Engineering a chimeric pyrroloquinoline quinone glucose dehydrogenase: improvement of EDTA tolerance, thermal stability and substrate specificity

An engineered Escherichia coli PQQ glucose dehydrogenase (PQQGDH) with improved enzymatic characteristics was constructed by substituting and combining the gene-encoding protein regions responsible for EDTA tolerance, thermal stability and substrate specificity. The protein region responsible for complete EDTA tolerance in Acinetobacter calcoaceticus, which is recognized as the indicator of high stability in co-factor binding, was elucidated. The region is located between 32 and 59% from the N-terminus of A. calcoaceticus PQQGDH(A27 region) and also corresponds to the same position from 32 to 59% from the N-terminus in E. coli PQQGDH, though E. coli PQQGDH is EDTA sensitive. We previously reported that the C-terminal 3% region of A. calcoaceticus (A3 region) played an important role in the increase of thermal stability, and that His775Asn substitution in E. coli PQQGDH resulted in an increase in the substrate specificity of E. coli PQQGDH towards glucose. Based on these findings, chimeric and/or mutated PQQGDHs, E97A3 H775N, E32A27E41 H782N, E32A27E38A3 and E32A27E38A3 H782N were constructed to investigate the compatibility of two protein regions and one amino acid substitution. His775 substitution to Asn corresponded to His782 substitution to Asn (H782N) in chimeric enzymes harbouring the A27 region. Since all the chimeric PQQGDHs harbouring the A27 region were EDTA tolerant, the A27 region was found to be compatible with the other region and substituted amino acid responsible for the improvement of enzymatic properties. The contribution of the A3 region to thermal stability complemented the decrease in the thermal stability due to the His775 or His782 substitution to Asn. E32A27E38A3 H782N, which harbours all the above mentioned three regions, showed improved EDTA tolerance, thermal stability and substrate specificity. These results suggested a strategy for the construction of a semi-artificial enzyme by substituting and combining the gene-encoding protein regions responsible for the improvement of enzyme characteristics. The characteristics of constructed chimeric PQQGDH are discussed based on the predicted model, beta-propeller structure.     

Construction of multi-chimeric pyrroloquinoline quinone glucose dehydrogenase with improved enzymatic properties and application in glucose monitoring

A multi-chimeric enzyme was constructed by combining the protein regions responsible for the enzymatic properties of Escherichia coli and Acinetobacter calcoaceticus pyrroloquinoline quinone glucose dehydrogenase (PQQGDH). The constructed multi-chimeric PQQGDH showed increased co-factor binding stability, thermal stability, an alteration in substrate specificity and a 10-fold increase in the K _m value for glucose compared with the wild-type E. coli PQQGDH. The cumulative effect of each introduced protein region on the improvement of enzymatic properties was observed. The application of the multi-chimeric PQQGDH in amperometric glucose sensor construction achieved an expanded dynamic range together with increased operational stability and narrower substrate specificity. The glucose sensor can measure glucose from 5 to 40 mM, suggesting its potential for the direct measurement of high blood-glucose levels in diabetic patients.     

Glu742 substitution to Lys enhances the EDTA tolerance ofEscherichia coli PQQ glucose dehydrogenase

Summary Based on homology analysis of the PQQ (pyrroloquinoline quinone) glucose dehydrogenase (PQQGDH) gene fromEscherichia coli andAcinetobacter calcoaceticus, Glu742 was substituted to Lys by site directed mutagenesis of theE. coli PQQGDH gene (gcd). The mutant enzyme, E742K showed higher tolerance towards EDTA inactivation than wild type PQQGDH. This is the first mutagenesis study of putative a PQQ binding site in PQQ enzyme.     

