An Unusually Rapid Protein Backbone Modification Stabilizes the Essential Bacterial Enzyme MurA

• Published in: Biochemistry (Easton) Vol. 59; no. 39; pp. 3683 - 3695
• Main Authors: Zhang, Tianze, Hansen, Kjetil, Politis, Argyris, Müller, Manuel M
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 06.10.2020
• Subjects: Alkyl and Aryl Transferases - chemistry; asparagine; aspartic acid; bacterial enzymes; Bacterial Proteins - chemistry; Enterobacter cloacae - chemistry; Enterobacter cloacae - enzymology; Enzyme Stability; Isoaspartic Acid - chemistry; Isomerism; isomers; mass spectrometry; Models, Molecular; mutants; Protein Aggregates; Protein Conformation; Protein Folding; protein unfolding; proteins; site-directed mutagenesis; stoichiometry
• DOI: 10.1021/acs.biochem.0c00502
• ISSN: 0006-2960; 1520-4995

Proteins are subject to spontaneous rearrangements of their backbones. Most prominently, asparagine and aspartate residues isomerize to their β-linked isomer, isoaspartate (isoAsp), on time scales ranging from days to centuries. Such modifications are typically considered “molecular wear-and-tear”, destroying protein function. However, the observation that some proteins, including the essential bacterial enzyme MurA, harbor stoichiometric amounts of isoAsp suggests that this modification can confer advantageous properties. Here, we demonstrate that nature exploits an isoAsp residue within a hairpin to stabilize MurA. We found that isoAsp formation in MurA is unusually rapid and critically dependent on folding status. Moreover, perturbation of the isoAsp-containing hairpin via site-directed mutagenesis causes aggregation of MurA variants. Structural mass spectrometry revealed that this effect is caused by local protein unfolding in MurA mutants. Our findings demonstrate that MurA evolved to “mature” via a spontaneous post-translational incorporation of a β-amino acid, which raises the possibility that isoAsp-containing hairpins may serve as a structural motif of biological importance.