Structure and mechanism of human UDP-glucose 6-dehydrogenase.

Elevated production of the matrix glycosaminoglycan hyaluronan is strongly implicated in epithelial tumor progression. Inhibition of synthesis of the hyaluronan precursor UDP-glucuronic acid (UDP-GlcUA) therefore presents an emerging target for cancer therapy. Human UDP-glucose 6-dehydrogenase (hUGDH) catalyzes, in two NAD(+)-dependent steps without release of intermediate aldehyde, the biosynthetic oxidation of UDP-glucose (UDP-Glc) to UDP-GlcUA. Here, we present a structural characterization of the hUGDH reaction coordinate using crystal structures of the apoenzyme and ternary complexes of the enzyme bound with UDP-Glc/NADH and UDP-GlcUA/NAD(+). The quaternary structure of hUGDH is a disc-shaped trimer of homodimers whose subunits consist of two discrete α/ß domains with the active site located in the interdomain cleft. Ternary complex formation is accompanied by rigid-body and restrained movement of the N-terminal NAD(+) binding domain, sequestering substrate and coenzyme in their reactive positions through interdomain closure. By alternating between conformations in and out of the active site during domain motion, Tyr(14), Glu(161), and Glu(165) participate in control of coenzyme binding and release during 2-fold oxidation. The proposed mechanism of hUGDH involves formation and breakdown of thiohemiacetal and thioester intermediates whereby Cys(276) functions as the catalytic nucleophile. Stopped-flow kinetic data capture the essential deprotonation of Cys(276) in the course of the first oxidation step, allowing the thiolate side chain to act as a trap of the incipient aldehyde. Because thiohemiacetal intermediate accumulates at steady state under physiological reaction conditions, hUGDH inhibition might best explore ligand binding to the NAD(+) binding domain.

Structural basis of fumarate hydratase deficiency.

Fumarate hydratase catalyzes the stereospecific hydration across the olefinic double bond in fumarate leading to L-malate. The enzyme is expressed in mitochondrial and cytosolic compartments, and participates in the Krebs cycle in mitochondria, as well as in regulation of cytosolic fumarate levels. Fumarate hydratase deficiency is an autosomal recessive trait presenting as metabolic disorder with severe encephalopathy, seizures and poor neurological outcome. Heterozygous mutations are associated with a predisposition to cutaneous and uterine leiomyomas and to renal cancer. The crystal structure of human fumarate hydratase shows that mutations can be grouped into two distinct classes either affecting structural integrity of the core enzyme architecture, or are localized around the enzyme active site. An interactive version of this manuscript (which may contain additional mutations appended after acceptance of this manuscript) may be found on the SSIEM website at: http://www.ssiem.org/resources/structures/FH .

The crystal structure of human GLRX5: iron-sulfur cluster co-ordination, tetrameric assembly and monomer activity.

Human GLRX5 (glutaredoxin 5) is an evolutionarily conserved thiol-disulfide oxidoreductase that has a direct role in the maintenance of normal cytosolic and mitochondrial iron homoeostasis, and its expression affects haem biosynthesis and erythropoiesis. We have crystallized the human GLRX5 bound to two [2Fe-2S] clusters and four GSH molecules. The crystal structure revealed a tetrameric organization with the [2Fe-2S] clusters buried in the interior and shielded from the solvent by the conserved ß1-α2 loop, Phe69 and the GSH molecules. Each [2Fe-2S] cluster is ligated by the N-terminal activesite cysteine (Cys67) thiols contributed by two protomers and two cysteine thiols from two GSH. The two subunits co-ordinating the cluster are in a more extended conformation compared with iron-sulfur-bound human GLRX2, and the intersubunit interactions are more extensive and involve conserved residues among monothiol GLRXs. Gel-filtration chromatography and analytical ultracentrifugation support a tetrameric organization of holo-GLRX5, whereas the apoprotein is monomeric. MS analyses revealed glutathionylation of the cysteine residues in the absence of the [2Fe-2S] cluster, which would protect them from further oxidation and possibly facilitate cluster transfer/acceptance. Apo-GLRX5 reduced glutathione mixed disulfides with a rate 100 times lower than did GLRX2 and was active as a glutathione-dependent electron donor for mammalian ribonucleotide reductase.

UDP-glucose dehydrogenase: structure and function of a potential drug target.

Biosynthesis of the glycosaminoglycan precursor UDP-α-D-glucuronic acid occurs through a 2-fold oxidation of UDP-α-D-glucose that is catalysed by UGDH (UDP-α-D-glucose 6-dehydrogenase). Structure-function relationships for UGDH and proposals for the enzymatic reaction mechanism are reviewed in the present paper, and structure-based sequence comparison is used for subclassification of UGDH family members. The eukaryotic group of enzymes (UGDH-II) utilize an extended C-terminal domain for the formation of complex homohexameric assemblies. The comparably simpler oligomerization behaviour of the prokaryotic group of enzymes (UGDH-I), in which dimeric forms prevail, is traced back to the lack of relevant intersubunit contacts and trimmings within the C-terminal region. The active site of UGDH contains a highly conserved cysteine residue, which plays a key role in covalent catalysis. Elevated glycosaminoglycan formation is implicated in a variety of human diseases, including the progression of tumours. The inhibition of synthesis of UDP-α-D-glucuronic acid using UGDH antagonists might therefore be a useful strategy for therapy.

