Cloning and characterization of a novel beta-transaminase from Mesorhizobium sp. strain LUK: a new biocatalyst for the synthesis of enantiomerically pure beta-amino acids.
A novel beta-transaminase gene was cloned from Mesorhizobium sp. strain LUK. By using N-terminal sequence and an internal protein sequence, a digoxigenin-labeled probe was made for nonradioactive hybridization, and a 2.5-kb gene fragment was obtained by colony hybridization of a cosmid library. Through Southern blotting and sequence analysis of the selected cosmid clone, the structural gene of the enzyme (1,335 bp) was identified, which encodes a protein of 47,244 Da with a theoretical pI of 6.2. The deduced amino acid sequence of the beta-transaminase showed the highest sequence similarity with glutamate-1-semialdehyde aminomutase of transaminase subgroup II. The beta-transaminase showed higher activities toward d-beta-aminocarboxylic acids such as 3-aminobutyric acid, 3-amino-5-methylhexanoic acid, and 3-amino-3-phenylpropionic acid. The beta-transaminase has an unusually broad specificity for amino acceptors such as pyruvate and alpha-ketoglutarate/oxaloacetate. The enantioselectivity of the enzyme suggested that the recognition mode of beta-aminocarboxylic acids in the active site is reversed relative to that of alpha-amino acids. After comparison of its primary structure with transaminase subgroup II enzymes, it was proposed that R43 interacts with the carboxylate group of the beta-aminocarboxylic acids and the carboxylate group on the side chain of dicarboxylic alpha-keto acids such as alpha-ketoglutarate and oxaloacetate. R404 is another conserved residue, which interacts with the alpha-carboxylate group of the alpha-amino acids and alpha-keto acids. The beta-transaminase was used for the asymmetric synthesis of enantiomerically pure beta-aminocarboxylic acids. (3S)-Amino-3-phenylpropionic acid was produced from the ketocarboxylic acid ester substrate by coupled reaction with a lipase using 3-aminobutyric acid as amino donor.
Kim
Juhan
J
School of Chemical Engineering, Seoul National University, Seoul 151-742, Korea.
Kyung
Dohyun
D
Yun
Hyungdon
H
Cho
Byung-Kwan
BK
Seo
Joo-Hyun
JH
Cha
Minho
M
Kim
Byung-Gee
BG
D020561:Alphaproteobacteria / Q000201:enzymology* / Q000235:genetics; D000596:Amino Acids / Q000096:biosynthesis*; D000613:Aminobutyrates / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000502:physiology; D015139:Blotting, Southern; D002208:Caproates / Q000378:metabolism; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D015723:Gene Library; D019751:Intramolecular Transferases / Q000235:genetics*; D007526:Isoelectric Point; D007656:Ketoglutaric Acids / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D016366:Open Reading Frames; D062907:Oxaloacetic Acid / Q000378:metabolism; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000378:metabolism; D019289:Pyruvic Acid / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D013379:Substrate Specificity
D000596:Amino Acids; D000613:Aminobutyrates; D002208:Caproates; D004269:DNA, Bacterial; D007656:Ketoglutaric Acids; C064718:3-amino-3-phenylpropionic acid; D062907:Oxaloacetic Acid; C047667:3-aminobutyric acid; D010649:Phenylalanine; D019289:Pyruvic Acid; D019751:Intramolecular Transferases; C061049:glutamate-1-semialdehyde 2,1-aminomutase