We localized the methionine aminopeptidase (map) gene on the Escherichia coli chromosome next to the rpsB gene at min 4. Genetically modified strains with the chromosomal map gene under lac promoter control grew only in the presence of the lac operon inducer isopropyl-p-thiogalactoside. Thus, methionine aminopeptidase is essential for cell growth.The map gene of Escherichia coli encodes the methionine aminopeptidase that preferentially removes the initiation methionine from certain intracellular proteins in vitro and in vivo (3). The substrate specificity of the methionine aminopeptidase is primarily determined by the residue adjacent to the methionine (3,5,11). The map location of the map gene and the biological significance of selective removal of the initiation methionine were not known. We report here the localization of the map gene on the E. coli genetic map and data indicating that methionine aminopeptidase performs an essential function in E. coli.The coding and flanking nucleotide sequences of the map gene have been reported elsewhere (3). We searched the GenBank DNA data base for E. coli sequences that matched the upstream sequence of map and identified the first 134 base pairs of the reported 5'-flanking sequence of the rpsBtsf operon (ribosomal protein S2 and translation elongation factor EF-Ts genes) (1). Thus, map is located 357 base pairs from the rpsB-tsf operon, and the two genes are transcribed divergently. The rpsB gene maps about 5 kilobases clockwise from the dapD gene at the 4-min position on the E. coli chromosome (2,4,8). The order of the four closely clustered genes is dapD-map-rpsB-tsf (Fig. IA). This conclusion is consistent with that deduced independently from the genomic restriction mapping data (9).We attempted by gene replacement, but failed, to substitute the chromosomal map+ gene with an inactivated copy of map (unpublished data). This led us to speculate that map is essential. Therefore, we substituted the chromosomal map+ gene with an altered map gene fragment that allowed us to conveniently manipulate its expression. We deleted the lacZ promoter sequence between the PvuII site and the EcoRI site in the pUC18 (13) (Fig. 1B), contains the map gene controlled by the lac promoter and operator fragment (lacZplo-map), the cat gene as a selectable marker, and flanking homologous sequences to allow double recombination for gene replacement.Plasmid pSYC1695 DNA was digested with Asp718 and SalI restriction enzymes, which cleave outside of the catlacZplo-map fragment. The DNA fragment was used to transform E. coli H205, an hsdR derivative from the recB recC sbcB strain JC7623 (12) that allows gene replacement through double homologous recombination between the linear transforming DNA and the chromosome (Fig. 1). Chloramphenicol-resistant (Cmr) transformants were selected on plates supplemented with the lac operon inducer isopropyl-,-thiogalactoside (IPTG) to induce and maintain the Map' phenotype in the transformants. One of these (designated EM1) was selected for further characterizati...

The removal of N-terminal translation initiator Met by methionine aminopeptidase (MetAP) is often crucial for the function and stability of proteins. On the basis of crystal structure and sequence alignment of MetAPs, we have engineered Escherichia coli MetAP by the mutation of three residues, Y168G, M206T, Q233G, in the substrate-binding pocket. Our engineered MetAPs are able to remove the Met from bulky or acidic penultimate residues, such as Met, His, Asp, Asn, Glu, Gln, Leu, Ile, Tyr, and Trp, as well as from small residues. The penultimate residue, the second residue after Met, was further removed if the antepenultimate residue, the third residue after Met, was small. By the coexpression of engineered MetAP in E. coli through the same or a separate vector, we have successfully produced recombinant proteins possessing an innate N terminus, such as onconase, an antitumor ribonuclease from the frog Rana pipiens. The N-terminal pyroglutamate of recombinant onconase is critical for its structural integrity, catalytic activity, and cytotoxicity. On the basis of N-terminal sequence information in the protein database, 85%-90% of recombinant proteins should be produced in authentic form by our engineered MetAPs.

Onconase, a cytotoxic ribonuclease from Rana pipiens, possesses pyroglutamate (Pyr) at the N-terminus and has a substrate preference for uridine-guanine (UG). To identify residues responsible for onconase's cytotoxicity, we cloned the rpr gene from genomic DNA and expressed it in Escherichia coli BL21(DE3). The recombinant onconase with Met at the N-terminus had reduced thermostability, catalytic activity and antigenicity. Therefore, we developed two methods to produce onconase without Met. One relied on the endogeneous E.coli methionine aminopeptidase and the other relied on the cleavage of a pelB signal peptide. The Pyr1 substitutional variants maintained similar secondary structures to wild-type onconase, but with less thermostability and specific catalytic activity for the innate substrate UG. However, the non-specific catalytic activity for total RNAs varied depending on the relaxation of base specificity. Pyr1 promoted the structural integrity by forming a hydrogen bond network through Lys9 in alpha1 and Val96 in beta6, and participated in catalytic activity by hydrogen bonds to Lys9 and P(1) catalytic phosphate. Residues Thr35 and Asp67 determined B(1) base specificity, and Glu91 determined B(2) base specificity. The cytotoxicity of onconase is largely determined by structural integrity and specific catalytic activity for UG through Pyr1, rather than non-specific activity for total RNAs.

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