Publications

• The catalytic role of aspartate in the active site of glutamate dehydrogenase.

  Dean J.L., Wang X.G., Teller J.K., Waugh M.L., Britton K.L., Baker P.J., Stillman T.J., Martin S.R., Rice D.W., Engel P.C.

  A putative catalytic aspartyl residue, Asp-165, in the active site of clostridial glutamate dehydrogenase has been replaced with serine by site-directed mutagenesis. The mutant enzyme is efficiently overexpressed in Escherichia coli as a soluble protein and can be successfully purified by the dye- ... >> More

  A putative catalytic aspartyl residue, Asp-165, in the active site of clostridial glutamate dehydrogenase has been replaced with serine by site-directed mutagenesis. The mutant enzyme is efficiently overexpressed in Escherichia coli as a soluble protein and can be successfully purified by the dye-ligand chromatographic procedure normally employed for the wild-type enzyme. By several criteria, including circular dichroism spectrum, sulphydryl reactivity with Ellman's reagent, crystallization and mobility in non-denaturing electrophoresis, the enzyme appears to be correctly folded. NAD+ protects the D165S mutant against modification by Ellman's reagent, suggesting unimpaired binding of coenzyme. In standard assays the specific activity is decreased 10(3)-fold in the reductive amination reaction and 10(5)-fold for oxidative deamination. Kinetic studies show that apparent Km values for NADH and 2-oxoglutarate are almost unchanged. The large reduction in the reaction rate coincides with a weakening of the affinity for ammonium ion (Km > 300 mM, compared with 60 mM for the wild-type). The data are entirely consistent with the direct involvement of D165 in catalysis rather than in the binding of coenzyme or 2-oxoglutarate. << Less

  Biochem. J. 301:13-16(1994) [PubMed] [EuropePMC]

• Biochemical characterization of two glutamate dehydrogenases with different cofactor specificities from a hyperthermophilic archaeon Pyrobaculum calidifontis.

  Wakamatsu T., Higashi C., Ohmori T., Doi K., Ohshima T.

  Two putative glutamate dehydrogenase (GDH) genes (pcal_1031 and pcal_1606) were found in a sulfur-dependent hyperthermophilic archaeon, Pyrobaculum calidifontis. The two genes were then expressed in Escherichia coli, and both of the recombinant gene products showed GDH activity. The two enzymes we ... >> More

  Two putative glutamate dehydrogenase (GDH) genes (pcal_1031 and pcal_1606) were found in a sulfur-dependent hyperthermophilic archaeon, Pyrobaculum calidifontis. The two genes were then expressed in Escherichia coli, and both of the recombinant gene products showed GDH activity. The two enzymes were then purified to homogeneity and characterized in detail. Although both purified GDHs had a hexameric structure and neither exhibited allosteric regulation, they showed different coenzyme specificities: one was specific for NAD(+), the other for NADP(+) and different heat activation mechanisms. In addition, there was little difference in the kinetic constants, optimal temperature, thermal stability, optimal pH and pH stability between the two enzymes. The overall sequence identity between the two proteins was very high (81%), but was not high in the region recognizing the 2' position of the adenine ribose moiety, which is responsible for coenzyme specificity. This is the first report on the identification of two GDHs with different coenzyme specificities from a single hyperthermophilic archaeon and the definition of their basic in vitro properties. << Less

  Extremophiles 17:379-389(2013) [PubMed] [EuropePMC]

• Probing the determinants of coenzyme specificity in Peptostreptococcus asaccharolyticus glutamate dehydrogenase by site-directed mutagenesis.

  Carrigan J.B., Engel P.C.

