutants DN/YW, DN/EA and YW/EA were over-expressed in Pichia pastoris and purified. Xylanase activities at different pH’s and temperatures were determined. All mutations increased the pH optimum by 0.5–1.5 pH units. All mutants have lower specific activities except the E141A mutant that exhibited a 50% increase in specific activity at pH 4.0 and had an overall catalytic efficiency higher than the wild-type enzyme. Thermal unfolding experiments show that both the wild-type and E141A mutant proteins have a T_m maximum at pH 3.5, the E141A mutant being slightly less stable than the wild-type enzyme. These mutations confirm the importance of these amino acids in the pH adaptation. Mutant E141A with its enhanced specific activity at pH 4.0 and improved overall catalytic efficiency is of possible interest for biotechnological applications.     

Kinetic evidence for surface residues influencing the active site of Coprinus cinereus peroxidase: analysis of the pH dependence of G154E, P90H and P90H-G154E substrate entrance mutants

Three mutants of Coprinus cinereus peroxidase (CIP) were made to mimic the substrate entrance histidine 82-glutamic acid 146 pair of the substrate channel in lignin peroxidase (LIP). Compound I formation of LIP has a low pH optimum around pH 3, while optimal formation of CIP compound I is obtained at pH 6-11. The mutants were glycine 154-->glutamic acid (G154E), proline 90-->histidine (P90H) and the double mutant P90H-G154E. All three showed kinetics of compound I formation similar to that of wt CIP between pH 3 and 9. However, the stability of compound I was strongly affected by these mutations. In wt CIP compound I is stable for approximately 30 min, while compound I of the mutants were stable for 5 s or less. The P90H and P90H-G154E mutants showed pK(a) values for the alkaline transition at least one pH unit lower than for wt CIP and the G154E mutant. We suggest that the changed electrostatic field results in destabilisation of the oxidised heme in compound I and II and that the P90H residue increases the electrostatic potential in the distal cavity thereby decreasing the pK(a) for the alkaline transition.     

Site-directed mutagenesis and functional analysis of an active site tryptophan of insect chitinase

Chitinase is an enzyme used by insects to degrade the structural polysaccharide, chitin, during the molting process. Tryptophan 145 (W145) of Manduca sexta (tobacco hornworm) chitinase is a highly conserved residue found within a second conserved region of family 18 chitinases. It is located between aspartate 144 (D144) and glutamate 146 (E146), which are putative catalytic residues. The role of the active site residue, W145, in M. sexta chitinase catalysis was investigated by site-directed mutagenesis. W145 was mutated to phenylalanine (F), tyrosine (Y), isoleucine (I), histidine (H), and glycine (G). Wild-type and mutant forms of M. sexta chitinases were expressed in a baculovirus-insect cell line system. The chitinases secreted into the medium were purified and characterized by analyzing their catalytic activity and substrate or inhibitor binding properties. The wild-type chitinase was most active in the alkaline pH range. Several of the mutations resulted in a narrowing of the range of pH over which the enzyme hydrolyzed the polymeric substrate, CM-Chitin-RBV, predominantly on the alkaline side of the pH optimum curve. The range was reduced by about 1 pH unit for W145I and W145Y and by about 2 units for W145H and W145F. The W145G mutation was inactive. Therefore, the hydrophobicity of W145 appears to be critical for maintaining an abnormal pKa of a catalytic residue, which extends the activity further into the alkaline range. All of the mutant enzymes bound to chitin, suggesting that W145 was not essential for binding to chitin. However, the small difference in Km's of mutated enzymes compared to Km values of the wild-type chitinase towards both the oligomeric and polymeric substrates suggested that W145 is not essential for substrate binding but probably influences the ionization of a catalytically important group(s). The variations in kcat's among the mutated enzymes and the IC50 for the transition state inhibitor analog, allosamidin, indicate that W145 also influences formation of the transition state during catalysis.     

