Syuhei Ishikura

Molecular cloning, expression and tissue distribution of hamster diacetyl reductase. Identity with L-xylulose reductase.

Using rapid amplification of cDNA ends PCR, a cDNA species for diacetyl reductase (EC 1.1.1.5) was isolated from hamster liver. The encoded protein consisted of 244 amino acids, and showed high sequence identity to mouse lung carbonyl reductase and hamster sperm P26h protein, which belong to the short-chain dehydrogenase/reductase family. The enzyme efficiently reduced L-xylulose as well as diacetyl, and slowly oxidized xylitol. The K(m) values for L-xylulose and xylitol were similar to those reported for L-xylulose reductase (EC 1.1.1.10) of guinea pig liver. The identity of diacetyl reductase with L-xylulose reductase was demonstrated by co-purification of the two enzyme activities from hamster liver and their proportional distribution in other tissues.

Identity of dimeric dihydrodiol dehydrogenase as NADP(+)-dependent D-xylose dehydrogenase in pig liver.

Dimeric dihydrodiol dehydrogenases (DDs, EC 1.3.1.20), which oxidize trans-dihydrodiols of aromatic hydrocarbons to the corresponding catechols, have been molecularly cloned from human intestine, monkey kidney, pig liver, dog liver, and rabbit lens. A comparison of the sequences with the DNA sequences in databases suggested that dimeric DDs constitute a novel protein family with 20 gene products. In addition, it was found that dimeric DD oxidizes several pentoses and hexoses, and the specificity resembles that of NADP(+)-dependent D-xylose dehydrogenase (EC 1.1.1.179) of pig liver. The inhibition of D-xylose dehydrogenase activity in the extracts of monkey kidney, dog liver and pig liver, its co-purification with dimeric DD activity from pig liver, and kinetic analysis of the D-xylose reduction by pig dimeric DD indicated that the two enzymes are the same protein.

Structure of the tetrameric form of human L-xylulose reductase : Probing the inhibitor-binding site with molecular modeling and site-directed mutagenesis

L‐Xylulose reductase (XR) is a member of the short‐chain dehydrogenase/reductase (SDR) superfamily. In this study we report the structure of the biological tetramer of human XR in complex with NADP+ and a competitive inhibitor solved at 2.3 Å resolution. A single subunit of human XR is formed by a centrally positioned, seven‐stranded, parallel β‐sheet surrounded on either side by two arrays of three α‐helices. Two helices located away from the main body of the protein form the variable substrate‐binding cleft, while the dinucleotide coenzyme‐binding motif is formed by a classical Rossmann fold. The tetrameric structure of XR, which is held together via salt bridges formed by the guanidino group of Arg203 from one monomer and the carboxylate group of the C‐terminal residue Cys244 from the neighboring monomer, explains the ability of human XR to prevent the cold inactivation seen in the rodent forms of the enzyme. The orientations of Arg203 and Cys244 are maintained by a network of hydrogen bonds and main‐chain interactions of Gln137, Glu238, Phe241, and Trp242. These interactions are similar to those defining the quaternary structure of the closely related carbonyl reductase from mouse lung. Molecular modeling and site‐directed mutagenesis identified the active site residues His146 and Trp191 as forming essential contacts with inhibitors of XR. These results could provide a structural basis in the design of potent and specific inhibitors for human XR. Proteins 2005.

Cloning and sequencing of the cDNA species for mammalian dimeric dihydrodiol dehydrogenases.

Cynomolgus and Japanese monkey kidneys, dog and pig livers and rabbit lens contain dimeric dihydrodiol dehydrogenase (EC 1.3.1.20) associated with high carbonyl reductase activity. Here we have isolated cDNA species for the dimeric enzymes by reverse transcriptase-PCR from human intestine in addition to the above five animal tissues. The amino acid sequences deduced from the monkey, pig and dog cDNA species perfectly matched the partial sequences of peptides digested from the respective enzymes of these animal tissues, and active recombinant proteins were expressed in a bacterial system from the monkey and human cDNA species. Northern blot analysis revealed the existence of a single 1.3 kb mRNA species for the enzyme in these animal tissues. The human enzyme shared 94%, 85%, 84% and 82% amino acid identity with the enzymes of the two monkey strains (their sequences were identical), the dog, the pig and the rabbit respectively. The sequences of the primate enzymes consisted of 335 amino acid residues and lacked one amino acid compared with the other animal enzymes. In contrast with previous reports that other types of dihydrodiol dehydrogenase, carbonyl reductases and enzymes with either activity belong to the aldo-keto reductase family or the short-chain dehydrogenase/reductase family, dimeric dihydrodiol dehydrogenase showed no sequence similarity with the members of the two protein families. The dimeric enzyme aligned with low degrees of identity (14-25%) with several prokaryotic proteins, in which 47 residues are strictly or highly conserved. Thus dimeric dihydrodiol dehydrogenase has a primary structure distinct from the previously known mammalian enzymes and is suggested to constitute a novel protein family with the prokaryotic proteins.

Crystallization and preliminary crystallographic analysis of human l-xylulose reductase

Human L-xylulose reductase was crystallized from buffered polyethylene glycol solutions using the hanging-drop vapour-diffusion method. The crystals diffract to 2.1 A resolution and belong to the orthorhombic P222 space group, with unit-cell parameters a = 72.9, b = 74.1, c = 87.9 A. This is the first crystallization report of a xylulose reductase that is identical to diacetyl reductase.

Crystallization and preliminary X-ray diffraction analysis of monkey dimeric dihydrodiol dehydrogenase

Dihydrodiol dehydrogenase catalyzes the NADP(+)-linked oxidation of trans-dihydrodiols of aromatic hydrocarbons to corresponding catechols and exists in multiple forms in mammalian tissues. The dimeric form of mammalian dihydrodiol dehydrogenase has a primary structure distinct from the previously known mammalian enzymes and may constitute a novel protein family with the prokaryotic proteins. Monkey kidney dimeric dihydrodiol dehydrogenase was crystallized from buffered ammonium phosphate solution using the hanging-drop vapour-diffusion method. The crystals diffract to 2.65 A resolution in the laboratory and belong to the hexagonal P6(1)22 or P6(5)22 space group, with unit-cell parameters a = b = 122.8, c = 121.3 A, alpha = beta = 90, gamma = 120 degrees.

Crystallization and preliminary X-ray diffraction analysis of mouse 3(17)α-hydroxysteroid dehydrogenase

Orthorhombic crystals of mouse 3(17)α-hydroxysteroid dehydrogenase were obtained from buffered polyethylene glycol solutions. The crystals diffracted to a resolution of 1.8 A at the Swiss Light Source beamline X06SA. The 3(17)α-hydroxysteroid dehydrogenase from mouse is involved in the metabolism of oestrogens, androgens, neurosteroids and xenobiotic compounds. The enzyme was crystallized by the hanging-drop vapour-diffusion method in space group P222{sub 1}, with unit-cell parameters a = 84.91, b = 84.90, c = 95.83 A. The Matthews coefficient (V{sub M}) and the solvent content were 2.21 A{sup 3} Da{sup −1} and 44.6%, respectively, assuming the presence of two molecules in the asymmetric unit. Diffraction data were collected to a resolution of 1.8 A at the Swiss Light Source beamline X06SA using a MAR CCD area detector and gave a data set with an overall R{sub merge} of 6.8% and a completeness of 91.1%.