METHIONINE AMINOPEPTIDASE FROM THE HYPERTHERMOPHILIC ARCHAEON PYROCOCCUS-FURIOSUS - MOLECULAR-CLONING AND OVEREXPRESSION IN ESCHERICHIA-COLI OF THE GENE, AND CHARACTERISTICS OF THE ENZYME

A gene for a methionine aminopeptidase (MAP; EC 3.4.11.18), which cata lyzes the removal of amino-terminal methionine from the growing peptid e chain on the ribosome, has been cloned from the hyperthermophilic Ar chaeon, Pyrococcus furiosus, by a novel method effectively using its c osmid protein library, sequenced and expressed in Escherichia coli. Th e DNA sequence encodes a protein containing 295 amino acid residues wi th methionine at the N-terminus. From protein analyses of the recombin ant protein expressed in E. coli, by using both amino acid sequence an alysis from the N-terminus by automated Edman degradation and analyses of molecular masses of the peptides generated by two enzymatic cleava ges performed independently, digestions with lysylendopeptidase and En doproteinase Asp-N, with ionspray mass spectrometry, the primary struc ture of the protein has been elucidated to be completely identical wit h that deduced from its DNA sequence. Comparison of the amino acid seq uence of P. furiosus MAP (P.f. MAP) with those of other MAPs from Euka rya and Bacteria showed that the protein has a high degree of sequence homology in the stretches surrounding the five cobalt-binding residue s fully preserved in all of MAPs determined so far, but P.f. MAP belon gs to Type II because it has an extra long insertion of about 60 amino acid residues between the fourth and fifth cobalt-binding ligands, si milar to MAPs from human and rat, and to Met-AP2 from Saccharomyces ce revisiae, in comparison to Type I MAPs from Bacteria. Therefore, P.f. MAP seems to be rather close to those from Eukarya, although it is dis tinct in lacking the N-terminal extension of about 90-150 residues uni versally found in MAPs from Eukarya. These findings suggest that P.f. MAP is evolutionally located at the Eukarya-Bacteria boundary. The enz yme expressed in E. coli exhibits a considerable thermostability, with a half-life of approximately 4.5 h at 90 degrees C and an optimum tem perature of around 90 degrees C.