Kinetic evidence for surface residues influencing the active site of Coprinus cinereus peroxidase: analysis of the pH dependence of G154E,P90H and P90H–G154E substrate entrance mutants

Three mutants of Coprinus cinereus peroxidase (CIP) were made to mimic the substrate entrance histidine 82–glutamic acid 146 pair of the substrate channel in lignin peroxidase (LIP). Compound I formation of LIP has a low pH optimum around pH 3, while optimal formation of CIP compound I is obtained at pH 6–11. The mutants were glycine 154→glutamic acid (G154E), proline 90→histidine (P90H) and the double mutant P90H–G154E. All three showed kinetics of compound I formation similar to that of wt CIP between pH 3 and 9. However, the stability of compound I was strongly affected by these mutations. In wt CIP compound I is stable for approximately 30 min, while compound I of the mutants were stable for 5 s or less. The P90H and P90H–G154E mutants showed pK_a values for the alkaline transition at least one pH unit lower than for wt CIP and the G154E mutant. We suggest that the changed electrostatic field results in destabilisation of the oxidised heme in compound I and II and that the P90H residue increases the electrostatic potential in the distal cavity thereby decreasing the pK_a for the alkaline transition.     

Structural and functional effects of mutations altering the subunit interface of mitochondrial malate dehydrogenase

Among highly conserved residues in eucaryotic mitochondrial malate dehydrogenases are those with roles in maintaining the interactions between identical monomeric subunits that form the dimeric enzymes. The contributions of two of these residues, Asp-43 and His-46, to structural stability and catalytic function were investigated by construction of mutant enzymes containing Asn-43 and Leu-46 substitutions using in vitro mutagenesis of the Saccharomyces cerevisiae gene (MDH1) encoding mitochondrial malate dehydrogenase. The mutant enzymes were expressed in and purified from a yeast strain containing a disruption of the chromosomal MDH1 locus. The enzyme containing the H46L substitution, as compared to the wild type enzyme, exhibits a dramatic shift in the pH profile for catalysis toward an optimum at low pH values. This shift corresponds with an increased stability of the dimeric form of the mutant enzyme, suggesting that His-46 may be the residue responsible for the previously described pH-dependent dissociation of mitochondrial malate dehydrogenase. The D43N substitution results in a mutant enzyme that is essentially inactive in in vitro assays and that tends to aggregate at pH 7.5, the optimal pH for catalysis for the dimeric wild type enzyme.     

Cold adaptation of xylose isomerase from Thermus thermophilus through random PCR mutagenesis. Gene cloning and protein characterization.

Random PCR mutagenesis was applied to the Thermus thermophilus xylA gene encoding xylose isomerase. Three cold-adapted mutants were isolated with the following amino-acid substitutions: E372G, V379A (M-1021), E372G, F163L (M-1024) and E372G (M-1026). The wild-type and mutated xylA genes were cloned and expressed in Escherichia coli HB101 using the vector pGEM-T Easy, and their physicochemical and catalytic properties were determined. The optimum pH for xylose isomerization activity for the mutants was approximately 7.0, which is similar to the wild-type enzyme. Compared with the wild-type, the mutants were active over a broader pH range. The mutants exhibited up to nine times higher catalytic rate constants (k(cat)) for d-xylose compared with the wild-type enzyme at 60 degrees C, but they did not show any increase in catalytic efficiency (k(cat)/K(m)). For d-glucose, both the k(cat) and the k(cat)/K(m) values for the mutants were increased compared with the wild-type enzyme. Furthermore, the mutant enzymes exhibited up to 255 times higher inhibition constants (K(i)) for xylitol than the wild-type, indicating that they are less inhibited by xylitol. The thermal stability of the mutated enzymes was poorer than that of the wild-type enzyme. The results are discussed in terms of increased molecular flexibility of the mutant enzymes at low temperatures.     

