Heuts Dominic P H M, van Hellemond Erik W, Janssen Dick B, Fraaije Marco W
Laboratory of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
J Biol Chem. 2007 Jul 13;282(28):20283-91. doi: 10.1074/jbc.M610849200. Epub 2007 May 21.
A gene encoding an alditol oxidase was found in the genome of Streptomyces coelicolor A3(2). This newly identified oxidase, AldO, was expressed at extremely high levels in Escherichia coli when fused to maltose-binding protein. AldO is a soluble monomeric flavoprotein with subunits of 45.1 kDa, each containing a covalently bound FAD cofactor. From sequence alignments with other flavoprotein oxidases, it was found that AldO contains a conserved histidine (His(46)) that is typically involved in covalent FAD attachment. Covalent FAD binding is not observed in the H46A AldO mutant, confirming its role in covalent attachment of the flavin cofactor. Steady-state kinetic analyses revealed that wild-type AldO is active with several polyols. The alditols xylitol (K(m) = 0.32 mm, k(cat) = 13 s(-1)) and sorbitol (K(m) = 1.4 mm, k(cat) = 17 s(-1)) are the preferred substrates. From pre-steady-state kinetic analyses, using xylitol as substrate, it can be concluded that AldO mainly follows a ternary complex kinetic mechanism. Reduction of the flavin cofactor by xylitol occurs at a relatively high rate (99 s(-1)), after which a second kinetic event is observed, which is proposed to represent ring closure of the formed aldehyde product, yielding the hemiacetal of d-xylose. Reduced AldO readily reacts with molecular oxygen (1.7 x 10(5) m(-1) s(-1)), which confirms that the enzyme represents a true flavoprotein oxidase.
在天蓝色链霉菌A3(2)的基因组中发现了一个编码醛糖醇氧化酶的基因。这种新鉴定的氧化酶AldO与麦芽糖结合蛋白融合后在大肠杆菌中以极高水平表达。AldO是一种可溶性单体黄素蛋白，亚基分子量为45.1 kDa，每个亚基含有一个共价结合的FAD辅因子。通过与其他黄素蛋白氧化酶的序列比对发现，AldO含有一个保守的组氨酸（His(46)），它通常参与FAD的共价连接。在H46A AldO突变体中未观察到共价FAD结合，证实了其在黄素辅因子共价连接中的作用。稳态动力学分析表明，野生型AldO对几种多元醇有活性。木糖醇（K(m)=0.32 mM，k(cat)=13 s(-1)）和山梨醇（K(m)=1.4 mM，k(cat)=17 s(-1)）是其优选底物。从前稳态动力学分析来看，以木糖醇为底物，可以得出AldO主要遵循三元复合物动力学机制的结论。木糖醇对黄素辅因子的还原以相对较高的速率（99 s(-1)）发生，之后观察到第二个动力学事件，该事件被认为代表所形成醛产物的环化，生成d-木糖的半缩醛。还原型AldO很容易与分子氧反应（1.7×10(5) M(-1) s(-1)），这证实该酶是一种真正的黄素蛋白氧化酶。
