Zhang Xiaomei, Chen Guanjun, Liu Weifeng
The State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Shandong, China.
FEMS Microbiol Lett. 2009 Apr;293(2):214-9. doi: 10.1111/j.1574-6968.2009.01529.x. Epub 2009 Feb 23.
Genetic improvements of Zymomonas mobilis for pentose utilization have a huge potential in fuel ethanol production. The production of xylitol and the resulting growth inhibition by xylitol phosphate have been considered to be one of the important factors affecting the rates and yields from xylose metabolism by the recombinant Z. mobilis, but the mechanism of xylitol formation is largely unknown. Here, we reported that glucose-fructose oxidoreductase (GFOR), a periplasmic enzyme responsible for sorbitol production, catalyzed the reduction of xylose to xylitol in vitro, operating via a ping-pong mechanism similar to that in the formation of sorbitol. However, the specific activity of GFOR for sorbitol was higher than that for xylitol (68.39 vs. 1.102 micromol min(-1) mg(-1)), and an apparent substrate-induced positive cooperativity occurred during the catalyzed formation of xylitol, with the Hill coefficient being about 2. While a change of the potential acid-base catalyst Tyr269 to Phe almost completely abolished the activity toward xylose as well as fructose, mutant S116D, which has been shown to lose tight cofactor binding, displayed an even slower catalytic process against xylose.
运动发酵单胞菌在戊糖利用方面的基因改良在燃料乙醇生产中具有巨大潜力。木糖醇的产生以及由此产生的磷酸木糖醇对生长的抑制作用被认为是影响重组运动发酵单胞菌木糖代谢速率和产量的重要因素之一，但木糖醇形成的机制在很大程度上尚不清楚。在此，我们报道了葡萄糖 - 果糖氧化还原酶（GFOR），一种负责山梨醇产生的周质酶，在体外催化木糖还原为木糖醇，其作用机制类似于山梨醇形成过程中的乒乓机制。然而，GFOR对山梨醇的比活性高于对木糖醇的比活性（68.39对1.102微摩尔·分钟⁻¹·毫克⁻¹），并且在催化木糖醇形成过程中出现了明显的底物诱导正协同性，希尔系数约为2。虽然潜在的酸碱催化剂Tyr269突变为Phe几乎完全消除了对木糖以及果糖的活性，但已证明失去紧密辅因子结合的突变体S116D对木糖的催化过程甚至更慢。