On the mechanism of Rhodotorula gracilis D-amino acid oxidase: role of the active site serine 335

Serine 335 at the active site of D-amino acid oxidase from the yeast Rhodotorula gracilis (RgDAAO) is not conserved in other DAAO sequences. To assess its role in catalysis, it was mutated to Gly, the residue present in mammalian DAAO, an enzyme with a 35-fold lower turnover number with D-alanine. The spectral and ligand binding properties of the S335G mutant are similar to those of wild-type enzyme, suggesting an active site with minimally altered electrostatic properties. The S335G mutant is catalytically active, excluding an essential role of S335 in catalysis. However, S335-OH contributes to the high efficiency of the mutant enzyme since the catalytic activity of the latter is lower due to a decreased rate of flavin reduction relative to wild-type RgDAAO. Catalytic rates are pH-dependent and appear to converge to very low, but finite and similar values at low pH for both wild-type and S335G RgDAAO. While this dependence exhibits two apparent pKs with wild-type RgDAAO, with the S335G mutant a single, apparent pK approximately 8 is observed, which is attributed to the ionization of the alphaNH2 group of the bound substrate. Removal of S335-OH thus suppresses an apparent pK approximately 6. Both wild-type RgDAAO and the S335G mutant exhibit a substantial deuterium solvent kinetic isotope effect (> or =4) at pH<7 that disappears with increasing pH and reflects a pKapp=6.9 +/- 0.4. Interestingly, the substitution suppresses the activity towards d-lactate, suggesting a role of the serine 335 in removal of the substrate alpha-OH hydrogen.     

Probing the active site of Corynebacterium callunae starch phosphorylase through the characterization of wild-type and His334-->Gly mutant enzymes

His334 facilitates catalysis by Corynebacterium callunae starch phosphorylase through selective stabilization of the transition state of the reaction, partly derived from a hydrogen bond between its side chain and the C-6 hydroxy group of the glucosyl residue undergoing transfer to and from phosphate. We have substituted His334 by a Gly and measured the disruptive effects of the site-directed replacement on active site function using steady-state kinetics and NMR spectroscopic characterization of the cofactor pyridoxal 5'-phosphate and binding of carbohydrate ligands. Purified H334G showed 0.05% and 1.3% of wild-type catalytic center activity for phosphorolysis of maltopentaose (kcatP = 0.033 s(-1)) and substrate binding affinity in the ternary complex with enzyme bound to phosphate (Km = 280 mm), respectively. The 31P chemical shift of pyridoxal 5'-phosphate in the wild-type was pH-dependent and not perturbed by binding of arsenate. At pH 7.25, it was not sensitive to the replacement His334-->Gly. Analysis of interactions of alpha-d-glucose 1-phosphate and alpha-d-xylose 1-phosphate upon binding to wild-type and H334G phosphorylase, derived from saturation transfer difference NMR experiments, suggested that disruption of enzyme-substrate interactions in H334G was strictly local, affecting the protein environment of sugar carbon 6. pH profiles of the phosphorolysis rate for wild-type and H334G were both bell-shaped, with the broad pH range of optimum activity in the wild-type (pH 6.5-7.5) being narrowed and markedly shifted to lower pH values in the mutant (pH 6.5-7.0). External imidazole partly restored the activity lost in the mutant, without, however, participating as an alternative nucleophile in the reaction. It caused displacement of the entire pH profile of H334G by + 0.5 pH units. A possible role for His334 in the formation of the oxocarbenium ion-like transition state is suggested, where the hydrogen bond between its side chain and the 6-hydroxyl polarizes and positions O-6 such that electron density in the reactive center is enhanced.     

