Publications

• An integrated study of threonine-pathway enzyme kinetics in Escherichia coli.

  Chassagnole C., Rais B., Quentin E., Fell D.A., Mazat J.P.

  We have determined the kinetic parameters of the individual steps of the threonine pathway from aspartate in Escherichia coli under a single set of experimental conditions chosen to be physiologically relevant. Our aim was to summarize the kinetic behaviour of each enzyme in a single tractable equ ... >> More

  We have determined the kinetic parameters of the individual steps of the threonine pathway from aspartate in Escherichia coli under a single set of experimental conditions chosen to be physiologically relevant. Our aim was to summarize the kinetic behaviour of each enzyme in a single tractable equation that takes into account the effect of the products as competitive inhibitors of the substrates in the forward reaction and also, when appropriate (e.g. near-equilibrium reactions), as substrates of the reverse reactions. Co-operative feedback inhibition by threonine and lysine was also included as necessary. We derived the simplest rate equations that describe the salient features of the enzymes in the physiological range of metabolite concentrations in order to incorporate them ultimately into a complete model of the threonine pathway, able to predict quantitatively the behaviour of the pathway under natural or engineered conditions. << Less

  Biochem. J. 356:415-423(2001) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Expression and purification of aspartate beta-semialdehyde dehydrogenase from infectious microorganisms.

  Moore R.A., Bocik W.E., Viola R.E.

  l-Aspartate-beta-semialdehyde dehydrogenase (ASA DH) lies at the first branch point in the aspartate metabolic pathway that leads to the formation of the amino acids lysine, isoleucine, methionine, and threonine in most plants, bacteria, and fungi. Since the aspartate pathway is not found in human ... >> More

  l-Aspartate-beta-semialdehyde dehydrogenase (ASA DH) lies at the first branch point in the aspartate metabolic pathway that leads to the formation of the amino acids lysine, isoleucine, methionine, and threonine in most plants, bacteria, and fungi. Since the aspartate pathway is not found in humans, but is necessary for bacterial cell wall biosynthesis, the enzymes in this pathway are potential targets for the development of new antibiotics. The asd gene that encodes for ASA DH has been obtained from several infectious organisms and ligated into a pET expression vector. ASA DHs from Haemophilus influenza, Pseudomonas aeruginosa, and Vibrio cholerae were expressed as soluble proteins in Escherichia coli, while ASA DH from Helicobacter pylori was obtained primarily as inclusion bodies. The V. cholerae genome contains two asd genes. Both enzymes have been expressed and purified, and each displays significant ASA DH activity. The purification of highly active ASA DH from each of these organisms has been achieved for the first time, in greater than 95% purity and high overall yield. Kinetic parameters have been determined for each purified enzyme, and the values have been compared to those of E. coli ASA DH. << Less

  Protein Expr. Purif. 25:189-194(2002) [PubMed] [EuropePMC]

• The role of substrate-binding groups in the mechanism of aspartate-beta-semialdehyde dehydrogenase.

  Blanco J., Moore R.A., Faehnle C.R., Coe D.M., Viola R.E.

  The reversible dephosphorylation of beta-aspartyl phosphate to L-aspartate-beta-semialdehyde (ASA) in the aspartate biosynthetic pathway is catalyzed by aspartate-beta-semialdehyde dehydrogenase (ASADH). The product of this reaction is a key intermediate in the biosynthesis of diaminopimelic acid, ... >> More

  The reversible dephosphorylation of beta-aspartyl phosphate to L-aspartate-beta-semialdehyde (ASA) in the aspartate biosynthetic pathway is catalyzed by aspartate-beta-semialdehyde dehydrogenase (ASADH). The product of this reaction is a key intermediate in the biosynthesis of diaminopimelic acid, an integral component of bacterial cell walls and a metabolic precursor of lysine and also a precursor in the biosynthesis of threonine, isoleucine and methionine. The structures of selected Haemophilus influenzae ASADH mutants were determined in order to evaluate the residues that are proposed to interact with the substrates ASA or phosphate. The substrate Km values are not altered by replacement of either an active-site arginine (Arg270) with a lysine or a putative phosphate-binding group (Lys246) with an arginine. However, the interaction of phosphate with the enzyme is adversely affected by replacement of Arg103 with lysine and is significantly altered when a neutral leucine is substituted at this position. A conservative Glu243 to aspartate mutant does not alter either ASA or phosphate binding, but instead results in an eightfold increase in the Km for the coenzyme NADP. Each of the mutations is shown to cause specific subtle active-site structural alterations and each of these changes results in decreases in catalytic efficiency ranging from significant (approximately 3% native activity) to substantial (<0.1% native activity). << Less

  Acta Crystallogr. D 60:1388-1395(2004) [PubMed] [EuropePMC]

• Cloning and characterization of aspartate-beta-semialdehyde dehydrogenase from Mycobacterium tuberculosis H37 Rv.

