Construction and properties of a fragmentary D-amino acid aminotransferase.
D-Amino acid aminotransferase [EC 2.6.1.21] catalyzes the inter-conversion between various D-amino acids and alpha-keto acids. The subunit of the homodimeric enzyme from Bacillus sp. YM-1 consists of two domains connected by a single loop, which has no direct contact with the active site residues or the cofactor, pyridoxal 5'-phosphate [Sugio, S., Petsko, G.A., Manning, J.M., Soda, K., and Ringe, D. (1995) Biochemistry 34, 9661-9669]. We constructed two plasmids, one encoding a polypeptide fragment corresponding to the N-terminal domain, and the other a fragment corresponding to the C-terminal domain. When both polypeptide fragments were expressed together in the same host cell, an active fragmentary enzyme consisting of two sets of the two polypeptide fragments was produced. When the two polypeptide fragments were expressed separately, each of them provided a soluble protein but with no activity. However, D-amino acid aminotransferase activity appeared upon incubation of a mixture of the two fragments. The active fragmentary enzyme was purified to homogeneity and characterized; it was found to be similar to the wild-type enzyme in various enzymological properties except substrate specificity, inhibition by alpha-ketoglutarate, and thermostability. The fragmentary enzyme showed higher catalytic activity toward several substrates, such as D-lysine and D-arginine, than the wild-type enzyme.
Fuchikami
Y
Y
Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto, 611-0011, Japan.
Yoshimura
T
T
Gutierrez
A
A
Soda
K
K
Esaki
N
N
D000410:Alanine Transaminase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D051263:D-Alanine Transaminase; D017931:DNA Primers; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D017931:DNA Primers; D011994:Recombinant Proteins; D000410:Alanine Transaminase; D051263:D-Alanine Transaminase