Vincenzo Carbone

Crystal structure of human L-xylulose reductase holoenzyme: probing the role of Asn107 with site-directed mutagenesis

L‐Xylulose reductase (XR), an enzyme in the uronate cycle of glucose metabolism, belongs to the short‐chain dehydrogenase/reductase (SDR) superfamily. Among the SDR enzymes, XR shows the highest sequence identity (67%) with mouse lung carbonyl reductase (MLCR), but the two enzymes show different substrate specificities. The crystal structure of human XR in complex with reduced nicotinamide adenine dinucleotide phosphate (NADPH) was determined at 1.96 Å resolution by using the molecular replacement method and the structure of MLCR as the search model. Features unique to human XR include electrostatic interactions between the N‐terminal residues of subunits related by the P‐axis, termed according to SDR convention, and an interaction between the hydroxy group of Ser185 and the pyrophosphate of NADPH. Furthermore, identification of the residues lining the active site of XR (Cys138, Val143, His146, Trp191, and Met200) together with a model structure of XR in complex with L‐xylulose, revealed structural differences with other members of the SDR family, which may account for the distinct substrate specificity of XR. The residues comprising a recently proposed catalytic tetrad in the SDR enzymes are conserved in human XR (Asn107, Ser136, Tyr149, and Lys153). To examine the role of Asn107 in the catalytic mechanism of human XR, mutant forms (N107D and N107L) were prepared. The two mutations increased Km for the substrate (>26‐fold) and Kd for NADPH (95‐fold), but only the N107L mutation significantly decreased kcat value. These results suggest that Asn107 plays a critical role in coenzyme binding rather than in the catalytic mechanism. Proteins 2004.

Structural and functional features of dimeric dihydrodiol dehydrogenase

Abstract.Dimeric dihydrodiol dehydrogenase (DD) catalyzes the NADP+-dependent oxidation of trans-dihydrodiols of aromatic hydrocarbons to their corresponding catechols. The tertiary structure of dimeric DD concists of a classical dinucleotide binding domain comprising two βαβαβ motifs at the N-terminus, and an eight-stranded, predominantly anti-parallel β-sheet, forming the C-terminal domain The aim of this review is to summarize the biochemical and structural properties of dimeric DD, compare it to enzymes that are structurally similar, and provide an insight into its catalytic mechanism and membership amongst a unique family of monomeric/oligomeric proteins that most likely share a common ancestry.

A Salicylic Acid-Based Analogue Discovered from Virtual Screening as a Potent Inhibitor of Human 20α-Hydroxysteroid Dehydrogenase

20alpha-hydroxysteroid dehydrogenase (AKR1C1) plays a key role in the metabolism of progesterone and other steroid hormones, thereby regulating their action at the pre-receptor level. AKR1C1 is implicated in neurological and psychiatric conditions such as catamenial epilepsy and depressive disorders. Increased activity of AKR1C1 is associated with termination of pregnancy and the development of breast cancer, endometriosis and endometrial cancer. Inhibition of the undesired activity of AKR1C1 will help reduce risks of premature birth, neurological disorders and the development of cancer. In order to identify potential leads for new inhibitors of AKR1C1 we adopted a virtual screening-based approach using the automated DOCK program. Approximately 250,000 compounds from the NCI database were screened for potential ligands based on their chemical complementarity and steric fit within the active site of AKR1C1. Kinetic analysis revealed 3,5-diiodosalicylic acid, an analogue of salicylic acid, as a potent competitive inhibitor with respect to the substrate 5beta-pregnane-3alpha,20alpha-diol with a K(i) of 9 nM. Aspirin, which is a well known salicylic acid-based drug, was also found to inhibit AKR1C1 activity. This is the first report to show aspirin (IC(50)=21 microM) and its metabolite salicylic acid (IC(50)=7.8 microM) as inhibitors of AKR1C1.

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.

Correlation of binding constants and molecular modelling of inhibitors in the active sites of aldose reductase and aldehyde reductase

Aldose reductase (ALR2) belongs to the aldo-keto reductase (AKR) superfamily of enzymes, is the first enzyme involved in the polyol pathway of glucose metabolism and has been linked to the pathologies associated with diabetes. Molecular modelling studies together with binding constant measurements for the four inhibitors Tolrestat, Minalrestat, quercetin and 3,5-dichlorosalicylic acid (DCL) were used to determine the type of inhibition, and correlate inhibitor potency and binding energies of the complexes with ALR2 and the homologous aldehyde reductase (ALR1), another member of the AKR superfamily. Our results show that the four inhibitors follow either uncompetitive or non-competitive inhibition pattern of substrate reduction for ALR1 and ALR2. Overall, there is correlation between the IC(50) (concentration giving 50% inhibition) values of the inhibitors for the two enzymes and the binding energies (DeltaH) of the enzyme-inhibitor complexes. Additionally, the results agree with the detailed structural information obtained by X-ray crystallography suggesting that the difference in inhibitor binding for the two enzymes is predominantly mediated by non-conserved residues. In particular, Arg312 in ALR1 (missing in ALR2) contributes favourably to the binding of DCL through an electrostatic interaction with the inhibitors electronegative halide atom and undergoes a conformational change upon Tolrestat binding. In ALR2, Thr113 (Tyr116 in ALR1) forms electrostatic interactions with the fluorobenzyl moiety of Minalrestat and the 3- and 4-hydroxy groups on the phenyl ring of quercetin. Our modelling studies suggest that Minalrestats binding to ALR1 is accompanied by a conformational change including the side chain of Tyr116 to achieve the selectivity for ALR1 over ALR2.

