Jordan Douglas B, Braker Jay D, Bowman Michael J, Vermillion Karl E, Moon Jaewoong, Liu Z Lewis
US Department of Agriculture, National Center of Agricultural Utilization Research, Peoria, IL, USA.
Biochim Biophys Acta. 2011 Dec;1814(12):1686-94. doi: 10.1016/j.bbapap.2011.08.011. Epub 2011 Aug 26.
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 NADPH-dependent conversion to corresponding alcohols, furfuryl alcohol (FFOH) and 5-hydroxymethylfurfuryl alcohol (HMFOH). At pH 7.0 and 25°C, purified Ari1p catalyzes the NADPH-dependent reduction of substrates with the following values (k(cat) (s(-1)), k(cat)/K(m) (s(-1)mM(-1)), K(m) (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 k(cat) of HMF and dl-glyceraldehyde that are independent of [NADPH] and (K(ia)(NADPH)/k(cat)) 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 k(cat) in the forward direction. Equilibrium constants, [NADP(+)][FFOH]/[NADPH][FFA][H(+)]=5600×10(7)M(-1) and [NADP(+)][HMFOH]/[NADPH][HMF][H(+)]=4200×10(7)M(-1), favor the physiological direction mirrored by the slowness of hydride transfer in the non-physiological direction, NADP(+)-dependent oxidation of alcohols (k(cat) (s(-1)), k(cat)/K(m) (s(-1)mM(-1)), K(m) (mM)): FFOH (0.221, 0.00158, 140) and HMFOH (0.0105, 0.000104, 101).
对于实现木质纤维素生物质高效发酵生产乙醇及其他产品而言，一种有效缓解呋喃醛（糠醛，FFA）和5-羟甲基糠醛（HMF）对发酵生物体毒性的方法至关重要。酿酒酵母中的醛还原酶Ari1p已被证明可通过催化NADPH依赖的反应将FFA和HMF转化为相应的醇类，即糠醇（FFOH）和5-羟甲基糠醇（HMFOH），从而减轻其毒性。在pH 7.0和25°C条件下，纯化后的Ari1p催化NADPH依赖的底物还原反应，其参数如下（k(cat) （s⁻¹），k(cat)/K(m) （s⁻¹mM⁻¹），K(m) （mM））：FFA（23.3，1.82，12.8），HMF（4.08，0.173，23.6），以及dl-甘油醛（2.40，0.0650，37.0）。当作用于HMF和dl-甘油醛时，该酶通过平衡有序动力学机制发挥作用。在反应的生理方向上，NADPH首先结合，NADP⁺最后从酶上解离，这通过HMF和dl-甘油醛的k(cat)与[NADPH]无关以及在饱和HMF或dl-甘油醛浓度下（K(ia)(NADPH)/k(cat)）外推至零得以证明。通过应用稳态、预稳态、动力学同位素效应和动力学建模方法，测定了HMF反应（HMF + NADPH ↔ HMFOH + NADP⁺）的微观动力学参数。产物HMFOH和NADP⁺的释放对正向反应的k(cat)而言，速率限制为84%。平衡常数，[NADP⁺][FFOH]/[NADPH][FFA][H⁺]=5600×10⁷M⁻¹以及[NADP⁺][HMFOH]/[NADPH][HMF][H⁺]=4200×10⁷M⁻¹，有利于生理方向，这反映在非生理方向（NADP⁺依赖的醇类氧化）中氢化物转移的缓慢上，其参数如下（k(cat) （s⁻¹），k(cat)/K(m) （s⁻¹mM⁻¹），K(m) （mM））：FFOH（0.221，0.00158，140）和HMFOH（0.0105，0.000104，101）。