Targeting a uniquely nonspecific prenyl synthase with bisphosphonates to combat cryptosporidiosis.

Cryptosporidiosis is a neglected disease without a wholly effective drug. We present a study demonstrating nitrogen-containing bisphosphonates (N-BPs) to be capable of inhibiting Cryptosporidium parvum at low micromolar concentrations in infected MDCK cells. Predictably, the mechanism of action is based on inhibition of biosynthesis of isoprenoids but the target enzyme is unexpectedly a distinctive C. parvum enzyme dubbed nonspecific polyprenyl pyrophosphate synthase (CpNPPPS). This enzyme produces various isoprenoid products larger than FPP and is inhibited by N-BPs at subnanomolar concentrations. It is part of an isoprenoid pathway in Cryptosporidium distinctly different from other organisms. The proposed mechanism of action is corroborated by crystal structures of the enzyme with risedronate and zoledronate bound showing how this enzyme's unique chain length determinant region enables it to accommodate larger substrates and products. These results, combined with existing data on their clinical use, demonstrate that N-BPs are very promising anticryptosporidial drug candidates.

SALMON: solvent accessibility, ligand binding, and mapping of ligand orientation by NMR spectroscopy.

Quinone oxidoreductase 2 (NQO2) binds the prodrug tretazicar (also known as CB1954, 5-(aziridin-1-yl)-2,4-dinitrobenzamide), which exhibits a profound antitumor effect in human cancers when administered together with caricotamide. X-ray structure determination allowed for two possible orientations of the ligand. Here we describe a new NMR method, SALMON (solvent accessibility, ligand binding, and mapping of ligand orientation by NMR spectroscopy), based on waterLOGSY to determine the orientation of a ligand bound to a protein by mapping its solvent accessibility, which was used to unambiguously determine the orientation of CB1954 in NQO2.

Bisphosphonates: an update on mechanisms of action and how these relate to clinical efficacy.

The bisphosphonates (BPs) are well established as the treatments of choice for disorders of excessive bone resorption, including Paget's disease of bone, myeloma and bone metastases, and osteoporosis. There is considerable new knowledge about how BPs work. Their classical pharmacological effects appear to result from two key properties: their affinity for bone mineral and their inhibitory effects on osteoclasts. Mineral binding affinities differ among the clinically used BPs and may influence their differential distribution within bone, their biological potency, and their duration of action. The inhibitory effects of the nitrogen-containing BPs (including alendronate, risedronate, ibandronate, and zoledronate) on osteoclasts appear to result from their inhibition of farnesyl pyrophosphate synthase (FPPS), a key branch-point enzyme in the mevalonate pathway. FPPS generates isoprenoid lipids used for the posttranslational modification of small GTP-binding proteins essential for osteoclast function. Effects on other cellular pathways, such as preventing apoptosis in osteocytes, are emerging as other potentially important mechanisms of action. As a class, BPs share several common properties. However, as with other classes of drugs, there are obvious chemical, biochemical, and pharmacological differences among the various individual BPs. Each BP has a unique profile that may help to explain potential important clinical differences among the BPs, in terms of speed of onset of fracture reduction, antifracture efficacy at different skeletal sites, and the degree and duration of suppression of bone turnover. As we approach the 40th anniversary of the discovery of their biological effects, there remain further opportunities for using their properties for medical purposes.

SDR-type human hydroxysteroid dehydrogenases involved in steroid hormone activation.

Hydroxysteroid dehydrogenases catalyze the NAD(P)(H)-dependent oxidoreduction of hydroxyl and oxo-functions at distinct positions of steroid hormones. This reversible reaction constitutes an important pre-receptor control mechanism for nuclear receptor ligands of the androgen, estrogen and glucocorticoid classes, since the conversion "switches" between receptor ligands and their inactive metabolites. The major reversible activities found in mammals acting on steroid hormones comprise 3alpha-, 11beta- and 17beta-hydroxysteroid dehydrogenases, and for each group several distinct isozymes have been described. The enzymes differ in their expression pattern, nucleotide cofactor preference, steroid substrate specificity and subcellular localization, and thus constitute a complex system ensuring cell-specific adaptation and regulation of steroid hormone levels. Several isoforms constitute promising drug targets, of particular importance in cancer, metabolic diseases, neurodegeneration and immunity.

Reversible sequestration of active site cysteines in a 2Fe-2S-bridged dimer provides a mechanism for glutaredoxin 2 regulation in human mitochondria.