Mutational analysis of Thermus caldophilus GK24 beta-glycosidase: role of His119 in substrate binding and enzyme activity

Three amino acid residues (His119, Glu164, and Glu338) in the active site of Thermus caldophilus GK24 beta- glycosidase (Tca beta-glycosidase), a family 1 glycosyl hydrolase, were mutated by site-directed mutagenesis. To verify the key catalytic residues, Glu164 and Glu338 were changed to Gly and Gln, respectively. The E164G mutation resulted in drastic reductions of both beta-galactosidase and beta-glucosidase activities, and the E338Q mutation caused complete loss of activity, confirming that the two residues are essential for the reaction process of glycosidic linkage hydrolysis. To investigate the role of His119 in substrate binding and enzyme activity, the residue was substituted with Gly. The H119G mutant showed 53-fold reduced activity on 5 mM p-nitrophenyl beta-Dgalactopyranoside, when compared with the wild type; however, both the wild-type and mutant enzymes showed similar activity on 5 mM p-nitrophenyl beta-D-glucopyranoside at 75degreeC. Kinetic analysis with p-nitrophenyl beta-D-galactopyranoside revealed that the kcat value of the H119G mutant was 76.3-fold lower than that of the wild type, but the Km of the mutant was 15.3-fold higher than that of the wild type owing to the much lower affinity of the mutant. Thus, the catalytic efficiency (kcat/Km) of the mutant decreased to 0.08% to that of the wild type. The kcat value of the H119G mutant for p-nitrophenyl beta- D-glucopyranoside was 5.1-fold higher than that of the wild type, but the catalytic efficiency of the mutant was 2.5% of that of the wild type. The H119G mutation gave rise to changes in optima pH (from 5.5-6.5 to 5.5) and temperature (from 90 degrees C to 80-85 degrees C). This difference of temperature optima originated in the decrease of H119G's thermostability. These results indicate that His119 is a crucial residue in beta- galactosidase and beta-glucosidase activities and also influences the enzyme's substrate binding affinity and thermostability.     

Role of Asp222 in the catalytic mechanism of Escherichia coli aspartate aminotransferase: the amino acid residue which enhances the function of the enzyme-bound coenzyme pyridoxal 5'-phosphate.

Asp222 is an invariant residue in all known sequences of aspartate aminotransferases from a variety of sources and is located within a distance of strong ionic interaction with N(1) of the coenzyme, pyridoxal 5'-phosphate (PLP), or pyridoxamine 5'-phosphate (PMP). This residue of Escherichia coli aspartate aminotransferase was replaced by Ala, Asn, or Glu by site-directed mutagenesis. The PLP form of the mutant enzyme D222E showed pH-dependent spectral changes with a pKa value of 6.44 for the protonation of the internal aldimine bond, slightly lower than that (6.7) for the wild-type enzyme. In contrast, the internal aldimine bond in the D222A or D222N enzyme did not titrate over the pH range 5.3-9.5, and a 430-nm band attributed to the protonated aldimine persisted even at high pH. The binding affinity of the D222A and D222N enzymes for PMP decreased by 3 orders of magnitude as compared to that of the wild-type enzyme. Pre-steady-state half-transamination reactions of all the mutant enzymes with substrates exhibited anomalous progress curves comprising multiphasic exponential processes, which were accounted for by postulating several kinetically different enzyme species for both the PLP and PMP forms of each mutant enzyme. While the replacement of Asp222 by Glu yielded fairly active enzyme species, the replacement by Ala and Asn resulted in 8600- and 20,000-fold decreases, respectively, in the catalytic efficiency (kmax/Kd value for the most active species of each mutant enzyme) in the reactions of the PLP form with aspartate. In contrast, the catalytic efficiency of the PMP form of the D222A or D222N enzyme with 2-oxoglutarate was still retained at a level as high as 2-10% of that of the wild-type enzyme. The presteady-state reactions of these two mutant enzymes with [2-2H]aspartate revealed a deuterium isotope effect (kH/kD = 6.0) greater than that [kH/kD = 2.2; Kuramitsu, S., Hiromi, K., Hayashi, H., Morino, Y., & Kagamiyama, H. (1990) Biochemistry 29, 5469-5476] for the wild-type enzyme. These findings indicate that the presence of a negatively charged residue at position 222 is particularly critical for the withdrawal of the alpha-proton of the amino acid substrate and accelerates this rate-determining step by about 5 kcal.mol-1. Thus it is concluded that Asp222 serves as a protein ligand tethering the coenzyme in a productive mode within the active site and stabilizes the protonated N(1) of the coenzyme to strengthen the electron-withdrawing capacity of the coenzyme.     

