Cloning, expression, and characterization of a methionyl aminopeptidase from a hyperthermophilic archaeon Thermococcus sp. NA1.
Genomic analysis of a hyperthermophilic archaeon Thermococcus sp. NA1 revealed the presence of an 885-bp open reading frame encoding a protein of 295 amino acids with a calculated molecular mass of 32,981 Da. Analysis of the deduced amino acid sequence showed that amino acid residues important for catalytic activity and the metal binding ligands conserved in all of methionyl aminopeptidases (MetAP) were also conserved and belonged to type IIa MetAP. The protein, designated TNA1_MetAP (Thermococcus sp. NA1 MetAP), was cloned and expressed in Escherichia coli. The recombinant enzyme was a Mn(2+)-, Ni(2+)-, Fe(2+)-, or Co(2+)-dependent metallopeptidase. Optimal MetAP activity against L: -methionine p-nitroanilide (Met-pNA) (K (m) = 0.68 mM) occurred at pH 7.0 and 80 to 90 degrees C. The MetAP was very unstable compared to Pyrococcus furiosus MetAP, which was completely inactivated by heating at 80 degrees C for 5 min. It seemed likely that the cysteine residue (Cys53) played a critical role in regulating the thermostability of TNA1_MetAP.
Lee
H S
HS
Korean Ocean Research & Development Institute, Ansan, P.O. Box 29, Seoul, 425-600, Korea. jlee@kordi.re.kr
Kim
Y J
YJ
Bae
S S
SS
Jeon
J H
JH
Lim
J K
JK
Jeong
B C
BC
Kang
S G
SG
Lee
J-H
JH
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000096:biosynthesis* / Q000737:chemistry / Q000187:drug effects / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D008670:Metals / Q000494:pharmacology; D063208:Methionyl Aminopeptidases; D008969:Molecular Sequence Data; D009792:Oceans and Seas; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics* / Q000502:physiology; D013997:Time Factors
D017931:DNA Primers; D008670:Metals; D011994:Recombinant Proteins; D000626:Aminopeptidases; D063208:Methionyl Aminopeptidases