Permeabilized cells of flavocytochrome b2 over-producing recombinant yeast Hansenula polymorpha as biological recognition element in amperometric lactate biosensors

A L-lactate-selective microbial biosensor was developed using permeabilized cells of gene-engineered thermotolerant methylotrophic yeast Hansenula polymorpha, over-producing L-lactate:cytochrome c-oxidoreductase (EC 1.1.2.3, flavocytochrome b(2), FC b(2)). The construction of FC b(2)-producers by over-expression of the gene CYB2 H. polymorpha encoding FC b(2) is described. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in the frame of a plasmid for multicopy integration was transformed to the recipient strain H. polymorpha C-105 (gcr1 catX) impaired in glucose repression and devoid of catalase activity. The permeabilized cells were either immobilized on the graphite working electrode by physical entrapment of the cell suspension by means of a dialysis membrane or by integration of the cells in an electrochemically generated layer using a cathodic electrodeposition polymer. Phenazine methosulphate was used as a free-diffusing redox mediator. It was assumed that the mediator reacts with mitochondrial FC b(2) after entering the cells in the presence of L-lactate. The biosensor based on recombinant yeast cells exhibited a higher K(M)(app) value and hence expanded linear range toward L-lactate as compared to a similar sensor based on the initial cells of H. polymorpha C-105.     

Construction of flavocytochrome b2-overproducing strains of the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta)

L-Lactate cytochrome c oxidoreductase (flavocytochrome b2, FC b2) from the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta) is, unlike the enzyme form baker's yeast, a thermostable enzyme potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods. This paper describes the construction of flavocytochrome b2 producers with overexpression of the H. polymorpha CYB2 gene, encoding FC b2. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in a plasmid for multicopy integration was transformed into the recipient strain H. polymorpha C-105 (gcr1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b2 activity in permeabilized yeast cells. The optimal cultivation conditions providing for the maximal yield of the target enzyme were found. The constructed strain is a promising FC b2 producer characterized by a sixfold increased (to 3 micromol min(-1) mg(-1) protein in cell-free extract) activity of the enzyme.     

Mutant Hansenula polymorpha strain with constitutive alcohol oxidase and peroxisome biosynthesis

A mutant of the methylotrophic yeast Hansenula polymorpha with constitutive alcohol oxidase (AOX) and peroxisome biosynthesis was obtained after UV treatment followed by cell plating on a medium containing methanol and 2-deoxy-D-glucose (DOG). DOG-resistant colonies of mutants were insensitive to catabolic repression by glucose and methanol. A selection procedure is described that allows the isolation of a mutant exhibiting a constitutive phenotype of AOX involved in methanol utilization. Furthermore, additional features of the constitutive presence of peroxisomes are demonstrated. 562 DOG-resistant colonies were tested, 24 of them demonstrating constitutive AOX formation. Based on quantitative analysis, one of the strains--DOG-13 was selected and its growth, biochemical and ultrastructural characteristics were examined. Its specific enzyme activity when cultivated on a yeast nitrogen base + 1% glucose (YNB + 1% Glucose) was found to reach 145 nmol x min(-1) x mg(-1) protein (compared to zero of the parent strain) after he 20th hour of cultivation. This was confirmed by fine-structure analysis, showing typical peroxisomes, which number and size increased with the enzyme activity. This study demonstrates a constitutive AOX and peroxisome biosynthesis by the mutant strain H. polymorpha DOG-13 obtained.     

Purification and characterization of alcohol oxidase from a genetically constructed over-producing strain of the methylotrophic yeast Hansenula polymorpha

Alcohol oxidase (AOX) has been purified 8-fold from a genetically constructed over-producing strain of the methylotrophic yeast Hansenula polymorpha C-105 (gcr1 catX) with impaired glucose-induced catabolite repression and completely devoid of catalase. The final enzyme preparation was homogeneous as judged by polyacrylamide gel electrophoresis and HPLC. Some physicochemical and biochemical properties of AOX were studied in detail: molecular weight (approximately 620 kD), isoelectric point (pI 6.1), and UV-VIS, circular dichroism (CD), and fluorescence spectra. The content of different secondary structure motifs of the enzyme has been calculated from the CD spectra using a computer program. It was found that the native protein contains about 50% alpha-helix, 25% beta-sheet, and about 20% random structures. The kinetic parameters for different substrates, such as methanol, ethanol, and formaldehyde, were measured using a Clark oxygen electrode. The rate of enzymatic oxidation of formaldehyde by alcohol oxidase from H. polymorpha is only twice lower compared to the best substrate of the enzyme, methanol.     

