Crystal Structure Determination and Mutagenesis Analysis of the Ene Reductase NCR

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.

Enhanced Ene‐Reductase Activity through Alteration of Artificial Nicotinamide Cofactor Substituents

“…The conversions of OYE1 and OYE3 did not match the product formation of NCR. This proves the literature‐known excellent activity of NCR . MR displayed a preference for NADH for all substrates (Table S2 in the Supporting Information).…”

“…The best cofactor was HPNAH for all substrates, with excellent conversions of >82 % after 20 min (Table ). Previous work on NCR with the natural cofactor NADH already showed a preference for the smaller substrates 5 and 6 (conversions >68 %) and lower conversions (<47 %) for the citral isomers 7 and 8 . Interestingly, the synthetic cofactors did not display significantly lower activity with any of the substrates.…”

Revealing Additional Stereocomplementary Pairs of Old Yellow Enzymes by Rational Transfer of Engineered Residues

“…Alignment was created with Clustal Omega, and sequences are sorted by pairwise identity. Structures of TsER (3HGJ, green), PETNR (3P81, yellow) and NCR (4A3U, cyan) were aligned with PyMol. Residues at hotspot positions are shown as sticks, FMN as lines.…”

Structural insights into stereospecific reduction of α, β-unsaturated carbonyl substrates by old yellow enzyme from Gluconobacter oxydans

“…Furthermore, both Gox0502 and NCR display monomeric form in solution, which are confirmed by analytic ultracentrifugation (AUC) (Fig. S1) and dynamic light scattering (DLS) experiments, 39) respectively. The key residues involved in catalytic activity, such as His172, Asn175, Tyr177, and key residues involved in substrate determination, such as Trp66 and Trp100, are highly conserved in both Gox0502 and NCR structures ( Fig.…”

“…1(A)). In addition, the critical residues Pro245, Asp337, and Phe314, whose mutations could enhance the catalytic activities in NCR, 39) are all conserved in the Gox0502 sequence. Such observations suggest that Gox0502 might be an authentic NCR targeting to cyclohexenone with specific "R" selectivity.…”