6F54

CRYSTAL STRUCTURE OF KETOSTEROID ISOMERASE TRIPLE VARIANT V88I/L99VD103S

Summary for 6F54

• Entry DOI: 10.2210/pdb6f54/pdb
• Related: 6F4Y 6F50 6F53
• Descriptor: Steroid Delta-isomerase (2 entities in total)
• Functional Keywords: isomerase, ksi
• Biological source: Pseudomonas putida (Arthrobacter siderocapsulatus)
• Total number of polymer chains: 2
• Total formula weight: 27527.28

Authors

Dunstan, M.,Currin, A. (deposition date: 2017-11-30, release date: 2018-05-23, Last modification date: 2024-05-08)

Primary citation

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

Experimental method

X-RAY DIFFRACTION (1.08 Å)