Efficient bioconversion of ethanol to acetaldehyde using a novel mutant strain of the methylotrophic yeast Hansenula polymorpha

We report the isolation of mutant strains of the methylotrophic yeast Hansenula polymorpha that are able to efficiently oxidize ethanol to acetaldehyde in an intact cell system. The oxidation reaction is catalyzed by alcohol oxidase (AOX), a key enzyme in the methanol metabolic pathway that is typically present only in H. polymorpha cells growing on methanol. At least three mutations were introduced in the strains. Two of the mutations resulted in high levels of AOX in glucose-grown cells of the yeast. The third mutation introduced a defect in the cell's normal ability to degrade AOX in response to ethanol, and thus stabilizing the enzyme in the presence of this substrate. Using these strains, conditions for bioconversion of ethanol to acetaldehyde were examined. In addition to pH and buffer concentration, we found that the yield of acetaldehyde was improved by the addition of the proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF) and by permeabilization of the cells with digitonin. Under optimal shake-flask conditions using one of the H. polymorpha mutant strains, conversion of ethanol to acetaldehyde was nearly quantitative.     

Loop Residues and Catalysis in OMP Synthase

Residue-to-alanine mutations and a two-amino acid deletion have been made in the highly conserved catalytic loop (residues 100-109) of Salmonella typhimurium OMP synthase (orotate phosphoribosyltransferase, EC 2.4.2.10). As described previously, the K103A mutant enzyme exhibited a 10(4)-fold decrease in k(cat)/K(M) for PRPP; the K100A enzyme suffered a 50-fold decrease. Alanine mutations at His105 and Glu107 produced 40- and 7-fold decreases in k(cat)/K(M), respectively, and E101A, D104A, and G106A were slightly faster than the wild-type (WT) in terms of k(cat), with minor effects on k(cat)/K(M). Equilibrium binding of OMP or PRPP in binary complexes was affected little by loop mutation, suggesting that the energetics of ground-state binding have little contribution from the catalytic loop, or that a favorable binding energy is offset by costs of loop reorganization. Pre-steady-state kinetics for mutants showed that K103A and E107A had lost the burst of product formation in each direction that indicated rapid on-enzyme chemistry for WT, but that the burst was retained by H105A. Δ102Δ106, a loop-shortened enzyme with Ala102 and Gly106 deleted, showed a 10(4)-fold reduction of k(cat) but almost unaltered K(D) values for all four substrate molecules. The 20% (i.e., 1.20) intrinsic [1'-(3)H]OMP kinetic isotope effect (KIE) for WT is masked because of high forward and reverse commitment factors. K103A failed to express intrinsic KIEs fully (1.095 ± 0.013). In contrast, H105A, which has a smaller catalytic lesion, gave a [1'-(3)H]OMP KIE of 1.21 ± 0.0005, and E107A (1.179 ± 0.0049) also gave high values. These results are interpreted in the context of the X-ray structure of the complete substrate complex for the enzyme [Grubmeyer, C., Hansen, M. R., Fedorov, A. A., and Almo, S. C. (2012) Biochemistry 51 (preceding paper in this issue, DOI 10.1021/bi300083p )]. The full expression of KIEs by H105A and E107A may result from a less secure closure of the catalytic loop. The lower level of expression of the KIE by K103A suggests that in these mutant proteins the major barrier to catalysis is successful closure of the catalytic loop, which when closed, produces rapid and reversible catalysis.     

