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A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli
Published on: December 9, 2017
Structural basis for microcin C7 inactivation by the MccE acetyltransferase
Vinayak Agarwal1, Anastasiya Metlitskaya, Konstantin Severinov
1Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
Abstract:
The antibiotic microcin C7 (McC) acts as a bacteriocide by inhibiting aspartyl-tRNA synthetase and stalling the protein translation machinery. McC is synthesized as a heptapeptide-nucleotide conjugate, which is processed by cellular peptidases within target strains to yield the biologically active compound. As unwanted processing of intact McC can result in self-toxicity, producing strains utilize multiple mechanisms for autoimmunity against processed McC. We have shown previously that the mccE gene within the biosynthetic cluster can inactivate processed McC by acetylating the antibiotic. Here, we present the characterization of this acetylation mechanism through biochemical and structural biological studies of the MccE acetyltransferase domain (MccE(AcTase)). We have also determined five crystal structures of the MccE-acetyl-CoA complex with bound substrates, inhibitor, and reaction product. The structural data reveal an unexpected mode of substrate recognition through π-stacking interactions similar to those found in cap-binding proteins and nucleotidyltransferases. These studies provide a rationale for the observation that MccE(AcTase) can detoxify a range of aminoacylnucleotides, including those that are structurally distinct from microcin C7.
Insights
The antibiotic microcin C7 (McC) is inactivated by acetylation, a process studied via biochemical and structural methods. This research reveals how the MccE enzyme detoxifies McC and similar compounds.
Area of Science:
- Microbiology
- Structural Biology
- Biochemistry
Background:
- Microcin C7 (McC) is a bacteriocin that inhibits protein synthesis by targeting aspartyl-tRNA synthetase.
- McC is produced as a conjugate and activated by processing, posing a self-toxicity risk to producing strains.
- The mccE gene product, MccE, inactivates processed McC via acetylation, contributing to bacterial autoimmunity.
Purpose of the Study:
- To characterize the acetylation mechanism of processed microcin C7 by the MccE enzyme.
- To elucidate the structural basis of MccE's substrate recognition and acetylation activity.
Main Methods:
- Biochemical assays to study the MccE acetyltransferase domain (MccE(AcTase)) activity.
- X-ray crystallography to determine the structures of MccE(AcTase) in complex with substrates, inhibitors, and products.
- Analysis of protein-ligand interactions, including π-stacking interactions.
Main Results:
- Detailed characterization of the MccE(AcTase) enzymatic mechanism.
- Determination of five crystal structures providing insights into substrate binding.
- Identification of an unexpected substrate recognition mode involving π-stacking interactions.
- Demonstration that MccE(AcTase) can detoxify various aminoacylnucleotides beyond microcin C7.
Conclusions:
- The MccE enzyme utilizes a unique π-stacking interaction for substrate recognition, similar to cap-binding proteins.
- Structural insights explain MccE's broad substrate specificity in detoxifying aminoacylnucleotides.
- This study provides a molecular understanding of antibiotic self-immunity mechanisms in bacteria.