High‐level production of soluble pyrroloquinoline quinone‐dependent glucose dehydrogenase in Escherichia coli

Pyrroloquinoline quinone‐dependent glucose dehydrogenase (EC1.1.5.2, PQQGDH) has attracted progressive attention due to its application in glucose detection in clinic diagnosis and industrial bioprocess controls. To satisfy its increasing demand, improvement of PQQGDH production derived from Acinetobacter calcoaceticus L.M.D. 79.41 in recombinant Escherichia coli is necessary and is therefore the focus of the current study. Different carbon sources as well as induction conditions were investigated for overexpression of soluble PQQGDH. The results indicate that the target protein was optimally produced with 20 g/L glucose as the substrate. Moreover, the highest expression level (1530 kU/L) was achieved by a novel two‐temperature cultivation strategy in the 10‐L fermentor. This presents a sixfold improvement over previously reported values. After Ni‐NTA affinity chromatography purification, high‐purity enzyme with the specific activity of 5811 U/mg was obtained with a purification yield of 55%. The purified recombinant PQQGDH showed thermal stability and substrate specificity as the native enzyme. In summary, this work provides an alternative production process to overexpress PQQGDH and shows high applicability for large‐scale production of this important glucose dehydrogenase.     

Functions of amino acid residues in the active site of Escherichia coli pyrroloquinoline quinone-containing quinoprotein glucose dehydrogenase

Several mutants of quinoprotein glucose dehydrogenase (GDH) in Escherichia coli, located around its cofactor pyrroloquinoline quinone (PQQ), were constructed by site-specific mutagenesis and characterized by enzymatic and kinetic analyses. Of these, critical mutants were further characterized after purification or by different amino acid substitutions. H262A mutant showed reduced affinities both for glucose and PQQ without significant effect on glucose oxidase activity, indicating that His-262 occurs very close to PQQ and glucose, but is not the electron acceptor from PQQH(2). W404A and W404F showed pronounced reductions of affinity for PQQ, and the latter rather than the former had equivalent glucose oxidase activity to the wild type, suggesting that Trp-404 may be a support for PQQ and important for the positioning of PQQ. D466N, D466E, and K493A showed very low glucose oxidase activities without influence on the affinity for PQQ. Judging from the enzyme activities of D466E and K493A, as well as their absorption spectra of PQQ during glucose oxidation, we conclude that Asp-466 initiates glucose oxidation reaction by abstraction of a proton from glucose and Lys-493 is involved in electron transfer from PQQH(2).     

Identification of omega-aminotransferase from Caulobacter crescentus and site-directed mutagenesis to broaden substrate specificity

A putative aminotransferase gene, cc3143 (aptA), from Caulobacter crescentus was screened by bioinformatical tools and overexpressed in E. coli, and the substrate specificity of the aminotransferase was investigated. AptA showed high activity for short-chain beta-amino acids. It showed the highest activity for 3-amino-n-butyric acid. It showed higher activity toward aromatic amines than aliphatic amines. The 3D model of the aminotransferase was constructed by homology modeling using a dialkylglycine decarboxylase PDB ID: 1DGE) as a template. Then, the aminotransferase was rationally redesigned to increase the activity for 3-amino- 3-phenylpropionic acid. The mutants N285A and V227G increased the relative activity for 3-amino-3-phenylpropionic acid to 3-amino-n-butyric acid by 11-fold and 3-fold, respectively, over that of wild type.     

Properties of a chimeric glucose dehydrogenase improved by site directed mutagenesis

Glucose dehydrogenase, a membrane bound enzyme oxidizing glucose to gluconic acid in the periplasmic space of Gram-negative bacteria plays a key role in mineral phosphate solubilization and is also an industrially important enzyme, being used as a glucose biosensor. A chimeric glucose dehydrogenase (ES chimera) encoding the N-terminal transmembrane domain from Escherichia coli and the C-terminal periplasmic domain from Serratia marcescens was constructed and the expression was studied on MacConkey glucose medium. The phosphate solubilizing ability of the chimeric GDH was also evaluated, substantiating the role of GDH in mineral phosphate solubilization (MPS). Four mutants of ES chimeric GDH were generated by site directed mutagenesis and the enzyme properties studied. Though the substrate affinity was unaltered for E742K and Y771M, the affinity of H775A and EYH/KMA to glucose and galactose decreased marginally and the affinity to maltose increased. Though Y771M showed a decreased GDH activity there was an increase in the heat tolerance. All the mutants showed an increase in the EDTA tolerance. The triple mutant EYH/KMA showed improved heat and EDTA tolerance and also an increase in affinity to maltose over the ES chimeric GDH.     

