Aldose reductases (ARs) belonging to the aldo‐keto reductase (AKR) superfamily catalyze the conversion of carbonyl substrates into their respective alcohols. Here we report the crystal structures of the yeast Debaryomyces nepalensis xylose reductase (DnXR, AKR2B10) in the apo form and as a ternary complex with a novel NADP‐DTT adduct. Xylose reductase, a key enzyme in the conversion of xylose to xylitol, has several industrial applications. The enzyme displayed the highest catalytic efficiency for l‐threose (138 ± 7 mm−1·s−1) followed by d‐erythrose (30 ± 3 mm−1·s−1). The crystal structure of the complex reveals a covalent linkage between the C4N atom of the nicotinamide ring of the cosubstrate and the S1 sulfur atom of DTT and provides the first structural evidence for a protein mediated NADP–low‐molecular‐mass thiol adduct. We hypothesize that the formation of the adduct is facilitated by an in‐crystallo Michael addition of the DTT thiolate to the specific conformation of bound NADPH in the active site of DnXR. The interactions between DTT, a four‐carbon sugar alcohol analog, and the enzyme are representative of a near‐cognate product ternary complex and provide significant insights into the structural basis of aldose binding and specificity and the catalytic mechanism of ARs. Database Structural data are available in the PDB under the accession numbers http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5ZCI and http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5ZCM.

Aldose reductase (AR) catalyzes the conversion of aldoses to their corresponding polyols in yeasts and filamentous fungi. ARs have the potential to be exploited for the enzymatic production of xylitol, thus the identification and characterization of ARs from novel strains have gained interest. In this study, we chose the novel yeast Debaryomyces nepalensis as a source for an AR gene. For the first time, here we isolated the AR gene from D. nepalensis (DnAR) that encodes a protein of 320 amino acids with a predicted molecular weight of 36.7 kDa using the RACE technique. It was heterologously expressed in Escherichia coli as a His-tagged fusion protein and purified. The enzyme showed strict NADPH dependence and broad substrate specificity with high catalytic efficiency for arabinose, xylose and 3-nitro benzaldehyde.Remarkably, it was active and stable in the presence of high concentrations of salts (KCl/NaCl), thus exhibiting halotolerance. It showed 75% and 45% activity at 0.5 and 1 M concentration of salts respectively. Enzyme half-lifetime at 1 M KCl and 1 M NaCl was found to be 30 h and 16.5 h respectively. Furthermore, to explore the structural basis of its halotolerance, we built a homology model of DnAR.Surprisingly, we found that the existence of a uniform negative electrostatic potential over the protein surface, which is one of the known mechanisms governing protein halotolerance. Therefore, DnAR could be exploited as a biocatalyst to develop an enzyme based bioprocess for xylitol production from lignocelluloses. Moreover, this is the first report providing the genetic sequence and biochemical characteristics of a halotolerant aldose reductase.

Lignocellulosic materials are one of the most abundant renewable resources whose exploitation for the production of biochemicals and biofuels is the major challenge in the area of industrial biotechnology due to inhibition of growth and product formation by the toxic compounds released upon their hydrolysis. Indeed the bioprocess that can produce industrial products from hemicellulose hydrolysates in the presence of toxic compounds is economical than the process which involves detoxification. In this study, the ability of halotolerant strain Debaryomyces nepalensis NCYC 3413 to convert non-detoxified xylose enriched hemicellulose hydrolysates from corn cobs, rice straw, sugarcane bagasse and wheat straw to xylitol was evaluated. It was found that this strain has the capability to grow in all hemicellulose hydrolysates and convert xylose to xylitol without detoxification of hydrolysates. The maximum xylitol concentration of 14.6 g L -1 was obtained from corn cobs and wheat straw with productivities of 0.16 and 0.20 g L -1 h -1 respectively at a yield of 0.30 g g -1 . Whereas sugarcane bagasse and rice straw gave xylitol yields of 0.31 and 0.32 g g -1 respectively with 14.2 g L -1 maximum xylitol and productivities were calculated to be 0.20 and 0.15 g L -1 h -1respectively. The presence of high glucose hindered xylitol production by producing ethanol. Based on our findings, we suggest that (i) D. nepalensis is a promising strain for ecofriendly xylitol production as it exhibits broad specificity to lignocellulose substrates, fermentation of mixed sugars and (ii) tolerance towards lignocellulosic inhibitors making the process more economical.

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