Aldose reductase, which catalyzes the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of a wide variety of aromatic and aliphatic carbonyl compounds, is implicated in the development of diabetic and galactosemic complications involving the lens, retina, nerves, and kidney. A 1.65 angstrom refined structure of a recombinant human placenta aldose reductase reveals that the enzyme contains a parallel beta 8/alpha 8-barrel motif and establishes a new motif for NADP-binding oxidoreductases. The substrate-binding site is located in a large, deep elliptical pocket at the COOH-terminal end of the beta barrel with a bound NADPH in an extended conformation. The highly hydrophobic nature of the active site pocket greatly favors aromatic and apolar substrates over highly polar monosaccharides. The structure should allow for the rational design of specific inhibitors that might provide molecular understanding of the catalytic mechanism, as well as possible therapeutic agents.

Three SUMO (small ubiquitin-related modifier) genes have been identified in humans, which tag proteins to modulate subcellular localization and/or enhance protein stability and activity. We report the identification of a novel intronless SUMO gene, SUMO-4, that encodes a 95-amino acid protein having an 86% amino acid homology with SUMO-2. In contrast to SUMO-2, which is highly expressed in all of the tissues examined, SUMO-4 mRNA was detected mainly in the kidney. A single nucleotide polymorphism was detected in SUMO-4, substituting a highly conserved methionine with a valine residue (M55V). In HepG2 (liver carcinoma) cells transiently transfected with SUMO-4 expression vectors, Met-55 was associated with the elevated levels of activated heat shock factor transcription factors as compared with Val-55, whereas the levels of NF-B were suppressed to an identical degree. The SUMO-4M (Met) variant is associated with type I diabetes mellitus susceptibility in families (p ‫؍‬ 4.0 ؋ 10 ؊4 ), suggesting that it may be involved in the pathogenesis of type I diabetes. SUMO-11 is involved in the post-translational modification of cellular proteins and regulates various cellular processes such as nuclear transport, oncogenesis, stress response, inflammation, and the response to viral infection (1). SUMO-1 is related to ubiquitin, and although both share only an 18% sequence homology, they have a similar three-dimensional structure (1). In contrast to ubiquitin, which tags proteins for degradation, SUMO-1 seems to enhance protein stability and modulate the subcellular localization. The list of mammalian proteins, which are known to be sumoylated, is growing continually and includes RAN GTPase-activating protein 1 (nuclear import) (2), IB␣ (NF-B inhibition) (3), c-Jun (transcription factor), p53 (tumor suppressor) (4), GLUT1 and GLUT4 (glucose transport) (5), heat shock transcription factors 1 and 2 (HSF1 and 2) (6, 7), and FAS/APO-1 (cell death domain/ apoptosis) (8).SUMO-1 is expressed initially as an inactive precursor of 101 amino acids. A C-terminal proteolytic event exposes two glycine residues at the C terminus that are involved in the formation of a peptide bond with the ⑀-amino group of a lysine residue of a target protein. The activating enzymes E1 (AOS1 and UBA2) and the conjugating enzyme E2 (UBC9) are required for sumoylation in vitro (1). In vivo, an additional enzyme E3 is required for efficient sumoylation (9). The E2 enzyme, UBC9, is specific for sumoylation, and recently, two additional SUMO family members have been identified based on the interaction with UBC9, SUMO-2, and SUMO-3 (10, 11). SUMO-1 shares a 48% identity with SUMO-2 and a 46% with SUMO-3. Very little is known regarding the functions of SUMO-2 and SUMO-3. However, SUMO-2 and SUMO-3 contain a consensus SUMO modification site (Fig. 1), which enables self-sumoylation and the formation of polymeric chains (11), in contrast to SUMO-1. Thus, SUMO-2 and SUMO-3 may have different roles in cellular functions.Here, we identify and characterize a novel fou...