Active-site mutants of beta-lactamase: use of an inactive double mutant to study requirements for catalysis

We have studied the catalytic activity and some other properties of mutants of Escherichia coli plasmid-encoded RTEM beta-lactamase (EC 3.5.2.6) with all combinations of serine and threonine residues at the active-site positions 70 and 71. (All natural beta-lactamases have conserved serine-70 and threonine-71.) From the inactive double mutant Ser-70----Thr, Thr-71----Ser [Dalbadie-McFarland, G., Cohen, L. W., Riggs, A. D., Morin, C., Itakura, K., & Richards, J. H. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6409-6413], an active revertant, Thr-71----Ser (i.e., residue 70 in the double mutant had changed from threonine to the serine conserved at position 70 in the wild-type enzyme), was isolated by an approach that allows identification of active revertants in the absence of a background of wild-type enzyme. This mutant (Thr-71----Ser) has about 15% of the catalytic activity of wild-type beta-lactamase. The other possible mutant involving serine and threonine residues at positions 70 and 71 (Ser-70----Thr) shows no catalytic activity. The primary nucleophiles of a serine or a cysteine residue [Sigal, I. S., Harwood, B. G., & Arentzen, R. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7157-7160] at position 70 thus seem essential for enzymatic activity. Compared to wild-type enzyme, all three mutants show significantly reduced resistance to proteolysis; for the active revertant (Thr-71----Ser), we have also observed reduced thermal stability and reduced resistance to denaturation by urea.     

Hydrogen bonding and catalysis: a novel explanation for how a single amino acid substitution can change the pH optimum of a glycosidase

The pH optima of family 11 xylanases are well correlated with the nature of the residue adjacent to the acid/base catalyst. In xylanases that function optimally under acidic conditions, this residue is aspartic acid, whereas it is asparagine in those that function under more alkaline conditions. Previous studies of wild-type (WT) Bacillus circulans xylanase (BCX), with an asparagine residue at position 35, demonstrated that its pH-dependent activity follows the ionization states of the nucleophile Glu78 (pKa 4.6) and the acid/base catalyst Glu172 (pKa 6.7). As predicted from sequence comparisons, substitution of this asparagine residue with an aspartic acid residue (N35D BCX) shifts its pH optimum from 5.7 to 4.6, with an approximately 20% increase in activity. The bell-shaped pH-activity profile of this mutant enzyme follows apparent pKa values of 3.5 and 5.8. Based on 13C-NMR titrations, the predominant pKa values of its active-site carboxyl groups are 3.7 (Asp35), 5.7 (Glu78) and 8.4 (Glu172). Thus, in contrast to the WT enzyme, the pH-activity profile of N35D BCX appears to be set by Asp35 and Glu78. Mutational, kinetic, and structural studies of N35D BCX, both in its native and covalently modified 2-fluoro-xylobiosyl glycosyl-enzyme intermediate states, reveal that the xylanase still follows a double-displacement mechanism with Glu78 serving as the nucleophile. We therefore propose that Asp35 and Glu172 function together as the general acid/base catalyst, and that N35D BCX exhibits a "reverse protonation" mechanism in which it is catalytically active when Asp35, with the lower pKa, is protonated, while Glu78, with the higher pKa, is deprotonated. This implies that the mutant enzyme must have an inherent catalytic efficiency at least 100-fold higher than that of the parental WT, because only approximately 1% of its population is in the correct ionization state for catalysis at its pH optimum. The increased efficiency of N35D BCX, and by inference all "acidic" family 11 xylanases, is attributed to the formation of a short (2.7 A) hydrogen bond between Asp35 and Glu172, observed in the crystal structure of the glycosyl-enzyme intermediate of this enzyme, that will substantially stabilize the transition state for glycosyl transfer. Such a mechanism may be much more commonly employed than is generally realized, necessitating careful analysis of the pH-dependence of enzymatic catalysis.     