Alcohol oxidase is a novel pathogenicity factor for Cladosporium fulvum, but aldehyde dehydrogenase is dispensable

Cladosporiumfulvum is a mitosporic ascomycete pathogen of tomato. A study of fungal genes expressed during carbon starvation in vitro identified several genes that were up regulated during growth in planta. These included genes predicted to encode acetaldehyde dehydrogenase (Aldh1) and alcohol oxidase (Aox1). An Aldh1 deletion mutant was constructed. This mutant lacked all detectable ALDH activity, had lost the ability to grow with ethanol as a carbon source, but was unaffected in pathogenicity. Aox1 expression was induced by carbon starvation and during the later stages of infection. The alcohol oxidase enzyme activity has broadly similar properties (Km values, substrate specificity, pH, and heat stability) to yeast enzymes. Antibodies raised to Hansenula polymorpha alcohol oxidase (AOX) detected antigens in Western blots of starved C. fulvum mycelium and infected plant material. Antigen reacting with the antibodies was localized to organelles resembling peroxisomes in starved mycelium and infected plants. Disruption mutants of Aox1 lacked detectable AOX activity and had markedly reduced pathogenicity as assayed by two different measures of fungal growth. These results identify alcohol oxidase as a novel pathogenicity factor and are discussed in relation to peroxisomal metabolism of fungal pathogens during growth in planta.     

Isolation of an inulinase derepressed mutant of Pichia polymorpha for the production of fructose

Pichia polymorpha has inulinase activity and could be used for the production of fructose syrup from inulin. The application of immobilized P. polymorpha whole cells for the continuous hydrolysis of inulin is, however, limited since the biosynthesis of this enzyme system is repressed by the reaction products, dextrose and fructose. A derepressed mutant hyperproducer of inulinase was isolated after treatment with EMS followed by a selection step with deoxyglucose.     

Alcohol oxidases of Kloeckera sp. and Hansenula polymorpha. Catalytic properties and subunit structures.

1. Alcohol oxidase (alcohol: oxygen oxidoreductase) of a thermophilic methanol-utilizing yeast, Hansenula polymorpha DL-1, was isolated in crystalline form. 2. This alcohol oxidase of H. polymorpha was more stable to heat than was the enzyme of Kloeckera sp. This difference in heat stability is compatible with the difference in growth temperatures for both yeasts. 3. The crystalline alcohol oxidases of both yeast oxidized the lower primary alcohols (C-2 to C-4) as well as methanol. The apparent Km values for the methanol of Kloeckera and H. polymorpha enzymes were 0.44 and 0.23 mM, respectively. The enzymes could also oxidize formaldehyde to formate, and were inactivated by relatively low concentrations of hydrogen peroxide. 4. The molecular weight for both enzymes was calculated to be about 670000. Each enzyme is composed of eight identical subunits (molecular weight 83000) and contains eight moles of FAD as the prosthetic group. The NH2-terminal and COOH-terminal amino acids of H. polymorpha enzyme were identified as alanine and phenylalanine, respectively. The octameric subunits model of each enzyme was confirmed by electron micrographs, which showed an octad aggregate, composed of two tetragons face to face.     

Kinetic, stereochemical, and structural effects of mutations of the active site arginine residues in 4-oxalocrotonate tautomerase.