  Shafiani S., Sharma P., Vohra R.M., Tewari R.

  <h4>Aims</h4>To clone and characterize the aspartate-beta-semialdehyde dehydrogenase of Mycobacterium tuberculosis H37Rv.<h4>Methods and results</h4>The asd gene of M. tuberculosis H37Rv was cloned in pGEM-T Easy vector, subcloned in expression vector pQE30 having a T5 promoter, and overexpressed ... >> More

  <h4>Aims</h4>To clone and characterize the aspartate-beta-semialdehyde dehydrogenase of Mycobacterium tuberculosis H37Rv.<h4>Methods and results</h4>The asd gene of M. tuberculosis H37Rv was cloned in pGEM-T Easy vector, subcloned in expression vector pQE30 having a T5 promoter, and overexpressed in Escherichia coli. The ASD enzyme was expressed to levels of 40% but was found to be inactive. Functional ASD was obtained by altering induction and growth conditions and the enzyme was purified to near homogeneity using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. The K(m) and V(max) values for the three substrates L-ASA, NADP and Pi, the turnover number and specific activity of the enzyme were determined.<h4>Conclusions</h4>Functional ASD enzyme of M. tuberculosis was obtained by gene cloning and protein purification using affinity chromatography. The K(cat) and specific activity of the enzyme were 8.49 s(-1) and 13.4 micromol min(-1) microg(-1) respectively.<h4>Significance and impact of the study</h4>The ASD enzyme is a validated drug target. We characterized this enzyme from M. tuberculosis and future work would focus on deducing the three-dimensional structure of the enzyme and design of inhibitors, which could be used as drugs against TB. << Less

  J. Appl. Microbiol. 98:832-838(2005) [PubMed] [EuropePMC]

• Aspartate-beta-semialdehyde dehydrogenase from Escherichia coli. Purification and general properties.

  Biellmann J.F., Eid P., Hirth C., Jornvall H.

  Aspartate-beta-semialdehyde dehydrogenase, from an Escherichia coli mutant derepressed for the biosynthesis of L-lysine, has been purified to homogeneity. Its isoelectric point is pH 4.3. This enzyme has a molecular weight of 77000 and is composed of two identical or highly similar subunits of mol ... >> More

  Aspartate-beta-semialdehyde dehydrogenase, from an Escherichia coli mutant derepressed for the biosynthesis of L-lysine, has been purified to homogeneity. Its isoelectric point is pH 4.3. This enzyme has a molecular weight of 77000 and is composed of two identical or highly similar subunits of molecular weight 38000 +/-2000. Their N-terminal amino-acid sequence is Met-Lys-Asx-Val-Gly-. Three cysteine residues per subunit were detected: two are reactive in the native enzyme and one is partially protected by the substrate. Formation of an acyl-enzyme intermediate was also detected. Correlation of the 1H nucleár magnetic resonance spectrum of [4-2H]NADPH produced from [4-2H]NADP+ indicated that aspartate beta-semialdehyde dehydrogenase transfers the pro-S hydrogen from NADPH (class B dehydrogenase). A short comparison with the corresponding yeast enzyme is given. << Less

  Eur. J. Biochem. 104:53-58(1980) [PubMed] [EuropePMC]

• Purification, crystallization and preliminary X-ray diffraction analysis of aspartate semialdehyde dehydrogenase (Rv3708c) from Mycobacterium tuberculosis.

  Vyas R., Kumar V., Panjikar S., Karthikeyan S., Kishan K.V., Tewari R., Weiss M.S.

  Aspartate semialdehyde dehydrogenase from Mycobacterium tuberculosis (Asd, ASADH, Rv3708c), which is the second enzyme in the lysine/homoserine-biosynthetic pathways, has been expressed heterologously in Escherichia coli. The enzyme was purified using affinity and gel-filtration chromatographic te ... >> More

  Aspartate semialdehyde dehydrogenase from Mycobacterium tuberculosis (Asd, ASADH, Rv3708c), which is the second enzyme in the lysine/homoserine-biosynthetic pathways, has been expressed heterologously in Escherichia coli. The enzyme was purified using affinity and gel-filtration chromatographic techniques and crystallized in two different crystal forms. Preliminary diffraction data analysis suggested the presence of up to four monomers in the asymmetric unit of the orthorhombic crystal form A and of one or two monomers in the cubic crystal form B. << Less

  Acta Crystallogr. F 64:167-170(2008) [PubMed] [EuropePMC]