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Title:   Author  item
                                            Jordan, Douglasitem
                                            Braker, Jayitem
                                            Bowman, Michaelitem
                                            Vermillion, Karlitem
                                            Moon, Jaewoongitem
                                            Liu, Zonglin
  Submitted to: Biochimica et Biophysica Acta
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 8/2/2011
                                    Publication  Date: 11/1/2011
                                    Citation: Jordan, D.B., Braker, J.D., Bowman, M.J., Vermillion, K., Moon, J., Liu, Z. 2011. Kinetic mechanism of an aldehyde reductase of Saccharomyces cerevisiae that relieves toxicity of furfural and 5-hydroxymethylfurfural. Biochimica et Biophysica Acta. 1814:1686-1694.Interpretive Summary: Agricultural biomass including crop residues, grain processing byproducts, dedicated energy crops [e.g. switchgrass], etc., represent abundant, renewable feedstocks for production of ethanol and other valuable products if practical conversion technologies can be developed.  These materials are rich in complex carbohydrates that must first be broken down to simple sugars that can be fermented by microorganisms to ethanol and other products.  A barrier to achieving production of ethanol is the occurrence of furan aldehydes in biomass preparations, particularly those that have been exposed to thermal and acidic conditions. The aldehydes are toxic to fermenting organisms, including yeast, and at sublethal concentrations, the aldehydes interfere with fermentation. Previously, we have reported cloning and production of an enzyme that catalyzes reduction of furan aldehydes, producing the corresponding alcohol which is much less toxic.  Here, we provide detailed analysis on how the enzyme operates, which will aid future attempts in finding improvements.
                                Technical Abstract: An effective means of relieving the toxicity of furan aldehydes, furfural (FFA) and 5-hydroxymethylfurfural (HMF), on fermenting organisms is essential for achieving efficient fermentation of lignocellulosic biomass to ethanol and other products. Ari1p, an aldehyde reductase from Saccharomyces cerevisiae, has been shown to mitigate the toxicity of FFA and HMF by catalyzing the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent conversion to corresponding alcohols, furfuryl alcohol (FFOH) and 5-hydroxymethylfurfuryl alcohol (HMFOH). At pH 7.0 and 25 deg C, purified Ari1p catalyzes the NADPH-dependent reduction of substrates with the following values (kcat (s**-1), kcat/Km (s**-1mM**-1), Km (mM)): FFA (23.3, 1.82, 12.8), HMF (4.08, 0.173, 23.6), and DL-glyceraldehyde (2.40, 0.0650, 37.0). When acting on HMF and DL-glyceraldehyde, the enzyme operates through an equilibrium ordered kinetic mechanism. In the physiological direction of the reaction, NADPH binds first and NADP**+ dissociates from the enzyme last, demonstrated by kcat of HMF and DL-glyceraldehyde that are independent of [NADPH] and (Kia**NADPH/kcat) that extrapolate to zero at saturating HMF or DL-glyceraldehyde concentration. Microscopic kinetic parameters were determined for the HMF reaction (HMF + NADPH - HMFOH + NADP**+), by applying steady-state, presteady-state, kinetic isotope effects, and dynamic modeling methods. Release of products, HMFOH and NADP**+, is 84% rate limiting to kcat in the forward direction. Equilibrium constants, [NADP**+][FFOH]/[NADPH][FFA][H**+]=5600 x 10**7 M**-1 and [NADP**+][HMFOH]/[NADPH][HMF][H**+]=4200 x 10**7 M**-1, favor the physiological direction mirrored by the slowness of hydride transfer in the non-physiological direction, NADP**+-dependent oxidation of alcohols (kcat (s**-1), kcat/Kmb (s**-1mM**-1), Km (mM)): FFOH (0.221, 0.00158, 140) and HMFOH (0.0105, 0.000104, 101).


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