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

Abstract

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.

Fig. 1.

Chemical structures of microcin C7 and analogs and demonstration of MccF hydrolysis activity. (A) Chemical structures of microcin C7 (1) and its processed form (2), along with synthetic sulfamoyl adenylates of aspartate (3), glutamate (4), and phenylalanine (5). The site of hydrolysis by wild-type MccF is marked by an arrow. (B) Hydrolysis of DSA by wild-type MccF enzyme demonstrated by HPLC separation the substrate and reaction product, sulfamoyl adenosine. (C) Michaelis–Menten kinetic curves for the hydrolysis of DSA (●) and ESA (○) by wild-type MccF enzyme.

Fig. 2.

Overall three-dimensional crystal structure of MccF. (A) Structure of MccF monomer showing the three domains: amino terminal domain (blue), carboxy-terminal domain (pink), and catalytic loop (green). (B) Structure of the MccF homodimer showing the relative positions of the three domains with one monomer colored as above and the second monomer colored gray.

Fig. 3.

Stereoviews of MccF-S118A active site with substrates bound. (A) Stereoview showing the active site features of the MccF-S118A in complex with processed McC7. The MccF carbon atoms are shown in yellow ball-and-stick and the McC7 carbon atoms are colored in green. Superimposed is a difference Fourier electron density map (contoured at 2.7σ over background in blue) calculated with coefficients |F_obs| - |F_calc| and phases from the final refined model with the coordinates of McC7 deleted prior to one round of refinement. (B) Stereoview showing the active site features of the MccF-S118A in complex with DSA. Superimposed is a difference Fourier electron density map (contoured at 2.5σ) calculated as above. (C) Stereoview showing the active site features of the MccF-S118A in complex with ESA. Superimposed is a difference Fourier electron density map (contoured at 2.5σ over background) calculated as above.

Fig. 4.

Cleavage of hydrophobic sulfamoyl adenosine by mutant forms of the MccF enzyme. (A) Time-dependent cleavage of substrate FSA (Inset) by MccF-N220L/K247L mutant enzyme demonstrated by separation of substrate and product peaks by HPLC. (B) Percentage hydrolysis of FSA plotted against reaction time for MccF-N220L/K247L enzyme.

Fig. 5.

Putative mechanism for McC7 hydrolysis by MccF. Hydrogen bond interactions are shown as dashes. MccF catalytic triad residue side-chain atoms are colored blue, the scissile peptide bond atoms are colored green, and the atoms of the phosphomoiety of the phosphoramidate bond are colored red.