5MPV

Crystal structure of a Mycobacterium tuberculosis chorismate mutase optimized for high autonomous activity by directed evolution

5MPV の概要

• エントリーDOI: 10.2210/pdb5mpv/pdb
• 分子名称: Intracellular chorismate mutase (2 entities in total)
• 機能のキーワード: chorismate mutase, directed evolution, mycobacterium tuberculosis, isomerase
• 由来する生物種: Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 10063.80

構造登録者

Thorbjornsrud, H.V.,Kamarauskaite, J.,Kast, P.,Krengel, U. (登録日: 2016-12-19, 公開日: 2018-08-01, 最終更新日: 2024-02-07)

主引用文献

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

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: 33453995
DOI: 10.1074/jbc.RA120.014924

実験手法

X-RAY DIFFRACTION (1.49 Å)