title: Molecular discrimination of type-I over type-II methionyl aminopeptidases.
abstract: Two residues that are conserved in type-I methionyl aminopeptidases (MetAPs) but are absent in all type-II MetAPs are the cysteine residues (Escherichia coli MetAP-I: C59 and C70) that reside at the back of the substrate recognition pocket. These Cys residues are 4.4 A apart and do not form a disulfide bond. Since bacteria and fungi contain only type-I MetAPs while all human cells contain both type-I and type-II MetAPs, type-I MetAPs represent a novel antibiotic/antifungal target if type-I MetAPs can be specifically targeted over type-II. Based on reaction of the thiol-specific binding reagent 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) with the type-I MetAP from E. coli and the type-II MetAP from Pyrococcus furiosus, the type-I MetAP can be selectively inhibited. Verification that DTNB covalently binds to C59 in EcMetAP-I was obtained by mass spectrometry (MS) from reaction of DTNB with the C59A and C70A mutant EcMetAP-I enzymes. In addition, two inhibitors of EcMetAP-I, 5-iodopentaphosphonic acid (1) and 6-phosphonohexanoic acid (2), were designed and synthesized. The first was designed as a selective-C59 binding reagent while the second was designed as a simple competitive inhibitor of EcMetAP. Indeed, inhibitor 1 forms a covalent interaction with C59 based on activity assays and MS measurements, while 2 does not. These data indicate that type-I MetAPs can be selectively targeted over type-II MetAPs, suggesting that type-I MetAPs represent a new enzymatic target for antibacterial or antifungal agents.
authors.affiliation: Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322-0300, USA.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Swierczek
authors.forename: Alicja J
authors.initials: AJ
authors.lastname: Copik
authors.forename: Sabina I
authors.initials: SI
authors.lastname: Swierczek
authors.forename: Richard C
authors.initials: RC
authors.lastname: Holz
mesh_terms: D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry / Q000145:classification* / Q000235:genetics / Q000378:metabolism*; D000890:Anti-Infective Agents / Q000494:pharmacology; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D063208:Methionyl Aminopeptidases; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D020027:Pyrococcus furiosus / Q000201:enzymology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000890:Anti-Infective Agents; D000626:Aminopeptidases; D063208:Methionyl Aminopeptidases; D003545:Cysteine