PMID:22721690

Citation	Liu, J, Asano, Y, Ikoma, K, Yamashita, S, Hirose, Y, Shimoyama, T, Takahashi, S, Nakayama, T and Nishino, T (2012) Purification, characterization, and primary structure of a novel N-acyl-D-amino acid amidohydrolase from Microbacterium natoriense TNJL143-2. J. Biosci. Bioeng. 114:391-7
Abstract	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.
Links	PubMed Online version:10.1016/j.jbiosc.2012.05.015
Keywords	Actinomycetales/enzymology; Amidohydrolases/chemistry; Amidohydrolases/genetics; Amidohydrolases/isolation & purification; Amidohydrolases/metabolism; Amino Acid Sequence; Amino Acids/biosynthesis; Amino Acids/chemistry; Amino Acids/genetics; Amino Acids/isolation & purification; Cloning, Molecular; Industrial Microbiology; Molecular Sequence Data; Molecular Weight; Phylogeny; Sequence Alignment; Temperature
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
9MICO:I7HFV7		GO:0004046: aminoacylase activity	ECO:0000314:		F		Figure 2	complete CACAO 9104

References

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