5YW4

Structure-Guided Engineering of Reductase: Efficient Attenuating Substrate Inhibition in Asymmetric Catalysis

Summary for 5YW4

• Entry DOI: 10.2210/pdb5yw4/pdb
• Descriptor: Protein induced by osmotic stress, NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE (3 entities in total)
• Functional Keywords: reductase, nadp+, asymmetric catalysis, oxidoreductase
• Biological source: Scheffersomyces stipitis (Yeast)
• Total number of polymer chains: 1
• Total formula weight: 37749.27

Authors

Shang, Y.P.,Chen, Q.,Li, A.T.,Yu, H.L.,Xu, J.H. (deposition date: 2017-11-28, release date: 2019-03-06, Last modification date: 2023-11-22)

Primary citation

Shang, Y.P.,Chen, Q.,Li, A.T.,Quan, S.,Xu, J.H.,Yu, H.L.
Attenuated substrate inhibition of a haloketone reductase via structure-guided loop engineering.
J.Biotechnol., 308:141-147, 2020

PubMed Abstract: Substrate inhibition of enzymes is one of the main obstacles encountered frequently in industrial biocatalysis. Haloketone reductase SsCR was seriously inhibited by substrate 2,2',4'-trichloroacetophenone. In this study, two essential loops were found that have a relationship with substrate binding by conducting X-ray crystal structure analysis. Three key residues were selected from the tips of the loops and substituted with amino acids with lower hydrophobicity to weaken the hydrophobic interactions that bridge the two loops, resulting in a remarkable reduction of substrate inhibition. Among these variants, L211H showed a significant attenuation of substrate inhibition, with a K of 16 mM, which was 16 times that of the native enzyme. The kinetic parameter k/K of L211H was 3.1 × 10 s mM, showing the comparable catalytic efficiency to that of the wild-type enzyme (WT). At the substrate loading of 100 mM, the space time yield of variant L211H in asymmetric reduction of the haloketone was 3-fold higher than that of the WT.

PubMed: 31866427
DOI: 10.1016/j.jbiotec.2019.12.011

Experimental method

X-RAY DIFFRACTION (2.047 Å)