Characterization of a bifunctional aminoacylase/carboxypeptidase from radioresistant bacterium Deinococcus radiodurans R1.
The gene encoding a Deinococcus radiodurans R1 bifunctional aminoacylase/carboxypeptidase (DR_ACY/CP) was amplified by polymerase chain reaction and cloned into pQE-30 to generate pQE-DRAC. The cloned gene consists of an open reading frame of 1197 bp encoding a protein with a molecular mass of 42,729 Da. The predicted amino acid sequence shows high homology with those of Geobacillus kaustophilus aminoacylase, Geobacillus stearothermophilus aminoacylase, Pyrococcus horikoshii carboxypeptidase/aminoacylase and Thermoanaerobacter tengcongensis aminoacylase/carboxypeptidase. The expressed enzyme was purified from the crude extract of IPTG-induced Escherichia coli M15 (pQE-DRAC) to homogeneity by nickel-chelate chromatography. The molecular mass of the purified enzyme was determined to be 43kDa by SDS-PAGE. Maximal aminoacylase activity with N-acetyl-methionine as the substrate occurred at pH 8.0 and 40 degrees C in the sodium phosphate buffer. The aminoacylase activity was strongly inhibited by metal-chelating agents, and was largely restored by divalent cations, such as Co(2+), Mn(2+) and Ni(2+). The purified enzyme had broad specificity toward N-acetylated L-amino acids as well as N-CBZ-peptides. Carboxypeptidase activity of DR_ACY/CP to N-CBZ-Gly-Ala exhibited K(m) and k(cat) values of 4.3mM and 28s(-1), respectively. The enzyme also had activity toward the cell wall-related substrates, D-Ala-Gly, D-Ala-Gly-Gly and L-Orn-L-Ala.
Lin
Long-Liu
LL
Department of Applied Chemistry, National Chiayi University, 600 Chiayi, Taiwan.
Chen
Mei-Hua
MH
Chien
Hung-Chien Roger
HC
Kan
Shu-Chen
SC
Chen
Chun-Chang
CC
Hu
Hui-Yu
HY
Hsu
Wen-Hwei
WH
D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D002268:Carboxypeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002413:Cations, Divalent / Q000378:metabolism; D034301:Deinococcus / Q000201:enzymology*; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity
D002413:Cations, Divalent; D002268:Carboxypeptidases; D000581:Amidohydrolases; C019672:aminoacylase I