What Drives Chorismate Mutase to Top Performance? Insights from a Combined In Silico and In Vitro Study
Abstract
Unlike typical chorismate mutases, the enzyme from Mycobacterium tuberculosis (MtCM) has only low activity on its own. Remarkably, its catalytic efficiency kcat/Km can be boosted more than 100-fold by complex formation with a partner enzyme. Recently, an autonomously fully active MtCM variant was generated using directed evolution, and its structure was solved by X-ray crystallography. However, key residues were involved in crystal contacts, challenging the functional interpretation of the structural changes. Here, we address these challenges by microsecond molecular dynamics simulations, followed up by additional kinetic and structural analyses of selected sets of specifically engineered enzyme variants. A comparison of wild-type MtCM with naturally and artificially activated MtCMs revealed the overall dynamic profiles of these enzymes as well as key interactions between the C-terminus and the active site loop. In the artificially evolved variant of this model enzyme, this loop is preorganized and stabilized by Pro52 and Asp55, two highly conserved residues in typical, highly active chorismate mutases. Asp55 stretches across the active site and helps to appropriately position active site residues Arg18 and Arg46 for catalysis. The role of Asp55 can be taken over by another acidic residue, if introduced at position 88 close to the C-terminus of MtCM, as suggested by molecular dynamics simulations and confirmed by kinetic investigations of engineered variants. Show more
Permanent link
https://doi.org/10.3929/ethz-b-000599925Publication status
publishedExternal links
Journal / series
BiochemistryVolume
Pages / Article No.
Publisher
American Chemical SocietySubject
Conformation; Crystal structure; Genetics; Monomers; Peptides and proteinsOrganisational unit
08816 - Kast, Peter (Tit.-Prof.)
Funding
182648 - Exploring structure, function, and mechanism of atypical bacterial chorismate mutases (SNF)
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