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.

iTRAQ-based quantitative proteomic analysis gives insight into sexually different metabolic processes of poplars under nitrogen and phosphorus deficiencies

“…Two serine carboxypeptidase (SCP) family proteins were up‐regulated in N‐ and P‐deficient females but not in males. SCPs play roles in the degradation of proteins and peptides and are involved in the remobilization of nitrogen resources during seed germination, wound stress and leaf senescence . There has been clear evidence showing that the expression of SCPs greatly affects the cell elongation and cell wall structure in tobacco and poplar .…”

Translation-Targeting RiPPs and Where to Find Them

“…Thus, McC is another example of a RiPP (together with GE2270A discussed earlier), which does not directly interact with the ribosome but blocks translation by inhibiting the supply of substrates required for protein synthesis. Although McC has been studied for more than 30 years and structures of multiple enzymes involved in its biosynthesis and immunity have been determined (Agarwal et al, 2011(Agarwal et al, , 2012Dong et al, 2019), we still lack structural information about the details of McC interaction with aspartyl-tRNA synthetase.…”

The structure of the iron-catecholate transporter Fiu suggests substrate import occurs via a 2-step mechanism

“…The ability of TBDTs to import large substrates comes at a cost: an evolutionary arms race exists between TBDT-producing bacteria and organisms seeking to kill them by hijacking these receptors [11][12][13][14][15]. Both small molecule and protein antibiotics mimic TBDT substrates, leading to their inadvertent import into the bacterial cell [12,[16][17][18][19][20][21]. Catecholates are one of the four recognized classes of siderophores [22], have a strong affinity for iron, and are abundant secretion products of both bacteria and fungi [23,24].…”

“…The Ferric Iron Uptake (Fiu) transporter is an TBDT responsible for the import of molecules containing the catecholate functional group [2,25]. In addition to its role in iron uptake, Fiu has also been shown to be important for sensitivity to antimicrobials that share the common feature of a catecholate functional group or the analogous dihydroxypyridine moiety [21,25,26]. It is thought that the presence of these chemical mimics leads to their inadvertent import into the bacterial cell [27].…”