  Glutamate dehydrogenase (EC 1.4.1.2-4) from Peptostreptococcus asaccharolyticus has a strong preference for NADH over NADPH as a coenzyme, over 1000-fold in terms of kcat/Km values. Sequence alignments across the wider family of NAD(P)-dependent dehydrogenases might suggest that this preference is ... >> More

  Glutamate dehydrogenase (EC 1.4.1.2-4) from Peptostreptococcus asaccharolyticus has a strong preference for NADH over NADPH as a coenzyme, over 1000-fold in terms of kcat/Km values. Sequence alignments across the wider family of NAD(P)-dependent dehydrogenases might suggest that this preference is mainly due to a negatively charged glutamate at position 243 (E243) in the adenine ribose-binding pocket. We have examined the possibility of altering coenzyme specificity of the Peptostreptococcus enzyme, and, more specifically, the role of residue 243 and neighbouring residues in coenzyme binding, by introducing a range of point mutations. Glutamate dehydrogenases are unusual among dehydrogenases in that NADPH-specific forms usually have aspartate at this position. However, replacement of E243 with aspartate led to only a nine-fold relaxation of the strong discrimination against NADPH. By contrast, replacement with a more positively charged lysine or arginine, as found in NADPH-dependent members of other dehydrogenase families, allows a more than 1000-fold shift toward NADPH, resulting in enzymes equally efficient with NADH or NADPH. Smaller shifts in the same direction were also observed in enzymes where a neighboring tryptophan, W244, was replaced by a smaller alanine (approximately six-fold) or Asp245 was changed to lysine (32-fold). Coenzyme binding studies confirm that the mutations result in the expected major changes in relative affinities for NADH and NADPH, and pH studies indicate that improved affinity for the extra phosphate of NADPH is the predominant reason for the increased catalytic efficiency with this coenzyme. The marked difference between the results of replacing E243 with aspartate and with positive residues implies that the mode of NADPH binding in naturally occurring NADPH-dependent glutamate dehydrogenases differs from that adopted in E243K or E243D and in other dehydrogenases. << Less

  FEBS J. 274:5167-5174(2007) [PubMed] [EuropePMC]

• Properties of the thermostable glutamate dehydrogenase of the mesophilic anaerobe Peptostreptoccus asaccharolyticus purified by a novel method after over-expression in an Escherichia coli host.

  Carrigan J.B., Coughlan S., Engel P.C.

  Peptostreptococcus asaccharolyticus glutamate dehydrogenase (L-glutamate: NAD+ oxidoreductase (deaminating); EC 1.4.1.2) overexpressed in Escherichia coli has been purified by two new methods. Enzyme made by the first method showed remarkable thermophilicity, with a temperature optimum of 60 degre ... >> More

  Peptostreptococcus asaccharolyticus glutamate dehydrogenase (L-glutamate: NAD+ oxidoreductase (deaminating); EC 1.4.1.2) overexpressed in Escherichia coli has been purified by two new methods. Enzyme made by the first method showed remarkable thermophilicity, with a temperature optimum of 60 degrees C, and also thermostability, which suggested the second, simpler method, incorporating a heat step. This produced 94 mg of homogeneous protein per litre culture medium. The basic kinetic parameters for P. asaccharolyticus glutamate dehydrogenase with all substrates are revealed at pH 7.0. The enzyme is highly specific for NAD+, with values for kcat/Km 405 times greater than for NADP+. In the reverse direction of reaction, the kcat/Km value for NADH is almost 1000-fold greater than for NADPH. << Less

  FEMS Microbiol Lett 244:53-59(2005) [PubMed] [EuropePMC]

• Molecular properties and enhancement of thermostability by random mutagenesis of glutamate dehydrogenase from Bacillus subtilis.

  Khan M.I., Ito K., Kim H., Ashida H., Ishikawa T., Shibata H., Sawa Y.