Three arginine residues (Arg-11, Arg-39, Arg-61) are found at the active site of 4-oxalocrotonate tautomerase in the X-ray structure of the affinity-labeled enzyme [Taylor, A. B., Czerwinski, R. M., Johnson, R. M., Jr., Whitman, C. P., and Hackert, M. L. (1998) Biochemistry 37, 14692-14700]. The catalytic roles of these arginines were examined by mutagenesis, kinetic, and heteronuclear NMR studies. With a 1,6-dicarboxylate substrate (2-hydroxymuconate), the R61A mutation showed no kinetic effects, while the R11A mutation decreased k(cat) 88-fold and increased K(m) 8.6-fold, suggesting both binding and catalytic roles for Arg-11. With a 1-monocarboxylate substrate (2-hydroxy-2,4-pentadienoate), no kinetic effects of the R11A mutation were found, indicating that Arg-11 interacts with the 6-carboxylate of the substrate. The stereoselectivity of the R11A-catalyzed protonation at C-5 of the dicarboxylate substrate decreased, while the stereoselectivity of protonation at C-3 of the monocarboxylate substrate increased in comparison with wild-type 4-OT, indicating the importance of Arg-11 in properly orienting the dicarboxylate substrate by interacting with the charged 6-carboxylate group. With 2-hydroxymuconate, the R39A and R39Q mutations decreased k(cat) by 125- and 389-fold and increased K(m) by 1.5- and 2.6-fold, respectively, suggesting a largely catalytic role for Arg-39. The activity of the R11A/R39A double mutant was at least 10(4)-fold lower than that of the wild-type enzyme, indicating approximate additivity of the effects of the two arginine mutants on k(cat). For both R11A and R39Q, 2D (1)H-(15)N HSQC and 3D (1)H-(15)N NOESY-HSQC spectra showed chemical shift changes mainly near the mutated residues, indicating otherwise intact protein structures. The changes in the R39Q mutant were mainly in the beta-hairpin from residues 50 to 57 which covers the active site. HSQC titration of R11A with the substrate analogue cis, cis-muconate yielded a K(d) of 22 mM, 37-fold greater than the K(d) found with wild-type 4-OT (0.6 mM). With the R39Q mutant, cis, cis-muconate showed negative cooperativity in active site binding with two K(d) values, 3.5 and 29 mM. This observation together with the low K(m) of 2-hydroxymuconate (0.47 mM) suggests that only the tight binding sites function catalytically in the R39Q mutant. The (15)Nepsilon resonances of all six Arg residues of 4-OT were assigned, and the assignments of Arg-11, -39, and -61 were confirmed by mutagenesis. The binding of cis,cis-muconate to wild-type 4-OT upshifts Arg-11 Nepsilon (by 0.05 ppm) and downshifts Arg-39 Nepsilon (by 1.19 ppm), indicating differing electronic delocalizations in the guanidinium groups. A mechanism is proposed in which Arg-11 interacts with the 6-carboxylate of the substrate to facilitate both substrate binding and catalysis and Arg-39 interacts with the 1-carboxylate and the 2-keto group of the substrate to promote carbonyl polarization and catalysis, while Pro-1 transfers protons from C-3 to C-5. This mechanism, together with the effects of mutations of catalytic residues on k(cat), provides a quantitative explanation of the 10(7)-fold catalytic power of 4-OT. Despite its presence in the active site in the crystal structure of the affinity-labeled enzyme, Arg-61 does not play a significant role in either substrate binding or catalysis.     

Cloning and characterization of glucokinase from a methylotrophic yeast Hansenula polymorpha: different effects on glucose repression in H. polymorpha and Saccharomyces cerevisiae

Glucokinase gene (HPGLK1) was cloned from a methylotrophic yeast Hansenula polymorpha by complementation of glucose-phosphorylation deficiency in a H. polymorpha double kinase-negative mutant A31-10 by a genomic library. An open reading frame of 1416 nt encoding a 471-amino-acid protein with calculated molecular weight 51.6 kDa was characterized in the genomic insert of the plasmid pH3. The protein sequence deduced from HPGLK1 exhibited 55 and 46% identity with glucokinases from Saccharomyces cerevisiae and Aspergillus niger, respectively. The enzyme phosphorylated glucose, mannose and 2-deoxyglucose, but not fructose. Transformation of HPGLK1 into A31-10 restored glucose repression of alcohol oxidase and catalase in the mutant. Transformation of HPGLK1 into S. cerevisiae triple kinase-negative mutant DFY632 showed that H. polymorpha glucokinase cannot transmit the glucose repression signal in S. CEREVSIAE: synthesis of invertase and maltase in respective transformants was insensitive to glucose repression similarly to S. cerevisiae DFY568 possessing only glucokinase.     

Adenosine deaminase converts purine riboside into an analogue of a reactive intermediate: a 13C NMR and kinetic study

The 13C NMR spectra of [2-13C]- and [6-13C]purine ribosides have been obtained free in solution and bound to the active site of adenosine deaminase. The positions of the resonances of the bound ligand are shifted relative to those of the free ligand as follows: C-2, -3.7 ppm; C-6, -73.1 ppm. The binary complexes are in slow exchange with free purine riboside on the NMR time scale, and the dissociation rate constant is estimated to be 13.5 s-1 from the slow exchange broadening of the free signal. In aqueous solution, protonation of purine riboside at N-1 results in changes in 13C chemical shift relative to those of the free base as follows: C-2, -4.9 ppm; C-6, -7.9 ppm. The changes in chemical shift that occur when purine riboside binds to the enzyme indicate that the hybridization of C-6 changes from sp2 to sp3 in the binary complex with formation of a new bond to oxygen or sulfur. A change in C-2 hybridization can be eliminated as can protonation at N-1 as the sole cause of the chemical shift changes. The kinetic constants for the adenosine deaminase catalyzed hydrolysis of 6-chloro- and 6-fluoropurine riboside have been compared, and the reactivity order implies that carbon-halogen bond breaking does not occur in the rate-determining step. These observations support a mechanism for the enzyme in which formation of a tetrahedral intermediate is the most difficult chemical step. Enzymic stabilization of this intermediate may be an important catalytic strategy used by the enzyme to lower the standard free energy of the preceding transition state.     

