Cloning, expression, and characterization of aminopeptidase P from the hyperthermophilic archaeon Thermococcus sp. strain NA1.
Genomic analysis of a hyperthermophilic archaeon, Thermococcus sp. strain NA1, revealed the presence of a 1,068-bp open reading frame encoding a protein consisting of 356 amino acids with a calculated molecular mass of 39,714 Da (GenBank accession no. DQ144132). Sequence analysis showed that it was similar to the putative aminopeptidase P (APP) of Thermococcus kodakaraensis KOD1. Amino acid residues important for catalytic activity and the metal binding ligands conserved in bacterial, nematode, insect, and mammalian APPs were also conserved in the Thermococcus sp. strain NA1 APP. The archaeal APP, designated TNA1_APP (Thermococcus sp. strain NA1 APP), was cloned and expressed in Escherichia coli. The recombinant enzyme hydrolyzed the amino-terminal Xaa-Pro bond of Lys(Nepsilon-Abz)-Pro-Pro-pNA and the dipeptide Met-Pro (Km, 0.96 mM), revealing its functional identity. Further enzyme characterization showed the enzyme to be a Co2+-, Mn2+-, or Zn2+-dependent metallopeptidase. Optimal APP activity with Met-Pro as the substrate occurred at pH 5 and a temperature of 100 degrees C. The APP was thermostable, with a half-life of >100 min at 80 degrees C. This study represents the first characterization of a hyperthermophilic archaeon APP.
Lee
Hyun Sook
HS
Korean Ocean Research & Development Institute, Ansan P.O. Box 29, Seoul 425-600, Korea.
Kim
Yun Jae
YJ
Bae
Seung Seob
SS
Jeon
Jeong Ho
JH
Lim
Jae Kyu
JK
Jeong
Byeong Chul
BC
Kang
Sung Gyun
SG
Lee
Jung-Hyun
JH
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006358:Hot Temperature; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
D019843:Archaeal Proteins; D000626:Aminopeptidases; C022750:X-Pro aminopeptidase