Improved substrate specificity of water-soluble pyrroloquinoline quinone glucose dehydrogenase by a peptide ligand

A new approach in altering the substrate specificity of enzyme is proposed using glucose dehydrogenase, with pyrroroquinoine quinone (PQQGDH) as co-factor, as the model. This approach is based on the selection of random peptide phage displayed library. Using an M13 phage-display random peptide library, we have selected peptide ligands. Among the peptide ligands, a 7-mer peptide, composed of Thr-Thr-Ala-Thr-Glu-Tyr-Ser, caused PQQGDH substrate specificity to decrease significantly toward disaccharides, such as maltose and lactose, while a smaller effect was observed toward glucose. Consequently, this peptide narrowed the substrate specificity of PQQGDH, without a significant loss of the enzyme activity.     

A conserved negatively charged cluster in the active site of creatine kinase is critical for enzymatic activity

Creatine kinase catalyzes the reversible transphosphorylation of creatine by MgATP. From the sequence homology and the molecular structure of creatine kinase isoenzymes, we have identified several highly conserved residues with a potential function in the active site: a negatively charged cluster (Glu(226), Glu(227), Asp(228)) and a serine (Ser(280)). Mutant proteins E226Q, E226L, E227Q, E227L, D228N, and S280A/S280D of human sarcomeric mitochondrial creatine kinase were generated by in vitro mutagenesis, expressed in Escherichia coli, and purified to homogeneity. Their overall structural integrity was confirmed by CD spectroscopy and gel filtration chromatography. The enzymatic activity of all proteins mutated in the negatively charged cluster was extremely low (0.002-0.4% of wild type) and showed apparent Michaelis constants (K(m)) similar to wild type, suggesting that most of the residual activity may be attributed to wild-type revertants. Mutations of Ser(280) led to higher residual activities and altered K(m) values; S280A showed an increase of K(m) for phosphocreatine (65-fold), creatine (6-fold), and ATP (6-fold); S280D showed a decrease of K(m) for creatine (6-fold). These results, together with the transition state structure of the homologous arginine kinase (Zhou, G., Somasundaram, T., Blanc, E., Parthasarathy G., Ellington, W. R., and Chapman, M. S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8449-8454), strongly suggest a critical role of Glu(226), Glu(227), and Asp(228) in substrate binding and catalysis and point to Glu(227) as a catalytic base.     

Characterization of 1-deoxy-D-xylulose 5-phosphate reductoisomerase, an enzyme involved in isopentenyl diphosphate biosynthesis, and identification of its catalytic amino acid residues

1-Deoxy-d-xylulose 5-phosphate (DXP) reductoisomerase, which simultaneously catalyzes the intramolecular rearrangement and reduction of DXP to form 2-C-methyl-d-erythritol 4-phosphate, constitutes a key enzyme of an alternative mevalonate-independent pathway for isopentenyl diphosphate biosynthesis. The dxr gene encoding this enzyme from Escherichia coli was overexpressed as a histidine-tagged protein and characterized in detail. DNA sequencing analysis of the dxr genes from 10 E. coli dxr-deficient mutants revealed base substitution mutations at four points: two nonsense mutations and two amino acid substitutions (Gly(14) to Asp(14) and Glu(231) to Lys(231)). Diethyl pyrocarbonate treatment inactivated DXP reductoisomerase, and subsequent hydroxylamine treatment restored the activity of the diethyl pyrocarbonate-treated enzyme. To characterize these defects, we overexpressed the mutant enzymes G14D, E231K, H153Q, H209Q, and H257Q. All of these mutant enzymes except for G14D were obtained as soluble proteins. Although the purified enzyme E231K had wild-type K(m) values for DXP and NADPH, the mutant enzyme had less than a 0.24% wild-type k(cat) value. K(m) values of H153Q, H209Q, and H257Q for DXP increased to 3.5-, 7.6-, and 19-fold the wild-type value, respectively. These results indicate that Glu(231) of E. coli DXP reductoisomerase plays an important role(s) in the conversion of DXP to 2-C-methyl-d-erythritol 4-phosphate, and that His(153), His(209), and His(257), in part, associate with DXP binding in the enzyme molecule.     