Structure of aldehyde reductase in ternary complex with a 5-arylidene-2,4-thiazolidinedione aldose reductase inhibitor

The structure of aldehyde reductase (ALR1) in ternary complex with the coenzyme NADPH and [5-(3-carboxymethoxy-4-methoxybenzylidene)-2,4-dioxothiazolidin-3-yl]acetic acid (CMD), a potent inhibitor of aldose reductase (ALR2), was determined at 1.99A resolution. The partially disordered inhibitor formed a tight network of hydrogen bonds with the active site residues (Tyr50 and His113) and coenzyme. Molecular modelling calculations and inhibitory activity measurements of CMD and [5-(3-hydroxy-4-methoxybenzylidene)-2,4-dioxothiazolidin-3-yl]acetic acid (HMD) indicated that pi-stacking interactions with several conserved active site tryptophan residues and hydrogen-bonding interactions with the non-conserved C-terminal residue Leu300 in ALR2 (Pro301 in ALR1) contributed to inhibitor selectivity. In particular for the potent inhibitor CMD, the rotameric state of the conserved residue Trp219 (Trp220 in ALR1) is important in forming a pi-stacking interaction with the inhibitor in ALR2 and contributes to the difference in the binding of the inhibitor to the enzymes.

Structures of dimeric dihydrodiol dehydrogenase apoenzyme and inhibitor complex: Probing the subunit interface with site‐directed mutagenesis

Dimeric dihydrodiol dehydrogenase (DD) catalyses the nicotinamide adenine dinucleotide phosphate (NADP+)‐dependent oxidation of trans‐dihydrodiols of aromatic hydrocarbons to their corresponding catechols. This is the first report of the crystal structure of the dimeric enzyme determined at 2.0 Å resolution. The tertiary structure is formed by a classical dinucleotide binding fold comprising of two βαβαβ motifs at the N‐terminus and an eight‐stranded, predominantly antiparallel β‐sheet at the C‐terminus. The active‐site of DD, occupied either by a glycerol molecule or the inhibitor 4‐hydroxyacetophenone, is located in the C‐terminal domain of the protein and maintained by a number of residues including Lys97, Trp125, Phe154, Leu158, Val161, Asp176, Leu177, Tyr180, Trp254, Phe279, and Asp280. The dimer interface is stabilized by a large number of intermolecular contacts mediated by the β‐sheet of each monomer, which includes an intricate hydrogen bonding network maintained in principal by Arg148 and Arg202. Site‐directed mutagenesis has demonstrated that the intact dimer is not essential for catalytic activity. The similarity between the quaternary structures of mammalian DD and glucose‐fructose oxidoreductase isolated from the prokaryotic organism Zymomonas mobilis suggests that both enzymes are members of a unique family of oligomeric proteins and may share a common ancestral gene. Proteins 2008.

Structure of aldehyde reductase in ternary complex with coenzyme and the potent 20α-hydroxysteroid dehydrogenase inhibitor 3,5-dichlorosalicylic acid: Implications for inhibitor binding and selectivity

The structure of aldehyde reductase (ALR1) in ternary complex with the coenzyme NADPH and 3,5-dichlorosalicylic acid (DCL), a potent inhibitor of human 20alpha-hydroxysteroid dehydrogenase (AKR1C1), was determined at a resolution of 2.41A. The inhibitor formed a network of hydrogen bonds with the active site residues Trp22, Tyr50, His113, Trp114 and Arg312. Molecular modelling calculations together with inhibitory activity measurements indicated that DCL was a less potent inhibitor of ALR1 (256-fold) when compared to AKR1C1. In AKR1C1, the inhibitor formed a 10-fold stronger binding interaction with the catalytic residue (Tyr55), non-conserved hydrogen bonding interaction with His222, and additional van der Waals contacts with the non-conserved C-terminal residues Leu306, Leu308 and Phe311 that contribute to the inhibitors selectivity advantage for AKR1C1 over ALR1.

Structure of 3(17)alpha-hydroxysteroid dehydrogenase (AKR1C21) holoenzyme from an orthorhombic crystal form: an insight into the bifunctionality of the enzyme.

Mouse 3(17)alpha-hydroxysteroid dehydrogenase (AKR1C21) is a bifunctional enzyme that catalyses the oxidoreduction of the 3- and 17-hydroxy/keto groups of steroid substrates such as oestrogens, androgens and neurosteroids. The structure of the AKR1C21-NADPH binary complex was determined from an orthorhombic crystal belonging to space group P2(1)2(1)2(1) at a resolution of 1.8 A. In order to identify the factors responsible for the bifunctionality of AKR1C21, three steroid substrates including a 17-keto steroid, a 3-keto steroid and a 3alpha-hydroxysteroid were docked into the substrate-binding cavity. Models of the enzyme-coenzyme-substrate complexes suggest that Lys31, Gly225 and Gly226 are important for ligand recognition and orientation in the active site.

Structure-based design of inhibitors of human L-xylulose reductase modelled into the active site of the enzyme

The program GRID was used to design potential inhibitors of human L-xylulose reductase based on a model of the holoenzyme in complex with n-butyric acid. The inclusion of phosphate or carboxylate functional groups in the ligand suggested an increase in the net binding energy of the complex up to 2.8- and 4.0-fold, respectively. This study may be useful in the development of potent and specific inhibitors of the enzyme.