Complete Reversal of Coenzyme Specificity of Xylitol Dehydrogenase and Increase of Thermostability by the Introduction of Structural Zinc

Abstract

Pichia stipitis NAD؉ -dependent xylitol dehydrogenase (XDH), a medium-chain dehydrogenase/reductase, is one of the key enzymes in ethanol fermentation from xylose. For the construction of an efficient biomass-ethanol conversion system, we focused on the two areas of XDH, 1) change of coenzyme specificity from NAD ؉ to NADP ؉ and 2) thermostabilization by introducing an additional zinc atom. Site-directed mutagenesis was used to examine the roles of Asp 207 ؉ -dependent XDH mutants constructed in this study decreased the thermostability compared with the wild-type enzyme, we attempted to improve the thermostability of XDH mutants by the introduction of an additional zinc atom. The introduction of three cysteine residues in wild-type XDH gave an additional zinc-binding site and improved the thermostability. The introduction of this mutation in D207A/I208R/F209S and D207A/I208R/F209S/N211R mutants increased the thermostability and further increased the catalytic activity with NADP ؉ .Xylose is one of the major components of hemicellulose, the second most abundant carbohydrate polymer in nature. Efficient utilization of xylose is required to develop economically viable processes for producing biofuels such as ethanol from biomass (for recent reviews, see Refs. 1-4). Yeasts have long been used for the production of alcoholic beverages such as wine, beer, and Japanese sake. In particular, Saccharomyces cerevisiae has been used widely because of the ability to produce high concentrations of ethanol and high inherent ethanol tolerance. The native strains, however, cannot ferment xylose as a carbon source. The major strategy for the generation of xylose-fermenting S. cerevisiae is to introduce genes involved in xylose metabolism from other organisms (Scheme 1). In xylosefermenting fungi such as Pichia stipitis, xylose is converted into xylulose by the sequential action of two oxidoreductases. First, xylose reductase (alditol:NADP ϩ 1-oxidoreductase, EC 1.1.1.21) catalyzes reduction of the C1 carbonyl group of xylose, yielding xylitol as the product. Xylitol is then oxidized by xylitol dehydrogenase (XDH 1 ; EC 1.1.1.9) to give xylulose. S. cerevisiae transformed with these two genes from P. stipitis could ferment xylose to ethanol. There is another problem; the excretion of xylitol occurs unless a co-metabolizable carbon source such as glucose is added. This is probably caused by several combined factors. In particular, intercellular redox imbalance due to a different coenzyme specificity of xylose reductase (with NADPH) and XDH (with NAD ϩ ) has been thought to be one of the main factors (2). The generation of an NADP ϩ -dependent XDH by protein engineering would avoid this problem.XDH is a medium-chain dehydrogenase/reductase (MDR), which constitutes a large enzyme superfamily consisting of about 1000 members (for reviews, see Refs. 5 and 6). The superfamily is classified into eight subfamilies based on amino acid sequence alignment and the structural similarity of substrates. XDH belongs to the polyol dehydrogenase ...

Computational design of Candida boidinii xylose reductase for altered cofactor specificity

“…Net charge, 65 hydrophobicity, 66 and side-chain volume 67 data for all amino acids were collected. A structural alignment was performed for the nicotinamide binding pockets targeted by mutational studies of the following proteins: glutathione reductase (GR), 31 ketol acid reductoisomerase (KARI), 32 p-hydroxybenzoate hydroxylase (PHBH), 34 2,5-diketo-D-gluconic acid (2,5-DKG), 29,30 1,5-anhydro-D-fructose reductase (1,5-AFR), 37 isocitrate dehydrogenase (IDH), 39 glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 46 P. stipitis xylitol dehydrogenase (PsXDH), 24 ferrodoxin: NADPþ reductase, 38 and L-lactate dehydrogenase (L-LDH). 42 The nicotinamide cofactor binding pockets of these proteins were aligned to the NADPH binding pocket of CtXR using Combinatorial Extension.…”

“…This double mutant showed potential as an efficient in vitro NAD(P)(H) regeneration system for reductive biocatalysis. 23 Watanabe et al 24 used site-directed mutagenesis to change cofactor specificity of a Pichia stipitis NAD þ -dependent xylitol dehydrogenase (PsXDH) from NAD þ to NADP þ as part of an efficient biomass-ethanol conversion system. Their designs yielded greater activity for NADP þ than NAD þ after redesign.…”

Generation of Xylose-Fermenting Saccharomyces Cerevisiae by Protein-Engineering

“…3) (Watanabe et al, 2005). Single substitutions produced a more positive effect on NADP + kinetics than those of NAD + ; their k cat /K m NADP values showed an approximately 5~48-fold increase (Fig.…”

Metabolic engineering of Saccharomyces cerevisiae for increased bioconversion of lignocellulose to ethanol

“…The glucose, xylose, xylitol, and ethanol concentrations were determined by high-performance liquid chromatography (HPLC) method as described by Walfridsson et al [12]. The separation was carried out with the using of an Aminex ion-exclusion HPX-87H cationexchange column (BioRad, USA) at 65°C with 5 mM H 2 SO 4 as the mobile phase.…”

“…The activity of XYL2 was determined by the method described previously [12]. The standard assay mixture for XYL2 activity contained 50 mM MgCl 2 and 300 mM xylitol in 50 mM Tris-HCl (pH 9.0) buffer.…”