Substrate and inhibitor specificity of Mycobacterium avium dihydrofolate reductase

Dihydrofolate reductase (EC 1.5.1.3) is a key enzyme in the folate biosynthetic pathway. Information regarding key residues in the dihydrofolate-binding site of Mycobacterium avium dihydrofolate reductase is lacking. On the basis of previous information, Asp31 and Leu32 were selected as residues that are potentially important in interactions with dihydrofolate and antifolates (e.g. trimethoprim), respectively. Asp31 and Leu32 were modified by site-directed mutagenesis, giving the mutants D31A, D31E, D31Q, D31N and D31L, and L32A, L32F and L32D. Mutated proteins were expressed in Escherichia coli BL21(DE3)pLysS and purified using His-Bind resin; functionality was assessed in comparison with the recombinant wild type by a standard enzyme assay, and growth complementation and kinetic parameters were evaluated. All Asp31 substitutions affected enzyme function; D31E, D31Q and D31N reduced activity by 80-90%, and D31A and D31L by > 90%. All D31 mutants had modified kinetics, ranging from three-fold (D31N) to 283-fold (D31L) increases in K(m) for dihydrofolate, and 12-fold (D31N) to 223 077-fold (D31L) decreases in k(cat)/K(m). Of the Leu32 substitutions, only L32D caused reduced enzyme activity (67%) and kinetic differences from the wild type (seven-fold increase in K(m); 21-fold decrease in k(cat)/K(m)). Only minor variations in the K(m) for NADPH were observed for all substitutions. Whereas the L32F mutant retained similar trimethoprim affinity as the wild type, the L32A mutation resulted in a 12-fold decrease in affinity and the L32D mutation resulted in a seven-fold increase in affinity for trimethoprim. These findings support the hypotheses that Asp31 plays a functional role in binding of the substrate and Leu32 plays a functional role in binding of trimethoprim.     

Single-turnover analysis of mutant human apurinic/apyrimidinic endonuclease

Apurinic/apyrimidinic endonuclease (AP endo) is a key enzyme in the repair of oxidatively damaged DNA. Using single-turnover conditions, we recently described substrate binding parameters for wild type human AP endo. In this study, we utilized four enzyme mutants, D283A, D308A, D283A/D308A, and H309N, and assayed them under steady state and single-turnover conditions. The turnover number of the single aspartate mutants was decreased 10-30-fold in comparison to that of the wild type. The decrease in the turnover number was accompanied by a 17- and 50-fold decrease in the forward rate constant (kon) for substrate binding by D308A and D283A, respectively. The dissociation rate constant for substrate (koff) was unchanged for the D308A mutant but was 10 times faster for the D283A mutant than for the wild type. The apparent Km values for both of the single aspartate mutants were about equal to their respective KD values. To account for the kinetic behavior of the D308A mutant, it was necessary to insert a conformational change into the kinetic scheme. In contrast to the single aspartate mutants, the turnover number for the double mutant was 500-fold lower than that of the wild type, its apparent Km was 2.5-fold higher, and binding to substrate was weak. Mutation of His309 caused the greatest decrease in activity, resulting in a turnover number that was more than 30000-fold lower than that of the wild type and an apparent Km that was 13-fold higher, supporting the notion that His309 is intimately involved in catalysis. Molecular dynamics simulation techniques suggested that conversion of either aspartate to alanine resulted in major shifts in the spatial localization of key amino acids. Despite the fact that the two aspartates flank His309, the movement they engendered was distinct, consistent with the differences in catalytic behavior. We suggest that the conformation of the active site is largely maintained by the two aspartates, which enable efficient binding and cleavage of abasic site-containing DNA.     

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.     

Development of a whole-cell biocatalyst co-expressing P450 monooxygenase and glucose dehydrogenase for synthesis of epoxyhexane

Oxygenases-based Escherichia coli whole-cell biocatalyst can be applied for catalysis of various commercially interesting reactions that are difficult to achieve with traditional chemical catalysts. However, substrates and products of interest are often toxic to E. coli, causing a disruption of cell membrane. Therefore, organic solvent-tolerant bacteria became an important tool for heterologous expression of such oxygenases. In this study, the organic solvent-tolerant Bacillus subtilis 3C5N was developed as a whole-cell biocatalyst for epoxidation of a toxic terminal alkene, 1-hexene. Comparing to other hosts tested, high level of tolerance towards 1-hexene and a moderately hydrophobic cell surface of B. subtilis 3C5N were suggested to contribute to its higher 1,2-epoxyhexane production. A systematic optimization of reaction conditions such as biocatalyst and substrate concentration resulted in a 3.3-fold increase in the specific rate. Co-expression of glucose dehydrogenase could partly restored NADPH-regenerating ability of the biocatalyst (up to 38?% of the wild type), resulting in approximately 53?% increase in specific rate representing approximately 22-fold increase in product concentration comparing to that obtained prior to an optimization.     

