Overexpression and characterization of a carboxypeptidase from the hyperthermophilic archaeon Thermococcus sp. NA1.
Genomic analysis of a hyperthermophilic archaeon, Thermococcus sp. NA1, revealed the presence of an 1,497 bp open reading frame, encoding a protein of 499 amino acids. The deduced amino acid sequence was similar to thermostable carboxypeptidase 1 from Pyrococcus furiosus, a member of peptidase family M32. Five motifs, including the HEXXH motif with two histidines coordinated with the active site metal, were conserved. The carboxypeptidase gene was cloned and overexpressed in Escherichia coli. Molecular masses assessed by SDS-PAGE and gel filtration were 61 kDa and 125 kDa respectively, which points to a dimeric structure for the recombinant enzyme, designated TNA1_CP. The enzyme showed optimum activity toward Z-Ala-Arg at pH 6.5 and 70-80 degrees C (k(cat)/K(m)=8.3 mM(-1) s(-1)). In comparison with that of P. furiosus CP (k(cat)/K(m)=667 mM(-1) s(-1)), TNA1_CP exhibited 80-fold lower catalytic efficiency. The enzyme showed broad substrate specificity with a preference for basic, aliphatic, and aromatic C-terminal amino acids. This broad specificity was confirmed by C-terminal ladder sequencing of porcine N-acetyl-renin substrate by TNA1_CP.
Lee
Hyun Sook
HS
Korea Ocean Research & Development Institute, Seoul, Korea. jlee@kordi.re.kr
Kim
Yun Jae
YJ
Bae
Seung Seob
SS
Jeon
Jeong Ho
JH
Lim
Jae Kyu
JK
Kang
Sung Gyun
SG
Lee
Jung-Hyun
JH
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D002268:Carboxypeptidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012083:Renin / Q000737:chemistry; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D019712:Thermococcus / Q000201:enzymology*
D019843:Archaeal Proteins; D002268:Carboxypeptidases; D012083:Renin