6F53

CRYSTAL STRUCTURE OF KETOSTEROID ISOMERASE QUADRUPLE VARIANT V88I/L99V/D103S/V101A

6F53 の概要

• エントリーDOI: 10.2210/pdb6f53/pdb
• 関連するPDBエントリー: 6F4Y 6F50
• 分子名称: Steroid Delta-isomerase (2 entities in total)
• 機能のキーワード: isomerase, ksi
• 由来する生物種: Pseudomonas putida (Arthrobacter siderocapsulatus)
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 13735.58

構造登録者

Dunstan, M.,Currin, A. (登録日: 2017-11-30, 公開日: 2018-05-23, 最終更新日: 2024-05-08)

主引用文献

Currin, A.,Dunstan, M.S.,Johannissen, L.O.,Hollywood, K.A.,Vinaixa, M.,Jervis, A.J.,Swainston, N.,Rattray, N.J.W.,Gardiner, J.M.,Kell, D.B.,Takano, E.,Toogood, H.S.,Scrutton, N.S.
Engineering the "Missing Link" in Biosynthetic (-)-Menthol Production: Bacterial Isopulegone Isomerase.
ACS Catal, 8:2012-2020, 2018

PubMed Abstract: The realization of a synthetic biology approach to microbial (1,2,5)-()-menthol () production relies on the identification of a gene encoding an isopulegone isomerase (IPGI), the only enzyme in the biosynthetic pathway as yet unidentified. We demonstrate that Δ5-3-ketosteroid isomerase (KSI) from can act as an IPGI, producing ()-(+)-pulegone (()-) from (+)--isopulegone (). Using a robotics-driven semirational design strategy, we identified a key KSI variant encoding four active site mutations, which confer a 4.3-fold increase in activity over the wild-type enzyme. This was assisted by the generation of crystal structures of four KSI variants, combined with molecular modeling of binding to identify key active site residue targets. The KSI variant was demonstrated to function efficiently within cascade biocatalytic reactions with downstream enzymes pulegone reductase and (-)-menthone:(-)-menthol reductase to generate from . This study introduces the use of a recombinant IPGI, engineered to function efficiently within a biosynthetic pathway for the production of in microorganisms.

PubMed: 29750129
DOI: 10.1021/acscatal.7b04115

実験手法

X-RAY DIFFRACTION (1.49 Å)