The Mechanism of Microcin C Resistance Provided by the MccF Peptidase

Tikhonov, Anton; Kazakov, Teymur; Semenova, Ekaterina; Serebryakova, Marina V.; Vondenhoff, Gaston; Reader, John S.; Govorun, Vadim M.; Severinov, Konstantin

doi:10.1074/jbc.m110.179135

Cited by 37 publications

(49 citation statements)

References 22 publications

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“…A catalytic triad, composed of Ser118, His311, and Glu243, is situated at the interface between the two domains ( Fig. 2A), consistent with the earlier report that mutation at Ser118 results in a loss of catalytic activity (8). The side chain of Glu243 is buried in a polar environment with one of the carboxylate oxygen atoms hydrogen bonded to the Nδ1 of His311 (2.9 Å) and the second carboxylate oxygen atom hydrogen bonded to the Oη of Tyr137 (2.5 Å) and to Nδ2 of Asn218 (3.0 Å).…”

Section: Resultssupporting

confidence: 77%

“…We had previously demonstrated that overexpression of the mccF gene results in McC7 resistance, and that recombinant MccF cleaved the amide bond that connects the peptidyl and nucleotidyl moieties of McC7 (8). Similar effects were also observed with the synthetic DSA and ESA (Fig.…”

Section: Resultssupporting

confidence: 65%

“…This catalytic loop is absent in the structurally homologous LD-carboxypeptidases. The lack of a cocrystal structure of the LD-carboxypeptidase with bound substrate precludes a comparison of the substrate binding sites of the two enzymes, though the absence of the catalytic loop provides a possible explanation for the reported inability of LD-carboxypeptidase to detoxify McC and its analogs (8,9). We propose that the presence of the catalytic loop can be used to distinguish proteins with MccF-like activity from LD-carboxypeptidases that have been misannotated in sequence databases (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…1A, 3) or glutamyl sulfamoyl adenylate (ESA) (Fig. 1A, 4) moiety conjugated to adenosine, with diminished activity also observed for seryl conjugated adenosine (8).…”

mentioning

confidence: 99%

“…The McC7 biosynthetic cluster does not encode a tRNA synthetase but instead, employs specialized enzymes that act on processed and/or intact McC7 in the producing cells. One such autoimmunity determinant is encoded by the mccF gene, whose product proteolytically hydrolyzes the amide bond connecting the terminal aspartate and modified AMP in McC7 (8). MccF was shown to inactivate both intact and processed forms of McC7.…”

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confidence: 99%

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Structure and function of a serine carboxypeptidase adapted for degradation of the protein synthesis antibiotic microcin C7

Agarwal

Tikhonov

Metlitskaya

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Several classes of naturally occurring antimicrobials exert their antibiotic activity by specifically targeting aminoacyl-tRNA synthetases, validating these enzymes as drug targets. The aspartyl tRNA synthetase “Trojan horse” inhibitor microcin C7 (McC7) consists of a nonhydrolyzable aspartyl-adenylate conjugated to a hexapeptide carrier that facilitates active import into bacterial cells through an oligopeptide transport system. Subsequent proteolytic processing releases the toxic compound inside the cell. Producing strains of McC7 must protect themselves against autotoxicity that may result from premature processing. The mccF gene confers resistance against endogenous and exogenous McC7 by hydrolyzing the amide bond that connects the peptide and nucleotide moieties of McC7. We present here crystal structures of MccF, in complex with various ligands. The MccF structure is similar to that of dipeptide ld -carboxypeptidase, but with an additional loop proximal to the active site that serves as the primary determinant for recognition of adenylated substrates. Wild-type MccF only hydrolyzes the naturally occurring aspartyl phosphoramidate McC7 and synthetic peptidyl sulfamoyl adenylates that contain anionic side chains. We show that substitutions of two active site MccF residues result in a specificity switch toward aromatic aminoacyl–adenylate substrates. These results suggest how MccF-like enzymes may be used to avert various toxic aminoacyl–adenylates that accumulate during antibiotic biosynthesis or in normal metabolism of the cell.

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Section: Resultssupporting

confidence: 77%

Section: Resultssupporting

confidence: 65%

Section: Discussionmentioning

confidence: 99%

“…1A, 3) or glutamyl sulfamoyl adenylate (ESA) (Fig. 1A, 4) moiety conjugated to adenosine, with diminished activity also observed for seryl conjugated adenosine (8).…”

mentioning

confidence: 99%

mentioning

confidence: 99%

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