Cloning, Expression, and Characterization of a Methionyl Aminopeptidase from a Hyperthermophilic Archaeon Thermococcus sp. NA1

Abstract

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.

Post-translation modification in Archaea: lessons fromHaloferax volcaniiand other haloarchaea

“…Among the archaea, MAPs from the hyperthermophiles P. furiosus (PfMAP) and Thermococcus sp. NA1 have been purified in enzymatically active forms (Tsunasawa et al, 1997;Tahirov et al, 1998;Lee et al, 2006). Of these two enzymes, PfMAP is the most extensively studied.…”

Proteolytic inventory of Thermococcus kodakaraensis

“…Methionine aminopeptidases are classified into two groups, types I and II, depending upon their structure. Bacteria contain type I, archaea have type II while eukaryotes contain both type I and II (Bradshaw et al, 1998 (Lee et al, 2006). Methionine aminopeptodases from E. coli, Staphylococcus aureus, Pyrococcus furiosus and Homo sapiens have been crystallographically characterized (Roderick and Mattews, 1993;Oefner et al, 2003;Tahirov et al, 1998;Liu et al, 1998).…”

“…T. kodakaraenssis has one putative ORF for methionine aminopeptidase with a highest homology of 85% with Thermococcus sp NA1. The homology comparison shows that both have two aspartic acid residues (Asp 83, Asp 94), two glutamic acid (Glu 188 Glu 281) and a histidine (His 154) as active site residues involved in metal ion coordination (Lee et al, 2006).…”

“…The other ORF exhibited highest homology of 73% with that of P. horikoshii. Carboxypeptidase Taq from Thermus aquaticus (Lee et al, 1994), Thermus thermophilus (Nagata et al, 2004), Thermococcus sp NA1 (Lee et al, 2006) and P. furiosus (Chang et al, 2001) have been characterized. Carboxypeptidase from Thermococcus sp NA1 shows broad substrate specificity which makes it a potential candidate in C-terminal sequencing.…”