Biochemical and molecular characterization of taurine:pyruvate aminotransferase from the anaerobe Bilophila wadsworthia.
Bilophila wadsworthia RZATAU is a Gram-negative bacterium which converts the sulfonate taurine (2-aminoethanesulfonate) to ammonia, acetate and sulfide in an anaerobic respiration. Taurine:pyruvate aminotransferase (Tpa) catalyses 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'-phosphate to all buffers during purification. SDS/PAGE revealed a single protein band with a molecular mass of 51 kDa. The apparent molecular mass of the native enzyme was 197 kDa as determined by gel filtration, which indicates a homotetrameric structure. The kinetic constants for taurine were: Km = 7.1 mM, Vmax = 1.20 nmol.s-1, and for pyruvate: Km = 0.82 mM, Vmax = 0.17 nmol.s-1. The purified enzyme was able to transaminate hypotaurine (2-aminosulfinate), taurine, beta-alanine and with low activity cysteine and 3-aminopropanesulfonate. In addition to pyruvate, 2-ketobutyrate and oxaloacetate were utilized as amino group acceptors. We have sequenced the encoding gene (tpa). It encoded a 50-kDa peptide, which revealed 33% identity to diaminopelargonate aminotransferase from Bacillus subtilis.
Laue
H
H
Fachbereich Biologie, Universität Konstanz, Konstanz, Germany. heike_laue@hotmail.com
Cook
A M
AM
D002087:Butyrates / Q000378:metabolism; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism; D020564:Deltaproteobacteria / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D062907:Oxaloacetic Acid / Q000378:metabolism; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D013654:Taurine / Q000031:analogs & derivatives* / Q000378:metabolism; D013997:Time Factors; D000637:Transaminases / Q000737:chemistry* / Q000235:genetics*; D014466:Ultraviolet Rays; D015091:beta-Alanine / Q000378:metabolism
D002087:Butyrates; D015091:beta-Alanine; D013654:Taurine; D062907:Oxaloacetic Acid; C001355:tramiprosate; C003949:hypotaurine; D011732:Pyridoxal Phosphate; C005087:alpha-ketobutyric acid; D000637:Transaminases; C022715:taurine pyruvate aminotransferase; D003545:Cysteine