IDENTIFICATION OF RETAINED N-FORMYLMETHIONINE IN BACTERIAL RECOMBINANT MAMMALIAN CYTOCHROME-P450 PROTEINS WITH THE N-TERMINAL SEQUENCE MALLLAVFL - ROLES OF RESIDUE-3-5 IN RETENTION AND MEMBRANE TOPOLOGY

An N-terminal block to Edman degradation was observed when any of five different mammalian cytochrome P450 (P450) proteins was expressed in Escherichia coli using the N-terminal sequence MALLLAVFL... This block was also seen in Salmonella typhimurium. With all proteins examined, the block could be removed by mild acid hydrolysis (0.6-6 N HCl, 23 de grees C) to expose Met as the N-terminus, suggesting N-formylMet reten tion. The N-terminal peptide of a modified P450 1A2 (''mutant 1'', con taining a thrombin-sensitive site inserted at residue 25) was released with thrombin and analyzed by electrospray mass spectrometry and foun d to yield the M(r) expected for the N-formyl derivative (+/-0.8 amu), The region of positions 3-5 was altered by random mutagenesis, and th ree P450 1A2-expressing clones were analyzed for nucleotide and amino acid sequences. The changes from LLL were to RER (P450 1A2a), VDS (P45 0 1A2b), and WRH (P450 1A2c); these all show slightly dissimilar hydro pathy plots compared to the MALLLAVFL... sequence. Mutant P450 1A2a ha d the N-terminal Met removed to yield N-terminal Ala; P450 1A2b contai ned an unmodified Met at the N-terminus; P450 1A2c had an similar to 8 0% block of the N-terminal Met, Experiments with bacterial membranes c ontaining expressed P450 1A2 mutant 1 and P450 1A2 mutant 2 (thrombin- sensitive site inserted at residue 46) suggest that thrombin site 2, b ut not 1, is sequestered in the membrane. Spheroplasts of bacteria exp ressing P450 1A2 and the mutants at positions 3-5 were treated with pr oteinase K; amino acid analysis indicated that no cleavage occurred. T hese results are interpreted in a model in which most of the mammalian P450 expressed in the bacterium is located in the cytosol, the region near residue 46 is in the inner membrane, the region near residue 25 is in the cytosol, and the N-terminus is either imbedded in the membra ne or free in the cytosolic space, depending upon the sequence. Howeve r, the possibility that the differences in N-terminal processing are t he result of direct changes in interactions with the deformylase and M et aminopeptidase cannot be excluded.