  The rocG gene encoding glutamate dehydrogenase from Bacillus subtilis (Bs-GluDH) was cloned, and expressed at considerable magnitude in Escherichia coli. The recombinant Bs-GluDH was purified to homogeneity and has been determined to have a hexameric structure (M(r) 270 kDa) with strict specificit ... >> More

  The rocG gene encoding glutamate dehydrogenase from Bacillus subtilis (Bs-GluDH) was cloned, and expressed at considerable magnitude in Escherichia coli. The recombinant Bs-GluDH was purified to homogeneity and has been determined to have a hexameric structure (M(r) 270 kDa) with strict specificity for 2-oxoglutarate and L-glutamate, requiring NADH and NAD+ as cofactors respectively. The enzyme showed low thermostability with T(m) = 41 degrees C due to dissociation of the hexamer. To improve the thermostability of this enzyme, we performed error-prone PCR, introducing random mutagenesis on cloned GluDH. Two single mutant enzymes, Q144R and E27F, were isolated from the final mutant library. Their T(m) values were 61 degrees C and 49 degrees C respectively. Furthermore, Q144R had a remarkably high k(cat) value (435 s(-1)) for amination reaction at 37 degrees C, 1.3 times higher than that of the wild-type. Thus, Q144R can be used as a template gene to modify the substrate specificity of Bs-GluDH for industrial use. << Less

  Biosci. Biotechnol. Biochem. 69:1861-1870(2005) [PubMed] [EuropePMC]

• The gdhB gene of Pseudomonas aeruginosa encodes an arginine-inducible NAD(+)-dependent glutamate dehydrogenase which is subject to allosteric regulation.

  Lu C.D., Abdelal A.T.

  The NAD(+)-dependent glutamate dehydrogenase (NAD-GDH) from Pseudomonas aeruginosa PAO1 was purified, and its amino-terminal amino acid sequence was determined. This sequence information was used in identifying and cloning the encoding gdhB gene and its flanking regions. The molecular mass predict ... >> More

  The NAD(+)-dependent glutamate dehydrogenase (NAD-GDH) from Pseudomonas aeruginosa PAO1 was purified, and its amino-terminal amino acid sequence was determined. This sequence information was used in identifying and cloning the encoding gdhB gene and its flanking regions. The molecular mass predicted from the derived sequence for the encoded NAD-GDH was 182.6 kDa, in close agreement with that determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme (180 kDa). Cross-linking studies established that the native NAD-GDH is a tetramer of equal subunits. Comparison of the derived amino acid sequence of NAD-GDH from P. aeruginosa with the GenBank database showed the highest homology with hypothetical polypeptides from Pseudomonas putida, Mycobacterium tuberculosis, Rickettsia prowazakii, Legionella pneumophila, Vibrio cholerae, Shewanella putrefaciens, Sinorhizobium meliloti, and Caulobacter crescentus. A moderate degree of homology, primarily in the central domain, was observed with the smaller tetrameric NAD-GDH (protomeric mass of 110 kDa) from Saccharomyces cerevisiae or Neurospora crassa. Comparison with the yet smaller hexameric GDH (protomeric mass of 48 to 55 kDa) of other prokaryotes yielded a low degree of homology that was limited to residues important for binding of substrates and for catalytic function. NAD-GDH was induced 27-fold by exogenous arginine and only 3-fold by exogenous glutamate. Primer extension experiments established that transcription of gdhB is initiated from an arginine-inducible promoter and that this induction is dependent on the arginine regulatory protein, ArgR, a member of the AraC/XyIS family of regulatory proteins. NAD-GDH was purified to homogeneity from a recombinant strain of P. aeruginosa and characterized. The glutamate saturation curve was sigmoid, indicating positive cooperativity in the binding of glutamate. NAD-GDH activity was subject to allosteric control by arginine and citrate, which function as positive and negative effectors, respectively. Both effectors act by influencing the affinity of the enzyme for glutamate. NAD-GDH from this organism differs from previously characterized enzymes with respect to structure, protomer mass, and allosteric properties indicate that this enzyme represents a novel class of microbial glutamate dehydrogenases. << Less

  J. Bacteriol. 183:490-499(2001) [PubMed] [EuropePMC]

• Functional dissection of a trigger enzyme: mutations of the bacillus subtilis glutamate dehydrogenase RocG that affect differentially its catalytic activity and regulatory properties.