Role of subsite +1 residues in pH dependence and catalytic activity of the glycoside hydrolase family 1 beta-glucosidase BGL1A from the basidiomycete Phanerochaete chrysosporium

The basidiomycete Phanerochaete chrysosporium produces two glycoside hydrolase family 1 intracellular beta-glucosidases, BGL1A and BGL1B, during the course of cellulose degradation. In order to clarify the catalytic difference between two enzymes, in spite of their high similarity in amino acid sequences (65%), five amino acids around the catalytic site of BGL1A were individually mutated to those of BGL1B (V173C, M177L, D229N, H231D, and K253A), and the effects of the mutations on cellobiose hydrolysis were evaluated. When the kinetic parameters (K(m) and k(cat)) were compared at the optimum pH for the wild-type enzyme, the kinetic efficiency was decreased in the cases of D229N, H231D, and K253A, but not V173C or M177L. The pH dependence of cellobiose hydrolysis showed a significantly more acidic pH profile for the D229N mutant, compared with the wild-type enzyme. Since D229 is located between K253 and the putative acid/base catalyst E170, we prepared the double mutant D229N/K253A, and found that its hydrolytic activity at neutral pH was restored to that of the wild-type enzyme. Our results indicate that the interaction between D229 and K253 is critical for the pH dependence and catalytic activity of BGL1A. Biotechnol. Bioeng.     

Conversion of a glutamate dehydrogenase into methionine/norleucine dehydrogenase by site-directed mutagenesis.

In earlier attempts to shift the substrate specificity of glutamate dehydrogenase (GDH) in favour of monocarboxylic amino-acid substrates, the active-site residues K89 and S380 were replaced by leucine and valine, respectively, which occupy corresponding positions in leucine dehydrogenase. In the GDH framework, however, the mutation S380V caused a steric clash. To avoid this, S380 has been replaced with alanine instead. The single mutant S380A and the combined double mutant K89L/S380A were satisfactorily overexpressed in soluble form and folded correctly as hexameric enzymes. Both were purified successfully by Remazol Red dye chromatography as routinely used for wild-type GDH. The S380A mutant shows much lower activity than wild-type GDH with glutamate. Activities towards monocarboxylic substrates were only marginally altered, and the pH profile of substrate specificity was not markedly altered. In the double mutant K89L/S380A, activity towards glutamate was undetectable. Activity towards L-methionine, L-norleucine and L-norvaline, however, was measurable at pH 7.0, 8.0 and 9.0, as for wild-type GDH. Ala163 is one of the residues that lines the binding pocket for the side chain of the amino-acid substrate. To explore its importance, the three mutants A163G, K89L/A163G and K89L/S380A/A163G were constructed. All three were abundantly overexpressed and showed chromatographic behaviour identical with that of wild-type GDH. With A163G, glutamate activity was lower at pH 7.0 and 8.0, but by contrast higher at pH 9.0 than with wild-type GDH. Activities towards five aliphatic amino acids were remarkably higher than those for the wild-type enzyme at pH 8.0 and 9.0. In addition, the mutant A163G used L-aspartate and L-leucine as substrates, neither of which gave any detectable activity with wild-type GDH. Compared with wild-type GDH, the A163 mutant showed lower catalytic efficiencies and higher K(m ) values for glutamate/2-oxoglutarate at pH 7.0, but a similar k(cat)/K(m) value and lower K(m) at pH 8.0, and a nearly 22-fold lower S(0.5) (substrate concentration giving half-saturation under conditions where Michaelis-Menten kinetics does not apply) at pH 9.0. Coupling the A163G mutation with the K89L mutation markedly enhanced activity (100-1000-fold) over that of the single mutant K89L towards monocarboxylic amino acids, especially L-norleucine and L-methionine. The triple mutant K89L/S380A/A163G retained a level of activity towards monocarboxylic amino acids similar to that of the double mutant K89L/A163G, but could no longer use glutamate as substrate. In terms of natural amino-acid substrates, the triple mutant represents effective conversion of a glutamate dehydrogenase into a methionine dehydrogenase. Kinetic parameters for the reductive amination reaction are also reported. At pH 7 the triple mutant and K89L/A163G show 5 to 10-fold increased catalytic efficiency, compared with K89L, towards the novel substrates. In the oxidative deamination reaction, it is not possible to estimate k(cat) and K(m) separately, but for reductive amination the additional mutations have no significant effect on k(cat) at pH 7, and the increase in catalytic efficiency is entirely attributable to the measured decrease in K(m). At pH 8 the enhancement of catalytic efficiency with the novel substrates was much more striking (e.g. for norleucine approximately 2000-fold compared with wild-type or the K89L mutant), but it was not established whether this is also exclusively due to more favourable Michaelis constants.     

