Biochemical and molecular characterization of taurine : pyruvate aminotransferase from the anaerobe Bilophila wadsworthia

Bilophila wadsworthia RZATAU is a Gram-negative bacterium which converts th e sulfonate taurine (2-aminoethanesulfonate) to ammonia, acetate and sulfid e in an anaerobic respiration. Taurine:pyruvate aminotransferase (Tpa) cata lyses the initial metabolic reaction yielding alanine and sulfoacetaldehyde . We purified Tpa 72-fold to apparent homogeneity with an overall yield of 89%. The purified enzyme did not require addition of pyridoxal 5'-phosphate , but highly active enzyme was only obtained by addition of pyridoxal 5'-ph osphate to all buffers during purification. SDS/PAGE revealed a single prot ein band with a molecular mass of 51 kDa. The apparent molecular mass of th e native enzyme was 197 kDa as determined by gel filtration, which indicate s a homotetrameric structure. The kinetic constants for taurine were: K-m = 7.1 mm, V-max = 1.20 nmol.s(-1), and for pyruvate: K-m = 0.82 mm, V-max = 0.17 nmol.s(-1). The purified enzyme was able to transaminate hypotaurine ( 2-aminosulfinate), taurine, beta -alanine and with low activity cysteine an d 3-aminopropanesulfonate. In addition to pyruvate, 2-ketobutyrate and oxal oacetate were utilized as amino group acceptors. We have sequenced the enco ding gene (tpa). It encoded a 50-kDa peptide, which revealed 33% identity t o diaminopelargonate aminotransferase from Bacillus subtilis.