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 (T 0.5 = 3 h at 90°C). The specific condensation of pyruvate and (S)-aspartate-b -semialdehyde was catalyzed optimally at 80°C at pH 8.0. Enzyme kinetics were determined at 60°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°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.

Biochemical studies and crystal structure determination of dihydrodipicolinate synthase from Pseudomonas aeruginosa

“…The biochemical characterization of PsDHDPS was carried out at its optimal conditions of pH (pH 8.0) and temperature (37 • C) where its pH value of pH 8.0 was found to be similar to those of EcDHDPS [35] and NmDHDPS [13] but the optimum temperature (37 • C) was 7 • C higher than those of EcDHDPS (30 • C) [32] and NmDHDPS (30 • C) [13]. The K m value of 0.90 ± 0.13 mM for PsDHDPS for the substrate pyruvate was similar to that of DHDPS from Thermoanaerobacter tengcongensis (0.85 mM) [36] which is approximately 4-fold higher than that of EcDHDPS (0.25 mM) [32] and 2-fold higher than that of NmDHDPS (0.50 mM) [13]. The K m value for the substrate (S)-ASA (0.17 ± 0.02 mM) was comparable to that of DHDPS of Thermotoga maritima (0.16 mM) [18] but higher than that observed in the case of EcDHDPS (0.11 mM) [32] and NmDHDPS (0.05 mM) [13].…”

Substrate-mediated Stabilization of a Tetrameric Drug Target Reveals Achilles Heel in Anthrax

“…1B), Thermotoga maritima (9), Thermoanaerobacter tengcongensis (10), Escherichia coli (11), Mycobacterium tuberculosis (12), Corynebacterium glutamicum (13), and Staphylococcus aureus (14,15). The enzyme usually assembles as a tetrameric protein (16), best described as a dimer of tight dimers (Fig.…”

Molecular evolution of an oligomeric biocatalyst functioning in lysine biosynthesis

“…In contrast to other enzymes in the DAP pathway, the expression of dapA in E. coli was not found to be regulated by cellular free lysine levels or any other stimuli (Butour et al 1974). In the last three decades, the dapA gene has been cloned and sequenced from a variety of other bacterial (Atkinson et al 2011(Atkinson et al , 2012aChen et al 1993;Cremer et al 1988;Devenish et al 2009;Dommaraju et al 2010;Evans et al 2011;García-Rodríguez et al 2000;Girish et al 2008;Gunji et al 2004;Kaur et al 2011;Pisabarro et al 1993;Siddiqui et al 2013;Skovpen and Palmer 2013;Wolterink-van Loo et al 2008;Wubben et al 2010) and plant species (Atkinson et al 2011(Atkinson et al , 2014Frisch et al 1991;Ghislain et al 1995;Kaneko et al 1990;Silk et al 1994;Vauterin and Jacobs 1994). Typically, bacteria have a single dapA gene that is 800-900 bp, whereas plants have two annotated dapA genes consisting of~1000 bp each.…”