The active site of human aldose reductase contains two residues, His110 and Tyr48, either of which could be the proton donor during catalysis. Tyr48 is a candidate since its hydroxyl group is in proximity to Lys77 and thus may have an abnormally low pKa value. To distinguish between these possibilities, we used site-directed mutagenesis to create the H110Q and H110A, the Y48F, Y48H, and Y48S, and the K77M mutant enzymes. The two His110 mutants resulted in a 1000-20,000-fold drop in kcat/Km, respectively, for the reduction of DL-glyceraldehyde at pH 7. The Y48F mutation caused total loss of activity, whereas the Y48H and Y48S mutants retained catalytic activity with kcat/Km reduced by 5 orders of magnitude. The K77M mutant is an inactive enzyme. Kinetic studies using xylose stereoisomers show that the wild-type enzyme distinguishes between D-xylose, L-xylose, and D-lyxose up to 150-fold better than the H110A or H110Q mutants. The His110 mutants do not effectively discriminate between these isomers (4-11-fold). The crystal structure of the Y48H mutant refined at 1.8-A resolution shows that the overall structure is not significantly different from the wild-type structure. Electron densities for the histidine side chain and a new water molecule fill the space occupied by Tyr48 in the wild-type enzyme. The water molecule is in hydrogen-bonding distance to the N zeta group of Lys77 and to the N epsilon of His48 and fills the space occupied by the hydroxyl group of tyrosine in the wild-type structure. These findings suggest that proton transfer is mediated in the Y48H mutant enzyme by the water molecule. The Y48H mutant shows large and equal primary deuterium isotope effects on kcat and kcat/Km (1.81 +/- 0.03), providing direct evidence for hydride transfer as the rate-determining step in this mutant. Deuterium solvent isotope effects indicate that the relative contribution of proton transfer to this step of the catalytic cascade is much less important for the Y48H mutant than for the wild-type enzyme [D2O(kcat/Km) = 1.06 +/- 0.02 and 4.73 +/- 0.23, respectively]. The kinetic and mutagenesis data, together with structural data, indicate that His 110 plays an important role in the orientation of substrates in the active site pocket, while Tyr48 is the proton donor during aldehyde reduction by aldose reductase.

Aldose reductase is a NADPH-dependent aldo-keto reductase involved in the pathogenesis of some diabetic and galactosemic complications. The published crystal structure of human aldose reductase [Wilson et al. (1992) Science 257, 81-84] contains a hitherto unexplained electron density positioned within the active site pocket facing the nicotinamide ring of the NADPH and other key active site residues (Tyr48, His110, and Cys298). In this paper we identify the electron density as citrate, which is present in the crystallization buffer (pH 5.0), and provide confirmatory evidence by both kinetic and crystallographic experiments. Citrate is an uncompetitive inhibitor in the forward reaction with respect to aldehyde (reduction of aldehyde), while it is a competitive inhibitor with respect to alcohol in the backward reaction (oxidation of alcohol), indicating that it interacts with the enzyme-NADP(+)-product complex. Citrate can be replaced in the crystalline enzyme complex by cacodylate or glucose 6-phosphate; the structure of each of these complexes shows the specific molecule bound in the active site. All of the structures have been determined to a nominal resolution of 1.76 A and refined to R-factors below 18%. While cacodylate can be bound within the active site under the crystallization conditions, it does not inhibit the wild-type enzyme in solution. Glucose 6-phosphate, however, is a substrate for aldose reductase. The similar location of the negative charges of citrate, cacodylate, and glucose 6-phosphate within the active site suggests an anion-binding site delineated by the C4N of nicotinamide, the OH of Tyr48, and the N epsilon of His110. The location of citrate binding in the active site leads to a plausible catalytic mechanism for aldose reductase.

We have used transient kinetic data for partial reactions of recombinant human aldose reductase and simulations of progress curves for D-xylose reduction with NADPH and for xylitol oxidation with NADP+ to estimate rate constants for the following mechanism at pH 8.0: E<-->E.NADPH<-->*E.NADPH<-->*E.NADPH.RCHO<-->*E.NADP+.RCH2OH <-->*E.NADP+<--> E.NADP+<-->E. The mechanism includes kinetically significant conformational changes of the two binary E.nucleotide complexes which correspond to the movement of a crystallographically identified nucleotide-clamping loop involved in nucleotide exchange. The magnitude of this conformational clamping is substantial and results in a 100- and 650-fold lowering of the nucleotide dissociation constant in the productive *E.NADPH and *E.NADP+ complexes, respectively. The transient reduction of D-xylose displays burst kinetics consistent with the conformational change preceding NADP+ release (*E.NADP+-->E.NADP+) as the rate-limiting step in the forward direction. The maximum burst rate also displays a large deuterium isotope effect (Dkburst = 3.6-4.1), indicating that hydride transfer contributes significantly to rate limitation of the sequence of steps up to and including release of xylitol. In the reverse reaction, no burst of NADPH production is observed because the hydride transfer step is overall 85% rate-limiting. Even so, the conformational change preceding NADPH release (*E.NADPH-->E.NADPH) still contributes 15% to the rate limitation for reaction in this direction. The estimated rate constant for hydride transfer from NADPH to the aldehyde of D-xylose (130 s-1) is only 5- to 10-fold lower than the corresponding rate constant determined for NADH-dependent carbonyl reduction catalyzed by lactate or liver alcohol dehydrogenase. Hydride transfer from alcohol to NADP+ (0.6 s-1), however, is at least 100- to 1000-fold slower than NAD(+)-dependent alcohol oxidation mediated by these two enzymes, resulting in a bound-state equilibrium constant for aldose reductase which greatly favors the forward reaction. The proposed kinetic model provides a basic set of rate constants for interpretation of kinetic results obtained with aldose reductase mutants generated for the purpose of examining structure-function relationships of different components of the native enzyme.