Purification, characterization, and molecular cloning of a novel amine:pyruvate transaminase from Vibrio fluvialis JS17.
A transaminase from Vibrio fluvialis JS17 showing activity toward chiral amines was purified to homogeneity and its enzymatic properties were characterized. The transaminase showed an apparent molecular mass of 100 kDa as determined by gel filtration chromatography and a subunit mass of 50 kDa by MALDI-TOF mass spectrometry, suggesting a dimeric structure. The enzyme had an isoelectric point of 5.4 and its absorption spectrum exhibited maxima at 320 and 405 nm. The optimal pH and temperature for enzyme activity were 9.2 and 37 degrees C, respectively. Pyruvate and pyridoxal 5'-phosphate increased enzyme stability whereas (S)-alpha-methylbenzylamine reversibly inactivated the enzyme. The transaminase gene was cloned from a V. fluvialis JS17 genomic library. The deduced amino acid sequence (453 residues) showed significant homology with omega-amino acid:pyruvate transaminases (omega-APT) from various bacterial strains (80 identical residues with four omega-APTs). However, of 159 conserved residues in the four omega-APTs, 79 were not conserved in the transaminase from V. fluvialis JS17. Taken together with the sequence homology results, and the lack of activity toward beta-alanine (a typical amino donor for the omega-APT), the results suggest that the transaminase is a novel amine:pyruvate transaminase that has not been reported to date.
Shin
J-S
JS
Bioengineering, Division of Engineering and Applied Science, California Institute of Technology, Pasadena, CA 91125, USA.
Yun
H
H
Jang
J-W
JW
Park
I
I
Kim
B-G
BG
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013379:Substrate Specificity; D013696:Temperature; D000637:Transaminases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D014733:Vibrio / Q000201:enzymology* / Q000235:genetics; D051278:beta-Alanine-Pyruvate Transaminase
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D000637:Transaminases; D051278:beta-Alanine-Pyruvate Transaminase