title: Characterization of a thermostable dihydrodipicolinate synthase from Thermoanaerobacter tengcongensis.
abstract: Dihydrodipicolinate synthase (DHDPS) catalyses the first reaction of the (S)-lysine biosynthesis pathway in bacteria and plants. The hypothetical gene for dihydrodipicolinate synthase (dapA) of Thermoanaerobacter tengcongensis was found in a cluster containing several genes of the diaminopimelate lysine-synthesis pathway. The dapA gene was cloned in Escherichia coli, DHDPS was subsequently produced and purified to homogeneity. The T. tengcongensis DHDPS was found to be thermostable (T0.5=3 h at 90 degrees C). The specific condensation of pyruvate and (S)-aspartate-beta -semialdehyde was catalyzed optimally at 80 degrees C at pH 8.0. Enzyme kinetics were determined at 60 degrees C, as close as possible to in vivo conditions. The established kinetic parameters were in the same range as for example E. coli dihydrodipicolinate synthase. The specific activity of the T. tengcongensis DHDPS was relatively high even at 30 degrees C. Like most dihydrodipicolinate synthases known at present, the DHDPS of T. tengcongensis seems to be a tetramer. A structural model reveals that the active site is well conserved. The binding site of the allosteric inhibitor lysine appears not to be conserved, which agrees with the fact that the DHDPS of T. tengcongensis is not inhibited by lysine under physiological conditions.
authors.affiliation: Laboratory of Microbiology, Wageningen University, Dreijenplein 10, 6703 HB Wageningen, The Netherlands.
authors.forename: Suzanne
authors.initials: S
authors.lastname: Wolterink-van Loo
authors.forename: Mark
authors.initials: M
authors.lastname: Levisson
authors.forename: Maud C
authors.initials: MC
authors.lastname: Cabrières
authors.forename: Maurice C R
authors.initials: MC
authors.lastname: Franssen
authors.forename: John
authors.initials: J
authors.lastname: van der Oost
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D003001:Cloning, Molecular; D004795:Enzyme Stability; D016680:Genome, Bacterial; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D019289:Pyruvic Acid / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D013696:Temperature; D046968:Thermoanaerobacter / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D001224:Aspartic Acid; C006644:aspartic semialdehyde; D019289:Pyruvic Acid; D006836:Hydro-Lyases; C020040:4-hydroxy-tetrahydrodipicolinate synthase; D008239:Lysine