Microbial volatile organic compound emissions from Stachybotrys chartarumgrowing on gypsum wallboard and ceiling tile

In neutralophilic bacteria, monovalent metal cation/H+ antiporters play a key role in pH homeostasis. In Escherichia coli, only four antiporters (NhaA, NhaB, MdfA and ChaA) are identified to function in maintenance of a stable cytoplasmic pH under conditions of alkaline stress. We hypothesised that the multidrug resistance protein MdtM, a recently characterised homologue of MdfA and a member of the major facilitator superfamily, also functions in alkaline pH homeostasis. Assays that compared the growth of an E. coli ΔmdtM deletion mutant transformed with a plasmid encoding wild-type MdtM or the dysfunctional MdtM D22A mutant at different external alkaline pH values (ranging from pH 8.5 to 10) revealed a potential contribution by MdtM to alkaline pH tolerance, but only when millimolar concentrations of sodium or potassium was present in the growth medium. Fluorescence-based activity assays using inverted vesicles generated from transformants of antiporter-deficient (ΔnhaA, ΔnhaB, ΔchaA) E. coli TO114 cells defined MdtM as a low-affinity antiporter that catalysed electrogenic exchange of Na+, K+, Rb+ or Li+ for H+. The K+/H+ antiport reaction had a pH optimum at 9.0, whereas the Na+/H+ exchange activity was optimum at pH 9.25. Measurement of internal cellular pH confirmed MdtM as contributing to maintenance of a stable cytoplasmic pH, acid relative to the external pH, under conditions of alkaline stress. Taken together, the results support a role for MdtM in alkaline pH tolerance. MdtM can therefore be added to the currently limited list of antiporters known to function in pH homeostasis in the model organism E. coli.     

长双歧杆菌N-乙酰氨基己糖1-位激酶的活性位点

【目的】研究长双歧杆菌(Bifidobacterium longum)JCM1217的N-乙酰氨基己糖1-位激酶(Nacetylhexosamine 1-kinase,Nah K)中对催化活性有影响的位点。【方法】利用点突变试剂盒,获得Nah K的4个位点的共10种单点突变体表达菌株。诱导表达并纯化野生型和突变体酶,用DNS法和NADH偶联的微孔板分光光度法检测野生型及突变体酶的最适p H和最适Mg~(2+)浓度,并测定酶促反应动力学参数。【结果】D208A、D208N、D208E和I24A四种突变体的催化活性几乎丧失。突变体H31A、H31V、F247A和I24V的最适p H由野生型的7.5变为7.0,突变体H31A和F247A的最适Mg~(2+)浓度由野生型的5 mmol/L变为10 mmol/L。反应动力学参数测定结果表明,突变体F247Y对底物Glc NAc/Gal NAc及ATP的催化活性均高于野生型。【结论】通过定点突变,确定了对Nah K催化活性有影响的4个位点,并且获得了一个催化效率提高的突变体(F247Y),为进一步对Nah K进行分子改造奠定了一定基础。