4A3U

X-structure of the old yellow enzyme homologue from zymomonas mobilis (NCR)

Summary for 4A3U

• Entry DOI: 10.2210/pdb4a3u/pdb
• Descriptor: NADH\:FLAVIN OXIDOREDUCTASE/NADH OXIDASE, FLAVIN MONONUCLEOTIDE, ACETATE ION, ... (7 entities in total)
• Functional Keywords: oxidoreductase
• Biological source: ZYMOMONAS MOBILIS
• Total number of polymer chains: 2
• Total formula weight: 80493.47

Authors

Hoeffken, H.W. (deposition date: 2011-10-04, release date: 2012-10-17, Last modification date: 2023-12-20)

Primary citation

Reich, S.,Hoeffken, H.W.,Rosche, B.,Nestl, B.M.,Hauer, B.
Crystal Structure Determination and Mutagenesis Analysis of the Ene Reductase Ncr.
Chembiochem, 13:2400-, 2012

PubMed Abstract: The crystal structure of the "ene" nicotinamide-dependent cyclohexenone reductase (NCR) from Zymomonas mobilis (PDB ID: 4A3U) has been determined in complex with acetate ion, FMN, and nicotinamide, to a resolution of 1.95 Å. To study the activity and enantioselectivity of this enzyme in the bioreduction of activated α,β-unsaturated alkenes, the rational design methods site- and loop-directed mutagenesis were applied. Based on a multiple sequence alignment of various members of the Old Yellow Enzyme family, eight single-residue variants were generated and investigated in asymmetric bioreduction. Furthermore, a structural alignment of various ene reductases predicted four surface loop regions that are located near the entrance of the active site. Four NCR loop variants, derived from loop-swapping experiments with OYE1 from Saccharomyces pastorianus, were analysed for bioreduction. The three enzyme variants, P245Q, D337Y and F314Y, displayed increased activity compared to wild-type NCR towards the set of substrates tested. The active-site mutation Y177A demonstrated a clear influence on the enantioselectivity. The loop-swapping variants retained reduction efficiency, but demonstrated decreased enzyme activity compared with the wild-type NCR ene reductase enzyme.

PubMed: 23033175
DOI: 10.1002/CBIC.201200404

Experimental method

X-RAY DIFFRACTION (1.7 Å)