Purification, properties and primary structure of alanine dehydrogenase involved in taurine metabolism in the anaerobe Bilophila wadsworthia

Abstract

Alanine dehydrogenase [L-alanine:NAD + oxidoreductase (deaminating), EC 1.4.1.4.] catalyses the reversible oxidative deamination of L-alanine to pyruvate and, in the anaerobic bacterium Bilophila wadsworthia RZATAU, it is involved in the degradation of taurine (2-aminoethanesulfonate). The enzyme regenerates the amino-group acceptor pyruvate, which is consumed during the transamination of taurine and liberates ammonia, which is one of the degradation end products. Alanine dehydrogenase seems to be induced during growth with taurine. The enzyme was purified about 24-fold to apparent homogeneity in a three-step purification. SDS-PAGE revealed a single protein band with a molecular mass of 42 kDa. The apparent molecular mass of the native enzyme was 273 kDa, as determined by gel filtration chromatography, suggesting a homo-hexameric structure. The N-terminal amino acid sequence was determined. The pH optimum was pH 9.0 for reductive amination of pyruvate and pH 9.0-11.5 for oxidative deamination of alanine. The apparent K m values for alanine, NAD + , pyruvate, ammonia and NADH were 1.6, 0.15, 1.1, 31 and 0.04 mM, respectively. The alanine dehydrogenase gene was sequenced. The deduced amino acid sequence corresponded to a size of 39.9 kDa and was very similar to that of the alanine dehydrogenase from Bacillus subtilis.

Protocatechuate 4,5-dioxygenase from Comamonas testosteroni T-2: biochemical and molecular properties of a new subgroup within class III of extradiol dioxygenases

“…SDS-PAGE was done with 12% separative gels (Scha¨gger and von Jagow 1987), and proteins were stained with colloidal Coomassie Brilliant Blue G-250 (Neuhoff et al 1988); a 10-kDa Protein Ladder (Gibco) was used for calibration. Proteins were subject to blotting and Nterminal sequencing, as described elsewhere (Laue and Cook 2000).…”

Inducible transcription of genes involved in taurine uptake and dissimilation by Silicibacter pomeroyi DSS-3T

“…The need for an alanine dehydrogenase (Ald) [EC 1.4.1.4] in the hypothesis in Fig. 1 is known (Laue and Cook 2000b;Denger et al 2004), as are roles for a sulfite dehydrogenase (sulfite oxidoreductase, Sor) and exporters of ammonium and sulfate ions, but candidate genes to encode the latter functions are either absent or unknown (Sor) or have not been confirmed . Thus, despite the logic of the scheme in Fig.…”

The sulfonated osmolyte N-methyltaurine is dissimilated by Alcaligenes faecalis and by Paracoccus versutus with release of methylamine

“…Further hypotheses on the dissimilation of N-methyltaurine involved the nature of removal of the methyl group, possibly by its oxygenation to yield taurine, followed by either taurine : pyruvate aminotransferase (EC 2.6.1.77) with alanine dehydrogenase (Laue & Cook, 2000;Denger et al, 2004a) to yield sulfoacetaldehyde, or by taurine : ferricytochrome-c oxidoreductase (deaminating) (taurine dehydrogenase, presumably EC 1.4.2.-) to yield sulfoacetaldehyde (Brüggemann et al, 2004). Alternatively, the reaction might proceed by hydrolytic removal of the methylamino moiety to yield sulfoacetaldehyde, analogous to reactions (e.g.…”