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.     

Functional studies of active-site mutants from Drosophila melanogaster deoxyribonucleoside kinase. Investigations of the putative catalytic glutamate-arginine pair and of residues responsible for substrate specificity

The catalytic reaction mechanism and binding of substrates was investigated for the multisubstrate Drosophila melanogaster deoxyribonucleoside kinase. Mutation of E52 to D, Q and H plus mutations of R105 to K and H were performed to investigate the proposed catalytic reaction mechanism, in which E52 acts as an initiating base and R105 is thought to stabilize the transition state of the reaction. Mutant enzymes (E52D, E52H and R105H) showed a markedly decreased k(cat), while the catalytic activity of E52Q and R105K was abolished. The E52D mutant was crystallized with its feedback inhibitor dTTP. The backbone conformation remained unchanged, and coordination between D52 and the dTTP-Mg complex was observed. The observed decrease in k(cat) for E52D was most likely due to an increased distance between the catalytic carboxyl group and 5'-OH of deoxythymidine (dThd) or deoxycytidine (dCyd). Mutation of Q81 to N and Y70 to W was carried out to investigate substrate binding. The mutations primarily affected the K(m) values, whereas the k(cat) values were of the same magnitude as for the wild-type. The Y70W mutation made the enzyme lose activity towards purines and negative cooperativity towards dThd and dCyd was observed. The Q81N mutation showed a 200- and 100-fold increase in K(m), whereas k(cat) was decreased five- and twofold for dThd and dCyd, respectively, supporting a role in substrate binding. These observations give insight into the mechanisms of substrate binding and catalysis, which is important for developing novel suicide genes and drugs for use in gene therapy.     

Surface charge engineering of PQQ glucose dehydrogenase for downstream processing

The ion-exchange chromatography behavior of recombinant glucose dehydrogenase harboring pyrroloquinoline quinone (PQQGDH) was modified to greatly simplify its purification. The surface charge of PQQGDH was engineered by either fusing a three-arginine tail to the C-terminus of PQQGDH (PQQGDH+Arg3) or by substituting three residues exposed on the surface of the enzyme to Arg by site-directed mutagenesis (3RPQQGDH). During cation exchange chromatography, both surface charge-engineered enzymes eluted at much higher salt concentrations than the wild-type enzyme. After the chromatography purification step, both PQQGDH+Arg3 and 3RPQQGDH appeared as single bands on SDS-PAGE, while extra bands appeared with the wild-type protein sample. Although all tested kinetic parameters of both engineered enzymes are similar to those of wild type, both modifications resulted in enzymes with increased thermal stability. Our achievements have resulted in the greater production of an improved quality PQQGDH by a simplified process.     

Analysis of the catalytic and binding residues of the diadenosine tetraphosphate pyrophosphohydrolase from Caenorhabditis elegans by site-directed mutagenesis

The contributions to substrate binding and catalysis of 13 amino acid residues of the Caenorhabditis elegans diadenosine tetraphosphate pyrophosphohydrolase (Ap(4)A hydrolase) predicted from the crystal structure of an enzyme-inhibitor complex have been investigated by site-directed mutagenesis. Sixteen glutathione S-transferase-Ap(4)A hydrolase fusion proteins were expressed and their k(cat) and K(m) values determined after removal of the glutathione S-transferase domain. As expected for a Nudix hydrolase, the wild type k(cat) of 23 s(-1) was reduced by 10(5)-, 10(3)-, and 30-fold, respectively, by replacement of the conserved P(4)-phosphate-binding catalytic residues Glu(56), Glu(52), and Glu(103) by Gln. K(m) values were not affected, indicating a lack of importance for substrate binding. In contrast, mutating His(31) to Val or Ala and Lys(83) to Met produced 10- and 16-fold increases in K(m) compared with the wild type value of 8.8 microm. These residues stabilize the P(1)-phosphate. H31V and H31A had a normal k(cat) but K83M showed a 37-fold reduction in k(cat). Lys(36) also stabilizes the P(1)-phosphate and a K36M mutant had a 10-fold reduced k(cat) but a relatively normal K(m). Thus both Lys(36) and Lys(83) may play a role in catalysis. The previously suggested roles of Tyr(27), His(38), Lys(79), and Lys(81) in stabilizing the P(2) and P(3)-phosphates were not confirmed by mutagenesis, indicating the absence of phosphate-specific binding contacts in this region. Also, mutating both Tyr(76) and Tyr(121), which clamp one substrate adenosine moiety between them in the crystal structure, to Ala only increased K(m) 4-fold. It is concluded that interactions with the P(1)- and P(4)-phosphates are minimum and sufficient requirements for substrate binding by this class of enzyme, indicating that it may have a much wider substrate range then previously believed.     

