5MPV
Crystal structure of a Mycobacterium tuberculosis chorismate mutase optimized for high autonomous activity by directed evolution
Summary for 5MPV
| Entry DOI | 10.2210/pdb5mpv/pdb |
| Descriptor | Intracellular chorismate mutase (2 entities in total) |
| Functional Keywords | chorismate mutase, directed evolution, mycobacterium tuberculosis, isomerase |
| Biological source | Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) |
| Total number of polymer chains | 1 |
| Total formula weight | 10063.80 |
| Authors | Thorbjornsrud, H.V.,Kamarauskaite, J.,Kast, P.,Krengel, U. (deposition date: 2016-12-19, release date: 2018-08-01, Last modification date: 2024-02-07) |
| Primary citation | Fahrig-Kamarauskait, J.,Wurth-Roderer, K.,Thorbjornsrud, H.V.,Mailand, S.,Krengel, U.,Kast, P. Evolving the naturally compromised chorismate mutase from Mycobacterium tuberculosis to top performance. J.Biol.Chem., 295:17514-17534, 2020 Cited by PubMed 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 k/K 270-fold to 5 × 10ms, 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. Pro and Asp) 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. PubMed: 33453995DOI: 10.1074/jbc.RA120.014924 PDB entries with the same primary citation |
| Experimental method | X-RAY DIFFRACTION (1.49 Å) |
Structure validation
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