Purification, characterization, and primary structure of a novel N-acyl-D-amino acid amidohydrolase from Microbacterium natoriense TNJL143-2.
A novel N-acyl-D-amino acid amidohydrolase (DAA) was purified from the cells of a novel species of the genus Microbacterium. The purified enzyme, termed AcyM, was a monomeric protein with an apparent molecular weight of 56,000. It acted on N-acylated hydrophobic D-amino acids with the highest preference for N-acetyl-D-phenylalanine (NADF). Optimum temperature and pH for the hydrolysis of NADF were 45°C and pH 8.5, respectively. The k(cat) and K(m) values for NADF were 41 s⁻¹ and 2.5 mM at 37°C and pH 8.0, although the enzyme activity was inhibited by high concentrations of NADF. Although many known DAAs are inhibited by 1 mM EDTA, AcyM displayed a 65% level of its full activity even in the presence of 20 mM EDTA. Based on partial amino acid sequences of the purified enzyme, the full-length AcyM gene was cloned and sequenced. It encoded a protein of 495 amino acids with a relatively low sequence similarity to a DAA from Alcaligenes faecalis DA1 (termed AFD), a binuclear zinc enzyme of the α/β-barrel amidohydrolase superfamily. The unique cysteine residue that serves as a ligand to the active-site zinc ions in AFD and other DAAs was not conserved in AcyM and was replaced by alanine. AcyM was the most closely related to a DAA of Gluconobacter oxydans (termed Gox1177) and phylogenetically distant from AFD and all other DAAs that have been biochemically characterized thus far. AcyM, along with Gox1177, appears to represent a new phylogenetic subcluster of DAAs.
Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba 6-6-11, Aramaki, Aoba-ku, Sendai, Miyagi 980-8579, Japan.
Jian
J
Liu
Yu
Y
Asano
Keiko
K
Ikoma
Satoshi
S
Yamashita
Yoshihiko
Y
Hirose
Takefumi
T
Shimoyama
Seiji
S
Takahashi
Toru
T
Nakayama
Tokuzo
T
Nishino
D000192:Actinomycetales / Q000201:enzymology*; D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D007218:Industrial Microbiology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D016415:Sequence Alignment; D013696:Temperature
D000596:Amino Acids; D000581:Amidohydrolases