Evaluation by mutagenesis of the roles of His309, His315, and His319 in the coenzyme site of pig heart NADP-dependent isocitrate dehydrogenase

Sequence alignment predicts that His(309) of pig heart NADP-dependent isocitrate dehydrogenase is equivalent to His(339) of the Escherichia coli enzyme, which interacts with the coenzyme in the crystal structure [Hurley et al. (1991) Biochemistry 30, 8671-8688], and porcine His(315) and His(319) are close to that site. The mutant porcine enzymes H309Q, H309F, H315Q, and H319Q were prepared by site-directed mutagenesis, expressed in E. coli, and purified. The H319Q mutant has K(m) values for NADP, isocitrate, and Mn(2+) similar to those of wild-type enzyme, and V(max) = 20.1, as compared to 37.8 micromol of NADPH min(-1) (mg of protein)(-1) for wild type. Thus, His(319) is not involved in coenzyme binding and has a minimal effect on catalysis. In contrast, H315Q exhibits a K(m) for NADP 40 times that of wild type and V(max) = 16.2 units/mg of protein, with K(m) values for isocitrate and Mn(2+) similar to those of wild type. These results implicate His(315) in the region of the NADP site. Replacement of His(309) by Q or F yields enzyme with no detectable activity. The His(309) mutants bind NADPH poorly, under conditions in which wild type and H319Q bind 1.0 mol of NADPH/mol of subunit, indicating that His(309) is important for the binding of coenzyme. The His(309) mutants bind isocitrate stoichiometrically, as do wild-type and the other mutant enzymes. However, as distinguished from the wild-type enzyme, the His(309) mutants are not oxidatively cleaved by metal isocitrate, implying that the metal ion is not bound normally. Since circular dichroism spectra are similar for wild type, H315Q, and H319Q, these amino acid substitutions do not cause major conformational changes. In contrast, replacement of His(309) results in detectable change in the enzyme's CD spectrum and therefore in its secondary structure. We propose that His(309) plays a significant role in the binding of coenzyme, contributes to the proper coordination of divalent metal ion in the presence of isocitrate, and maintains the normal conformation of the enzyme.     

Use of 3H and 14C double-labeled glucose to assess in vivo pathways of amino acid biosynthesis in Escherichia coli.

3H and 14C tracing data concerning amino acid biosynthetic pathways in Escherichia coli K12 are presented. Thirteen acidic and neutral amino acids were isolated from protein hydrolysates of wild type E. coli K12 grown aerobically or anaerobically in the presence of [U-14C]glucose together with [1-3H]glucose, [3-3H]glucose, [4-3H]glucose, or [6-3H]glucose. The observed 3H/14C counts of the amino acids were compared with the ratios expected on the basis of the input substrate specific activities and present understanding of biosynthetic pathways. For nine amino acids, serine, valine, leucine, threonine, isoleucine, glycine, glutamate, proline, and phenylalanine, the agreement between anticipated and observed specific activities was satisfactory. For the remaining four, methionine, alanine, aspartate, and (in cells labeled with [3-3H]glucose) tyrosine, the anticipated and observed specific activities differed markedly. For alanine, aspartate, and tyrosine, the differences are probably due to exchange of tritium in the course of biosynthesis; for methionine, it may be that there is a principle source of the methyl group other than carbon 3 of serine.