Processing of the initiation methionine from proteins: properties of the Escherichia coli methionine aminopeptidase and its gene structure

Abstract

Methionine aminopeptidase (MAP) catalyzes the removal of amino-terminal methionine from proteins. The Escherichia coli map gene encoding this enzyme was cloned; it consists of 264 codons and encodes a monomeric enzyme of 29,333 daltons. In vitro analyses with purified enzyme indicated that MAP is a metallooligopeptidase with absolute specificity for the amino-terminal methionine. The methionine residues from the amino-terminal end of the recombinant proteins interleukin-2 (Met-Ala-Pro-IL-2) and ricin A (Met-Ile-Phericin A) could be removed either in vitro with purified MAP enzyme or in vivo in MAP-hyperproducing strains of E. coli. In vitro analyses of the substrate preference of the E. coli MAP indicated that the residues adjacent to the initiation methionine could significantly influence the methionine cleavage process. This conclusion is consistent, in general, with the deduced specificity of the enzyme based on the analysis of known aminoterminal sequences of intracellular proteins (S. Tsunasawa, J. W. Stewart, and F. Sherman, J. Biol. Chem. 260:5382-5391, 1985).Protein synthesis is always initiated with either methionine or N-formylmethionine. The N-formyl moiety that is present on proteins from organelles and bacteria is removed by deformylases, leaving methionine at the amino terminus. For a significant fraction of the intracellular proteins, the amino-terminal methionine is removed enzymatically after the initiation of translation (1,2,5,10,24,33,34,38,40; for a review, see reference 28). Although other mechanisms may also be involved in the post-or cotranslational modifications of the amino-terminal residues of some proteins (e.g., signal peptidases that remove the secretory signal peptide sequences from certain transported proteins, transacetylases that acetylate the amino-terminal residues of some eucaryotic proteins), methionine is removed from the majority of proteins. However, very little is known about the structure and properties of the methionine aminopeptidase (MAP) that is responsible for removing the amino-terminal methionine residue from the cellular proteins. From a systematic analysis of the amino-terminal sequences data for various mutant forms of yeast iso-1-cytochrome c, as well as from the data for 82 mature intracellular proteins, Sherman et al. (28) For Escherichia coli and Salmonella typhimurium, many peptidase genes have been mapped, and their cognate peptidases have been characterized (6,9,22,23,29,30,32,35,36). However, none reported so far corresponds to the activity postulated for MAP. We took a direct approach to study the MAP of E. coli by molecular cloning of its gene (map) and identified the gene based on the postulated properties of the gene product. An E. coli strain deficient in five peptidases was transformed with a plasmid preparation * Corresponding author. containing a library of cloned genomic DNA fragments from the same strain. The lysates from individual transformants were then prepared and analyzed for the presence of elevated MAP activity. This allowed us to i...

Chitinase B from Serratia marcescens BJL200 is exported to the periplasm without processing

“…This contradicts an earlier report in which, on the basis of sequence analysis only, it was suggested that chitinase B possesses a 41 amino acid signal peptide (Harpster & Dunsmuir, 1989). The Nterminal methionine of the chitinase is probably removed by the action of methionyl-aminopeptidase, which has a preference for proteins with a small side chain in the penultimate position (Bem-Bassat et al, 1987 ;Hire1 et al, 1989). The present results show that chitinase B of S. marcescens B JL200(pMAY2-10) must be exported by a secretion mechanism that differs from the general, signalpeptide-based secretion mechanism observed in prokaryotes.…”

In vivo formation of allosteric aspartate transcarbamoylase containing circularly permuted catalytic polypeptide chains: Implications for protein folding and assembly

“…Less than 10% methionine processing was observed with cpc,22, in contrast to the estimated amount of 84%. This discrepancy can be attributed to Pro 123 because it has been found that a proline residue following the residue next to the N-terminal amino acid is inhibitory to methionine removal (Ben-Bassat et al, 1987;Hirel et al, 1989). For cpc222, the amount of methionine processing observed was about 40%, whereas the value estimated by Hirel et al (1989) was 84%.…”

Purification and sequencing of cytochrome b from potato reveals methionine cleavage of a mitochondriallly encoded protein

“…Cleavage occurs before Gly, Ala, Pro, Ser, Thr, Val and Cys, but never before His, Gln, Glu, Phe, Met, Lys, Tyr, Trp and Arg. The third residue of the polypeptide may influence these peptidases as well [2,40]. In Fig.…”

“…One of the mechanisms of protein modification is the removal of the amino-terminal methionine [l]. It is catalysed by methionine aminopeptidases which have been characterized in E. co& [2], ~ffl~onel~a [3,4] and yeast [5-71. Methionine specific aminopeptidases share similar substrate specificity in pro-and eukaryotes [8,9]. In prokaryotes an additional deformylase is required which makes the N-terminal methionine accessible to the cleavage reaction.…”