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An engineered old yellow enzyme that enables efficient synthesis of (4R,6R)-Actinol in a one-pot reduction system.

Horita S, Kataoka M, Kitamura N, Nakagawa T, Miyakawa T, Ohtsuka J, Nagata K, Shimizu S, Tanokura M
Chembiochem 16 440-5 (2015)
Cited: 10 times
EuropePMC logo PMID: 25639703

Abstract

(4R,6R)-Actinol can be stereo-selectively synthesized from ketoisophorone by a two-step conversion using a mixture of two enzymes: Candida macedoniensis old yellow enzyme (CmOYE) and Corynebacterium aquaticum (6R)-levodione reductase. However, (4S)-phorenol, an intermediate, accumulates because of the limited substrate range of CmOYE. To address this issue, we solved crystal structures of CmOYE in the presence and absence of a substrate analogue p-HBA, and introduced point mutations into the substrate-recognition loop. The most effective mutant (P295G) showed two- and 12-fold higher catalytic activities toward ketoisophorone and (4S)-phorenol, respectively, than the wild-type, and improved the yield of the two-step conversion from 67.2 to 90.1%. Our results demonstrate that the substrate range of an enzyme can be changed by introducing mutation(s) into a substrate-recognition loop. This method can be applied to the development of other favorable OYEs with different substrate preferences.

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Reviews citing this publication (2)

  1. What are the Limitations of Enzymes in Synthetic Organic Chemistry? Reetz MT. Chem Rec 16 2449-2459 (2016)
  2. Enzymes useful for chiral compound synthesis: structural biology, directed evolution, and protein engineering for industrial use. Kataoka M, Miyakawa T, Shimizu S, Tanokura M. Appl Microbiol Biotechnol 100 5747-5757 (2016)

Articles citing this publication (6)

  1. Discovery, Characterisation, Engineering and Applications of Ene Reductases for Industrial Biocatalysis. Toogood HS, Scrutton NS. ACS Catal 8 3532-3549 (2019)
  2. Revealing Additional Stereocomplementary Pairs of Old Yellow Enzymes by Rational Transfer of Engineered Residues. Nett N, Duewel S, Richter AA, Hoebenreich S. Chembiochem 18 685-691 (2017)
  3. Flavin-dependent biocatalysts in synthesis. Baker Dockrey SA, Narayan ARH. Tetrahedron 75 1115-1121 (2019)
  4. Selectivity through discriminatory induced fit enables switching of NAD(P)H coenzyme specificity in Old Yellow Enzyme ene-reductases. Iorgu AI, Hedison TM, Hay S, Scrutton NS. FEBS J 286 3117-3128 (2019)
  5. Structural basis of different substrate preferences of two old yellow enzymes from yeasts in the asymmetric reduction of enone compounds. Horita S, Kataoka M, Kitamura N, Miyakawa T, Ohtsuka J, Maejima Y, Shimomura K, Nagata K, Shimizu S, Tanokura M. Biosci Biotechnol Biochem 83 456-462 (2019)
  6. Structural investigation into the C-terminal extension of the ene-reductase from Ralstonia (Cupriavidus) metallidurans. Opperman DJ. Proteins 85 2252-2257 (2017)


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