The wood rot ascomycete Xylaria polymorpha produces a novel GH78 glycoside hydrolase that exhibits α-L-rhamnosidase and feruloyl esterase activities and releases hydroxycinnamic acids from lignocelluloses

Soft rot (type II) fungi belonging to the family Xylariaceae are known to substantially degrade hardwood by means of their poorly understood lignocellulolytic system, which comprises various hydrolases, including feruloyl esterases and laccase. In the present study, several members of the Xylariaceae were found to exhibit high feruloyl esterase activity during growth on lignocellulosic materials such as wheat straw (up to 1,675 mU g(-1)) or beech wood (up to 80 mU g(-1)). Following the ester-cleaving activity toward methyl ferulate, a hydrolase of Xylaria polymorpha was produced in solid-state culture on wheat straw and purified by different steps of anion-exchange and size-exclusion chromatography to apparent homogeneity (specific activity, 2.2 U mg(-1)). The peptide sequence of the purified protein deduced from the gene sequence and verified by de novo peptide sequencing shows high similarity to putative α-L-rhamnosidase sequences belonging to the glycoside hydrolase family 78 (GH78; classified under EC 3.2.1.40). The purified enzyme (98 kDa by SDS-PAGE, 103 kDa by size-exclusion chromatography; pI 3.7) converted diverse glycosides (e.g., α-L-rhamnopyranoside and α-L-arabinofuranoside) but also natural and synthetic esters (e.g., chlorogenic acid, hydroxycinnamic acid glycoside esters, veratric acid esters, or p-nitrophenyl acetate) and released free hydroxycinnamic acids (ferulic and coumaric acid) from arabinoxylan and milled wheat straw. These catalytic properties strongly suggest that X. polymorpha GH78 is a multifunctional enzyme. It is the first fungal enzyme that combines glycosyl hydrolase with esterase activities and may help this soft rot fungus to degrade lignocelluloses.     

Neisseria gonorrheae O-acetylpeptidoglycan esterase, a serine esterase with a Ser-His-Asp catalytic triad

O-Acetylpeptidoglycan esterase from Neisseria gonorrheae FA1090 is similar in sequence to family CE-3 carbohydrate esterases of the CAZy classification system, and it functions to release O-linked acetyl groups from the C-6 position of muramoyl residues in O-acetylated peptidoglycan. Here, we characterize the peptidoglycan of N. gonorrheae FA1090 as being O-acetylated and find that it serves as a substrate for the esterase. The influence of pH on the activity of O-acetylpeptidoglycan esterase was determined, and pKa values of 6.38 and 6.78 for the enzyme-substrate complex (VEt-1) and free enzyme (VEt-1KM-1), respectively, were calculated. The enzyme was inactivated by sulfonyl fluorides but not by EDTA. Multiple-sequence alignment of the O-acetylpeptidoglycan esterase family 1 enzymes with members of the CE-3 enzymes and protein modeling studies identified Ser80, Asp366, and His369 as three invariant amino acid residues that could potentially serve as a catalytic triad. Replacement of each with alanine was accomplished by site-directed mutagenesis, and the resulting mutant proteins were purified to apparent homogeneity. The specific activity of each of the three esterase derivatives was greatly reduced on O-acetylpeptidoglycan. Using the artificial substrate p-nitrophenyl acetate, a kinetic analysis revealed that the turnover number (VEt-1) but not KM was affected by the replacements. These data thus indicate that N. gonorrheae O-acetylpeptidoglycan esterase, and by analogy the CE-3 family of enzymes, function as serine esterases involving a Ser-His-Asp catalytic triad.     

