4RF2

Crystal structure of NADP+ bound ketoreductase from Lactobacillus kefir

Summary for 4RF2

• Entry DOI: 10.2210/pdb4rf2/pdb
• Related: 4RF3 4RF4 4RF5
• Descriptor: NADPH dependent R-specific alcohol dehydrogenase, NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE, MAGNESIUM ION, ... (4 entities in total)
• Functional Keywords: oxidoreductase
• Biological source: Lactobacillus kefiri
• Total number of polymer chains: 2
• Total formula weight: 59521.04

Authors

Tang, Y.,Tibrewal, N.,Cascio, D. (deposition date: 2014-09-24, release date: 2015-09-30, Last modification date: 2023-09-20)

Primary citation

Noey, E.L.,Tibrewal, N.,Jimenez-Oses, G.,Osuna, S.,Park, J.,Bond, C.M.,Cascio, D.,Liang, J.,Zhang, X.,Huisman, G.W.,Tang, Y.,Houk, K.N.
Origins of stereoselectivity in evolved ketoreductases.
Proc.Natl.Acad.Sci.USA, 112:E7065-E7072, 2015

PubMed Abstract: Mutants of Lactobacillus kefir short-chain alcohol dehydrogenase, used here as ketoreductases (KREDs), enantioselectively reduce the pharmaceutically relevant substrates 3-thiacyclopentanone and 3-oxacyclopentanone. These substrates differ by only the heteroatom (S or O) in the ring, but the KRED mutants reduce them with different enantioselectivities. Kinetic studies show that these enzymes are more efficient with 3-thiacyclopentanone than with 3-oxacyclopentanone. X-ray crystal structures of apo- and NADP(+)-bound selected mutants show that the substrate-binding loop conformational preferences are modified by these mutations. Quantum mechanical calculations and molecular dynamics (MD) simulations are used to investigate the mechanism of reduction by the enzyme. We have developed an MD-based method for studying the diastereomeric transition state complexes and rationalize different enantiomeric ratios. This method, which probes the stability of the catalytic arrangement within the theozyme, shows a correlation between the relative fractions of catalytically competent poses for the enantiomeric reductions and the experimental enantiomeric ratio. Some mutations, such as A94F and Y190F, induce conformational changes in the active site that enlarge the small binding pocket, facilitating accommodation of the larger S atom in this region and enhancing S-selectivity with 3-thiacyclopentanone. In contrast, in the E145S mutant and the final variant evolved for large-scale production of the intermediate for the antibiotic sulopenem, R-selectivity is promoted by shrinking the small binding pocket, thereby destabilizing the pro-S orientation.

PubMed: 26644568
DOI: 10.1073/pnas.1507910112

Experimental method

X-RAY DIFFRACTION (2.089 Å)