Crystal structure of L-arabinose 1-dehydrogenase as a short-chain reductase/dehydrogenase protein.

l-Arabinose 1-dehydrogenase (AraDH) catalyzes the NAD(P)+-dependent oxidation of l-arabinose to L-arabinono-1,4-lactone in the non-phosphorylative l-arabinose pathway, and is classified into glucose-fructose oxidoreductase and short-chain dehydrogenase/reductase (SDR). We herein report the crystal structure of a SDR-type AraDH (from Herbaspirillum huttiense) for the first time. The interactions between Asp49 and the 2'- and 3'-hydroxyl groups of NAD+ were consistent with strict specificity for NAD+. In a binding model for the substrate, Ser155 and Tyr168, highly conserved in the SDR superfamily, interacted with the C1 and/or C2 hydroxyl(s) of l-arabinose, whereas interactions between Asp107, Arg109, and Gln206 and the C2 and/or C3 hydroxyl(s) were unique to AraDH. Trp200 significantly contributed to the selectivities of the C4 hydroxyl and C6 methyl of substrates.

Crystal structure of bacterial L-arabinose 1-dehydrogenase in complex with L-arabinose and NADP.

L-Arabinose 1-dehydrogenase (AraDH) is responsible for the first step of the non-phosphorylative L-arabinose pathway from bacteria, and catalyzes the NAD(P)+-dependent oxidation of L-arabinose to L-arabinonolactone. This enzyme belongs to the so-called Gfo/Idh/MocA protein superfamily, but has a very poor phylogenetic relationship with other functional members. We previously reported the crystal structures of AraDH without a ligand and in complex with NADP+. To clarify the underlying catalytic mechanisms in more detail, we herein elucidated the crystal structure in complex with L-arabinose and NADP+. In addition to the previously reported five amino acid residues (Lys91, Glu147, His153, Asp169, and Asn173), His119, Trp152, and Trp231 interacted with L-arabinose, which were not found in substrate recognition by other Gfo/Idh/MocA members. Structure-based site-directed mutagenic analyses suggested that Asn173 plays an important role in catalysis, whereas Trp152, Trp231, and His119 contribute to substrate binding. The preference of NADP+ over NAD+ was significantly subjected by a pair of Ser37 and Arg38, whose manners were similar to other Gfo/Idh/MocA members.

Molecular evolutionary insight of structural zinc atom in yeast xylitol dehydrogenases and its application in bioethanol production by lignocellulosic biomass.

Xylitol dehydrogenase (XDH) catalyzes the NAD+-dependent oxidization of xylitol into D-xylulose, and belongs to a zinc-dependent medium-chain dehydrogenase/reductase family. This protein family consists of enzymes with one or two zinc atoms per subunit, among which catalytic zinc is necessary for the activity. Among many XDHs from yeast and fungi, XDH from Pichia stipitis is one of the key enzymes for bioethanol production by lignocellulosic biomass, and possesses only a catalytic zinc atom. Despite its importance in bioindustry, a structural data of XDH has not yet been available, and little insight into the role of a second zinc atom in this protein family is known. We herein report the crystal structure of XDH from P. stipitis using a thermostabilized mutant. In the refined structure, a second zinc atom clearly coordinated with four artificially introduced cysteine ligands. Homologous mutations in XDH from Saccharomyces cerevisiae also stabilized and enhanced activity. The substitution of each of the four cysteine ligands with an aspartate in XDH from Schizosaccharomyces pombe contributed to the significantly better maintenance of activity and thermostability than their substitution with a serine, providing a novel hypothesis for how this zinc atom was eliminated.

Crystal structure of l-rhamnose 1-dehydrogenase involved in the nonphosphorylative pathway of l-rhamnose metabolism in bacteria.

Several microorganisms can utilize l-rhamnose as a carbon and energy source through the nonphosphorylative metabolic pathway, in which l-rhamnose 1-dehydrogenase (RhaDH) catalyzes the NAD(P)+ -dependent oxidization of l-rhamnose to l-rhamnono-1,4-lactone. We herein investigated the crystal structures of RhaDH from Azotobacter vinelandii in ligand-free, NAD+ -bound, NADP+ -bound, and l-rhamnose- and NAD+ -bound forms at 1.9, 2.1, 2.4, and 1.6 Å resolution, respectively. The significant interactions with the 2'-phosphate group of NADP+ , but not the 2'-hydroxyl group of NAD+ , were consistent with a preference for NADP+ over NAD+ . The C5-OH and C6-methyl groups of l-rhamnose were recognized by specific residues of RhaDH through hydrogen bonds and hydrophobic contact, respectively, which contribute to the different substrate specificities from other aldose 1-dehydrogenases in the short-chain dehydrogenase/reductase superfamily.