  Gunka K., Newman J.A., Commichau F.M., Herzberg C., Rodrigues C., Hewitt L., Lewis R.J., Stulke J.

  Any signal transduction requires communication between a sensory component and an effector. Some enzymes engage in signal perception and transduction, as well as in catalysis, and these proteins are known as "trigger" enzymes. In this report, we detail the trigger properties of RocG, the glutamate ... >> More

  Any signal transduction requires communication between a sensory component and an effector. Some enzymes engage in signal perception and transduction, as well as in catalysis, and these proteins are known as "trigger" enzymes. In this report, we detail the trigger properties of RocG, the glutamate dehydrogenase of Bacillus subtilis. RocG not only deaminates the key metabolite glutamate to form alpha-ketoglutarate but also interacts directly with GltC, a LysR-type transcription factor that regulates glutamate biosynthesis from alpha-ketoglutarate, thus linking the two metabolic pathways. We have isolated mutants of RocG that separate the two functions. Several mutations resulted in permanent inactivation of GltC as long as a source of glutamate was present. These RocG proteins have lost their ability to catabolize glutamate due to a strongly reduced affinity for glutamate. The second class of mutants is exemplified by the replacement of aspartate residue 122 by asparagine. This mutant protein has retained enzymatic activity but has lost the ability to control the activity of GltC. Crystal structures of glutamate dehydrogenases that permit a molecular explanation of the properties of the various mutants are presented. Specifically, we may propose that D122N replacement affects the surface of RocG. Our data provide evidence for a correlation between the enzymatic activity of RocG and its ability to inactivate GltC, and thus give insights into the mechanism that couples the enzymatic activity of a trigger enzyme to its regulatory function. << Less

  J. Mol. Biol. 400:815-827(2010) [PubMed] [EuropePMC]

• Molecular gene cloning, expression, and characterization of bovine brain glutamate dehydrogenase.

  Kim D.W., Eum W.S., Jang S.H., Yoon C.S., Kim Y.H., Choi S.H., Choi H.S., Kim S.Y., Kwon H.Y., Kang J.H., Kwon O.-S., Cho S.-W., Park J., Choi S.Y.

  A cDNA of bovine brain glutamate dehydrogenase (GDH) was isolated from a cDNA library by recombinant PCR. The isolated cDNA has an open-reading frame of 1677 nucleotides, which codes for 559 amino acids. The expression of the recombinant bovine brain GDH enzyme was achieved in E. coli. BL21 (DE3) ... >> More

  A cDNA of bovine brain glutamate dehydrogenase (GDH) was isolated from a cDNA library by recombinant PCR. The isolated cDNA has an open-reading frame of 1677 nucleotides, which codes for 559 amino acids. The expression of the recombinant bovine brain GDH enzyme was achieved in E. coli. BL21 (DE3) by using the pET-15b expression vector containing a T7 promoter. The recombinant GDH protein was also purified and characterized. The amino acid sequence was found 90% homologous to the human GDH. The molecular mass of the expressed GDH enzyme was estimated as 50 kDa by SDS-PAGE and Western blot using monoclonal antibodies against bovine brain GDH. The kinetic parameters of the expressed recombinant GDH enzymes were quite similar to those of the purified bovine brain GDH. The Km and Vmax values for NAD+ were 0.1 mM and 1.08 micromol/min/mg, respectively. The catalytic activities of the recombinant GDH enzymes were inhibited by ATP in a concentration-dependent manner over the range of 10 - 100 microM, whereas, ADP increased the enzyme activity up to 2.3-fold. These results indicate that the recombinant-expressed bovine brain GDH that is produced has biochemical properties that are very similar to those of the purified GDH enzyme. << Less

  J. Biochem. Mol. Biol. 36:545-551(2003) [PubMed] [EuropePMC]