Orotidine-5'-monophosphate decarboxylase catalysis: kinetic isotope effects and the state of hybridization of a bound transition-state analogue

The enzymatic decarboxylation of orotidine 5'-monophosphate may proceed by an addition-elimination mechanism involving a covalently bound intermediate or by elimination of CO2 to generate a nitrogen ylide. In an attempt to distinguish between these two alternatives, 1-(phosphoribosyl)barbituric acid was synthesized with 13C at the 5-position. Interaction of this potential transition-state analogue inhibitor with yeast orotidine-5'-monophosphate decarboxylase resulted in a small (0.6 ppm) downfield displacement of the C-5 resonance, indicating no rehybridization of the kind that might have been expected to accompany 5,6-addition of an enzyme nucleophile. When the substrate orotidine 5'-monophosphate was synthesized with deuterium at C-5, no significant change in kcat (H/D = 0.99 +/- 0.06) or kcat/KM (H/D = 1.00 +/- 0.06) was found to result, suggesting that C-5 does not undergo significant changes in geometry before or during the step that determines the rate of the catalytic process. These results are consistent with a nitrogen ylide mechanism and offer no support for the intervention of covalently bound intermediates in the catalytic process.     

Biochemical characterization and substrate profiling of a new NADH-dependent enoate reductase from Lactobacillus casei

Carbon-carbon double bond of α,β-unsaturated carbonyl compounds can be reduced by enoate reductase (ER), which is an important reaction in fine chemical synthesis. A putative enoate reductase gene from Lactobacillus casei str. Zhang was cloned into pET-21a+ and expressed in Escherichia coli BL21 (DE3) host cells. The encoded enzyme (LacER) was purified by ammonium sulfate precipitation and treatment in an acidic buffer. This enzyme was identified as a NADH-dependent enoate reductase, which had a K(m) of 0.034 ± 0.006 mM and k(cat) of (3.2 ± 0.2) × 103 s?1 toward NADH using 2-cyclohexen-1-one as the substrate. Its K(m) and k(cat) toward substrate 2-cyclohexen-1-one were 1.94 ± 0.04 mM and (8.4 ± 0.2) × 103 s?1, respectively. The enzyme showed a maximum activity at pH 8.0-9.0. The optimum temperature of the enzyme was 50-55°C, and LacER was relatively stable below 60 °C. The enzyme was active toward aliphatic alkenyl aldehyde, ketones and some cyclic anhydrides. Substituted groups of cyclic α,β-unsaturated ketones and its ring size have positive or negative effects on activity. (R)-(-)-Carvone was reduced to (2R,5R)-dihydrocarvone with 99% conversion and 98% (diasteromeric excess: de) stereoselectivity, indicating a high synthetic potential of LacER in asymmetric synthesis.     

Identification of the catalytic residues in family 52 glycoside hydrolase,a beta-xylosidase from Geobacillus stearothermophilus T-6

beta-d-Xylosidases (EC 3.2.1.37) are exo-type glycoside hydrolases that hydrolyze short xylooligosaccharides to xylose units. The enzymatic hydrolysis of the glycosidic bond involves two carboxylic acid residues, and their identification, together with the stereochemistry of the reaction, provides crucial information on the catalytic mechanism. Two catalytic mutants of a beta-xylosidase from Geobacillus stearothermophilus T-6 were subjected to detailed kinetic analysis to verify their role in catalysis. The activity of the E335G mutant decreased approximately 106-fold, and this activity was enhanced 103-fold in the presence of external nucleophiles such as formate and azide, resulting in a xylosyl-azide product with an opposite anomeric configuration. These results are consistent with Glu335 as the nucleophile in this retaining enzyme. The D495G mutant was subjected to detailed kinetic analysis using substrates bearing different leaving groups (pKa). The mutant exhibited 103-fold reduction in activity, and the Br?nsted plot of log(kcat) versus pKa revealed that deglycosylation is the rate-limiting step, indicating that this step was reduced by 103-fold. The rates of the glycosylation step, as reflected by the specificity constant (kcat/Km), were similar to those of the wild type enzyme for hydrolysis of substrates requiring little protonic assistance (low pKa) but decreased 102-fold for those that require strong acid catalysis (high pKa). Furthermore, the pH dependence profile of the mutant enzyme revealed that acid catalysis is absent. Finally, the presence of azide significantly enhanced the mutant activity accompanied with the generation of a xylosyl-azide product with retained anomeric configuration. These results are consistent with Asp495 acting as the acid-base in XynB2.     

