Xylitol dehydrogenase

Carbohydrate abnormalities, such as renal glycosuria (a transport defect), pentosuria (enzyme deficiency, xylitol dehydrogenase I. lactase deficiencies, fructose intolerance, galactosemia, galacloki-nase deficiency, oxalosis, and several glycogenoses (von Gierke s, Forbes . Andersen s, Hers s. and Tarui s diseases). 

D-3) Essential pentosuria (deficiency of xylitol dehydrogenase). A benign accumulation of xylulose develops, which may be confused with glucose when detected in the urine. 

L-Xylulose reductase (xylitol dehydrogenase) is deficient in essential pentosuria. L-Xylulose (a pentose) appears in the urine and gives a positive reducing-sugar test. The condition is benign. 

Fig. 5. Assimilation of D-xylose, L-arabinose and D-arabinose. In yeasts and fungi, pentoses are assimilated by way of oxidoreductases. D-xylose. L-arabinose and D-arabinose are each reduced to their respective polyols by aldose reductases, designated here as Xor, Lar and Dar. Both D-xylose and L-xylose are reduced to xylitol, which is symmetrical. D-xylose and L-arabinose are the forms normally found in nature. D- and L-arabitol dehydrogenases (Dad and Lad) form D- and L-xylulose, respectively. D- and L-Xylitol dehydrogenase (Dxd and Lxd) mediate the formation of D- and L-xylulose from xylitol...

The Pichia xylitol dehydrogenase gene chosen by us to be cloned into the Saccharomyces yeast is known to produce a xylitol dehydrogenase that catalyzes a reversible reaction between xylitol and xylulose as shown in Fig. 1 but favors the formation of xylitol rather than xylulose. Thus, an extremely strong xylulokinase activity will help to direct the carbon flux towards the production of ethanol rather than the formation of byproduct xylitol. 

Ethanol fermentation from xylose by yeasts can be divided into four distinctive steps. The first step is the reduction of xylose to xylitol mediated by NADPH/ NADH-linked xylose reductase (XR). This is followed by the oxidation of xylitol to xylulose, mediated by NAD-linked xylitol dehydrogenase (XDH). Xylulose-5-phosphate, the key intermediate, is generated from the phosphorylation of xylulose by xylulose kinase. Xylulose-5-phosphate is then channeled into the pentose phosphate pathway for further metabolism (Fig. 9). 

Xylose reductase and xylitol dehydrogenase have different cofactor requirements. 

Different nitrogen source (organic vs. inorganic) affects the level of xylitol dehydrogenase activity [82]. 

Xylitol dehydrogenase converts xylitol to the 2-ketopentose xylulose and the tetrameric enzyme from Galactocandida mastotermitis has been shown to possess one essential Zn " per monomer. As expected, binding is ordered with the cofactor binding first however, binding of carbohydrate is so weak that a Theorell-Chance kinetic mechanism obtains (he. one in which there is a bimolecular reaction between E.NAD and xylitol, without detectable E.NAD ". xylitol or E.NADEI.xylulose complexes). 

In fungi, xylose is reduced to xylitol by NADH- or NADPH-dependent xylose reductase (XR) and thereafter is oxidized to xylulose by NAD -dependent xylitol dehydrogenase (XDH). The xylulose is phosphorylated, channeled into the pentose phosphate pathway [3]. XR of most fungi, including most yeasts, prefers NADPH to NADH. Because of the cofactor preference of XR (NADPH) and XDH (NAD, redox imbalance occurs under anaerobic condition [4]. Therefore, the oxygen-limited rather than anaerobic condition is ideal for bioconversion of xylose to ethanol, so that the accumulated reduced cofactor can be oxidized to reach redox balance. A critical level of oxygen should exist for the highest ethanol yield and productivity. 

C. guilliermondii produces the enzyme D-xylose reductase which catalyses a reaction where the proton carrier NADPH donates a hydrogen atom to D-xylose, and D-xylose is converted to xylitol as seen in Fig. 1. The xylitol can then be converted to D-xylulose, catalyzed by xylitol dehydrogenase, which is utilized in central metabolism [3]. Under semi-aerobic conditions, xylitol accumulation is favored compared to anaerobic or aerobic conditions. Under anaerobic conditions, the ratio of NAD(P)H to NAD(P) is low, and NAD(P)H is required for xylitol production. Under aerobic conditions, excess oxygen allows oxidation of NADH to NAD", and a resulting high NAD /NADH ratio results in a faster xylitol conversion rate to D-xylulose, eliminating the accumulation of xylitol [4, 5]. 

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