Furfural reduction mechanism of a zinc‐dependent alcohol dehydrogenase from Cupriavidus necator JMP134

Abstract

Summary FurX is a tetrameric Zn-dependent alcohol dehydrogenase (ADH) from Cupriavidus necator JMP134. The enzyme rapidly reduces furfural with NADH as the reducing power. For the first time among characterized ADHs, the high-resolution structures of all reaction steps were obtained in a time-resolved manner, thereby illustrating the complete catalytic events of NADH-dependent reduction of furfural and the dynamic Zn2+ coordination among Glu66, water, substrate and product. In the fully closed conformation of the NADH complex, the catalytic turnover proved faster than observed for the partially closed conformation due to an effective proton transfer network. The domain motion triggered by NAD(H) association/dissociation appeared to facilitate dynamic interchanges in Zn2+ coordination with substrate and product molecules, ultimately increasing the enzymatic turnover rate. NAD+ dissociation appeared to be a slow process, involving multiple steps in concert with a domain opening and reconfiguration of Glu66. This agrees with the report that the cofactor is not dissociated from FurX during ethanol-dependent reduction of furfural, in which ethanol reduces NAD+ to NADH that is subsequently used for furfural reduction.

The Enzyme Activity and Substrate Specificity of Two Major Cinnamyl Alcohol Dehydrogenases in Sorghum (Sorghum bicolor), SbCAD2 and SbCAD4

“…Upon binding of NADPH to the apo-form SbCAD4 or its substrate binary complex, the resulting domain closure with concomitant dissociation of the carboxyl oxygen of Glu-70 from the Zn 2+ brings the carbonyl oxygen of the substrate into a proper position for Zn 2+ coordination, establishing a productive ternary complex (step C). As shown in the crystal structure of SbCAD4 and other MDHs such as FurX (Kang et al, 2012), in the event of NADPH association to the apoform SbCAD2/SbCAD4, the two domains become closed and a water or other oxygenic molecule in the solution, as shown in our crystal structures, will replace the Glu-70's Zn 2+ coordination (step B2). In the closed state, diffused-in coniferaldehyde replaces this water molecule and locates its carbonyl oxygen in the Figure 7.…”

A polyextremophilic alcohol dehydrogenase from the Atlantis II Deep Red Sea brine pool

“…The thermophilic htADH , the mesophilic FurX , and the psychrophilic MADH are among the closest homologous structures (Table S3). A comparison with the homology model of ADH/A1a revealed high structural similarities (RMSDs: 0.63–0.7 Å; Fig.…”

Evolution of a Biomass-Fermenting Bacterium To Resist Lignin Phenolics

“…Growth lags are proposed to be due to alcohol dehydrogenase (ADH) reducing furan, causing NADH depletion and acetaldehyde accumulation (17). Additionally, the ADH protein Cphy1179 shares 28% amino acid identity with a furfuralreducing, Zn-dependent ADH (18). However, if C. phytofermentans detoxifies furans, this mechanism is abruptly overwhelmed at concentrations above 2 g liter Ϫ1 5-HMF and 1 g liter Ϫ1 furfural.…”