Catalytic role of histidine 147 in Escherichia coli thymidylate synthase

Nine mutant thymidylate synthases were isolated that only differed in sequence at position 147. The wild-type enzyme (which had a histidine residue at 147) and mutant enzymes were purified to near homogeneity and their kinetic properties were compared. Although the kcat values for the mutant enzymes were 10-10,000-fold lower than for the wild-type enzyme, the Km values for both 2'-deoxyuridylate and 5,10-methylenetetrahydrofolate were nearly identical for all the enzymes indicating that His-147 is not significantly involved in initial substrate binding. By comparing the wild-type (His-147) to the glycine (Gly-147) enzyme, the side chain of His-147 was estimated to lower the activation energy of the catalytic step by 1.6-2.9 kcal mol-1. In contrast to the wild-type enzyme, the activity of the Gly-147 enzyme decreased when the pH was raised above 7.5. The activity loss coincided with the deprotonation of a residue that had a pKa of 9.46 +/- 0.2 and an enthalpy of ionization (delta Hion) of 12.1 +/- 0.9. These values are consistent with the involvement of a lysine or an arginine residue in the catalytic process. An inspection of the rates of ternary complex formation among enzyme, 5-fluoro-2'-deoxyuridylate, and 5,10-methylenetetrahydrofolate for the mutant enzymes indicated that His-147 is not needed for the proton removal from C-5 of 2'-deoxyuridylate but rather participates in an initial catalytic step and alters the pKa value of a catalytically important lysine or arginine residue.     

Recombinant expression and enzymatic characterization of PttCel9A, a KOR homologue from Populus tremula x tremuloides

PttCel9A is a membrane-bound, family 9 glycosyl hydrolase from Populus tremula x tremuloides that is upregulated during secondary cell wall synthesis. The catalytic domain of PttCel9A, Delta(1-105)PttCel9A, was purified, and its activity was compared to TfCel9A and TfCel9B from Thermobifida fusca. Since aromatic amino acids involved in substrate binding at subsites -4, -3, and -2 are missing in PttCel9A, the activity of TfCel9A mutant enzymes W256S, W209A, and W313G was also investigated. Delta(1-105)PttCel9A hydrolyzed a comparatively narrow range of polymeric substrates, and the preferred substrate was (carboxymethyl)cellulose 4M. Moreover, Delta(1-105)PttCel9A did not hydrolyze oligosaccharides shorter than cellopentaose, whereas TfCel9A and TfCel9B hydrolyzed cellotetraose and cellotriose, respectively. These data suggest that the preferred substrates of PttCel9A are long, low-substituted, soluble cellulosic polymers. At 30 degrees C and pH 6.0, the kcat for cellohexaose of Delta(1-105)PttCel9A, TfCel9A, and TfCel9B were 0.023 +/- 0.001, 16.9 +/- 2.0, and 1.3 +/- 0.2, respectively. The catalytic efficiency (kcat/Km) of TfCel9B was 39% of that of TfCel9A, whereas the catalytic efficiency of Delta(1-105)PttCel9A was 0.04% of that of TfCel9A. Removing tryptophan residues at subsites -4, -3, and -2 decreased the efficiency of cellohexaose hydrolysis by TfCel9A. Mutation of W313 to G had the most drastic effect, producing a mutant enzyme with 1% of the catalytic efficiency of TfCel9A. The apparent narrow substrate range and catalytic efficiency of PttCel9A are correlated with a lack of aromatic amino acids in the substrate binding cleft and may be necessary to prevent excessive hydrolysis of cell wall polysaccharides during cell wall formation.     

Reversal of the extreme coenzyme selectivity of Clostridium symbiosum glutamate dehydrogenase

Active-site mutants of glutamate dehydrogenase from Clostridium?symbiosum have been designed and constructed and the effects on coenzyme preference evaluated by detailed kinetic measurements. The triple mutant F238S/P262S/D263K shows complete reversal in coenzyme selectivity from NAD(H) to NADP(H) with retention of high levels of catalytic activity for the new coenzyme. For oxidized coenzymes, k(cat) /K(m) ratios of the wild-type and triple mutant enzyme indicate a shift in preference of approximately 1.6?×?10(7) -fold, from ～?80?000-fold in favour of NAD(+) to ～?200-fold in favour of NADP(+) . For reduced coenzymes the corresponding figure is 1.7?×?10(4) -fold, from ～?1000-fold in favour of NADH to ～?17-fold in favour of NADPH. A fourth mutation (N290G), previously identified as having a potential bearing on coenzyme specificity, did not engender any further shift in preference when incorporated into the triple mutant, despite having a significant effect when expressed as a single mutant.