Leferink Nicole G H, Jose Mac Donald F, van den Berg Willy A M, van Berkel Willem J H
Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands.
FEBS Lett. 2009 Oct 6;583(19):3199-203. doi: 10.1016/j.febslet.2009.09.004. Epub 2009 Sep 6.
The flavoenzyme L-galactono-gamma-lactone dehydrogenase (GALDH) catalyzes the terminal step of vitamin C biosynthesis in plants. Little is known about the catalytic mechanism of GALDH and related aldonolactone oxidoreductases. Here we identified an essential Glu-Arg pair in the active site of GALDH from Arabidopsis thaliana. Glu386 and Arg388 variants show high K(m) values for L-galactono-1,4-lactone and low turnover rates. Arg388 is crucial for the stabilization of the anionic form of the reduced FAD cofactor. Glu386 is involved in productive substrate binding. The E386D variant has lost its specificity for L-galactono-1,4-lactone and shows the highest catalytic efficiency with L-gulono-1,4-lactone.
黄素酶L-半乳糖酸-γ-内酯脱氢酶（GALDH）催化植物中维生素C生物合成的最后一步。关于GALDH和相关醛糖酸内酯氧化还原酶的催化机制知之甚少。在这里，我们在拟南芥GALDH的活性位点鉴定出一对必需的Glu-Arg。Glu386和Arg388变体对L-半乳糖酸-1,4-内酯表现出高K(m)值和低周转率。Arg388对于还原型FAD辅因子阴离子形式的稳定至关重要。Glu386参与有效的底物结合。E386D变体失去了对L-半乳糖酸-1,4-内酯的特异性，对L-古洛糖酸-1,4-内酯表现出最高的催化效率。
