4M5P

OYE2.6 Y78W, I113C

Summary for 4M5P

• Entry DOI: 10.2210/pdb4m5p/pdb
• Related: 3TJL
• Descriptor: NADPH dehydrogenase, FLAVIN MONONUCLEOTIDE, MALONIC ACID, ... (6 entities in total)
• Functional Keywords: tim barrel, alkene reductase, oxidoreductase
• Biological source: Scheffersomyces stipitis (Yeast)
• Total number of polymer chains: 1
• Total formula weight: 46294.17

Authors

Sullivan, B.,Stewart, J.D. (deposition date: 2013-08-08, release date: 2014-06-25, Last modification date: 2024-10-16)

Primary citation

Walton, A.Z.,Sullivan, B.,Patterson-Orazem, A.C.,Stewart, J.D.
Residues Controlling Facial Selectivity in an Alkene Reductase and Semirational Alterations to Create Stereocomplementary Variants.
ACS catalysis, 4:2307-2318, 2014

PubMed Abstract: A systematic saturation mutagenesis campaign was carried out on an alkene reductase from (OYE 2.6) to develop variants with reversed stereoselectivities. Wild-type OYE 2.6 reduces three representative Baylis-Hillman adducts to the corresponding products with almost complete stereoselectivities and good catalytic efficiencies. We created and screened 13 first-generation, site-saturation mutagenesis libraries, targeting residues found near the bound substrate. One variant (Tyr78Trp) showed high selectivity toward one of the three substrates, but no change (cyclohexenone derivative) and no catalytic activity (acrylate derivative) for the other two. Subsequent rounds of mutagenesis retained the Tyr78Trp mutation and explored other residues that impacted stereoselectivity when altered in a wild-type background. These efforts yielded double and triple mutants that possessed inverted stereoselectivities for two of the three substrates (conversions >99% and at least 91% ee ()). To understand the reasons underlying the stereochemical changes, we solved crystal structures of two key mutants: Tyr78Trp and Tyr78Trp/Ile113Cys, the latter with substrate partially occupying the active site. By combining these experimental data with modeling studies, we have proposed a rationale that explains the impacts of the most useful mutations.

PubMed: 25068071
DOI: 10.1021/cs500429k

Experimental method

X-RAY DIFFRACTION (1.503 Å)