Evolving the naturally compromised chorismate mutase from Mycobacterium tuberculosis to top performance
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2020-12-18
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Journal Article
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Abstract
Chorismate mutase (CM), an essential enzyme at the branch-point of the shikimate pathway, is required for the biosynthesis of phenylalanine and tyrosine in bacteria, archaea, plants, and fungi. MtCM, the CM from Mycobacterium tuberculosis, has less than 1% of the catalytic efficiency of a typical natural CM and requires complex formation with 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase for high activity. To explore the full potential of MtCM for catalyzing its native reaction, we applied diverse iterative cycles of mutagenesis and selection, thereby raising kcat/Km 270-fold to 5 × 105 m−1s−1, which is even higher than for the complex. Moreover, the evolutionarily optimized autonomous MtCM, which had 11 of its 90 amino acids exchanged, was stabilized compared with its progenitor, as indicated by a 9 °C increase in melting temperature. The 1.5 Å crystal structure of the top-evolved MtCM variant reveals the molecular underpinnings of this activity boost. Some acquired residues (e.g. Pro52 and Asp55) are conserved in naturally efficient CMs, but most of them lie beyond the active site. Our evolutionary trajectories reached a plateau at the level of the best natural enzymes, suggesting that we have exhausted the potential of MtCM. Taken together, these findings show that the scaffold of MtCM, which naturally evolved for mediocrity to enable inter-enzyme allosteric regulation of the shikimate pathway, is inherently capable of high activity.
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published
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Journal / series
Volume
295 (51)
Pages / Article No.
17514 - 17534
Publisher
American Society for Biochemistry and Molecular Biology
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Subject
catalytic efficiency; conformational change; crystal structure; directed evolution; enzyme catalysis; enzyme mutation; molecular evolution; protein structure; structure-activity relationship; x-ray crystallography
Organisational unit
08816 - Kast, Peter (Tit.-Prof.)
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Funding
135651 - Exploring structure and function of unusual chorismate mutases from pathogens (SNF)
156453 - Exploring structure and function of atypical chorismate mutases from pathogens (SNF)
182648 - Exploring structure, function, and mechanism of atypical bacterial chorismate mutases (SNF)
156453 - Exploring structure and function of atypical chorismate mutases from pathogens (SNF)
182648 - Exploring structure, function, and mechanism of atypical bacterial chorismate mutases (SNF)