Human mitochondrial glutaredoxin 2 (GLRX2), which controls intracellular redox balance and apoptosis, exists in a dynamic equilibrium of enzymatically active monomers and quiescent dimers. Crystal structures of both monomeric and dimeric forms of human GLRX2 reveal a distinct glutathione binding mode and show a 2Fe-2S-bridged dimer. The iron-sulfur cluster is coordinated through the N-terminal active site cysteine, Cys-37, and reduced glutathione. The structures indicate that the enzyme can be inhibited by a high GSH/GSSG ratio either by forming a 2Fe-2S-bridged dimer that locks away the N-terminal active site cysteine or by binding non-covalently and blocking the active site as seen in the monomer. The properties that permit GLRX2, and not other glutaredoxins, to form an iron-sulfur-containing dimer are likely due to the proline-to-serine substitution in the active site motif, allowing the main chain more flexibility in this area and providing polar interaction with the stabilizing glutathione. This appears to be a novel use of an iron-sulfur cluster in which binding of the cluster inactivates the protein by sequestering active site residues and where loss of the cluster through changes in subcellular redox status creates a catalytically active protein. Under oxidizing conditions, the dimers would readily separate into iron-free active monomers, providing a structural explanation for glutaredoxin activation under oxidative stress.

The crystal structure of human geranylgeranyl pyrophosphate synthase reveals a novel hexameric arrangement and inhibitory product binding.

Modification of GTPases with isoprenoid molecules derived from geranylgeranyl pyrophosphate or farnesyl pyrophosphate is an essential requisite for cellular signaling pathways. The synthesis of these isoprenoids proceeds in mammals through the mevalonate pathway, and the final steps in the synthesis are catalyzed by the related enzymes farnesyl pyrophosphate synthase and geranylgeranyl pyrophosphate synthase. Both enzymes play crucial roles in cell survival, and inhibition of farnesyl pyrophosphate synthase by nitrogen-containing bisphosphonates is an established concept in the treatment of bone disorders such as osteoporosis or certain forms of cancer in bone. Here we report the crystal structure of human geranylgeranyl pyrophosphate synthase, the first mammalian ortholog to have its x-ray structure determined. It reveals that three dimers join together to form a propeller-bladed hexameric molecule with a mass of approximately 200 kDa. Structure-based sequence alignments predict this quaternary structure to be restricted to mammalian and insect orthologs, whereas fungal, bacterial, archaeal, and plant forms exhibit the dimeric organization also observed in farnesyl pyrophosphate synthase. Geranylgeranyl pyrophosphate derived from heterologous bacterial expression is tightly bound in a cavity distinct from the chain elongation site described for farnesyl pyrophosphate synthase. The structure most likely represents an inhibitory complex, which is further corroborated by steady-state kinetics, suggesting a possible feedback mechanism for regulating enzyme activity. Structural comparisons between members of this enzyme class give deeper insights into conserved features important for catalysis.

Structure and function of human 17beta-hydroxysteroid dehydrogenases.

17Beta-hydroxysteroid dehydrogenases (17beta-HSDs) catalyze the NAD(P)(H) dependent oxidoreduction at C17 oxo/beta-hydroxyl groups of androgen and estrogen hormones. This reversible reaction constitutes an important pre-receptor control mechanism for nuclear receptor ligands, since the conversion "switches" between the 17beta-OH receptor ligands and their inactive 17-oxo metabolites. At present, 14 mammalian 17beta-HSDs are described, of which at least 11 exist within the human genome, encoded by different genes. The enzymes differ in their expression pattern, nucleotide cofactor preference, steroid substrate specificity and subcellular localization, and thus constitute a complex system ensuring cell-specific adaptation and regulation of sex steroid hormone levels. Broad and overlapping substrate specificities with enzymes involved in lipid metabolism suggest interactions of several 17beta-HSDs with other metabolic pathways. Several 17beta-HSDs enzymes constitute promising drug targets, of particular importance in cancer, metabolic diseases, neurodegeneration and possibly immunity.

Structure of human phytanoyl-CoA 2-hydroxylase identifies molecular mechanisms of Refsum disease.

Refsum disease (RD), a neurological syndrome characterized by adult onset retinitis pigmentosa, anosmia, sensory neuropathy, and phytanic acidaemia, is caused by elevated levels of phytanic acid. Many cases of RD are associated with mutations in phytanoyl-CoA 2-hydroxylase (PAHX), an Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes the initial alpha-oxidation step in the degradation of phytenic acid in peroxisomes. We describe the x-ray crystallographic structure of PAHX to 2.5 A resolution complexed with Fe(II) and 2OG and predict the molecular consequences of mutations causing RD. Like other 2OG oxygenases, PAHX possesses a double-stranded beta-helix core, which supports three iron binding ligands (His(175), Asp(177), and His(264)); the 2-oxoacid group of 2OG binds to the Fe(II) in a bidentate manner. The manner in which PAHX binds to Fe(II) and 2OG together with the presence of a cysteine residue (Cys(191)) 6.7 A from the Fe(II) and two further histidine residues (His(155) and His(281)) at its active site distinguishes it from that of the other human 2OG oxygenase for which structures are available, factor inhibiting hypoxia-inducible factor. Of the 15 PAHX residues observed to be mutated in RD patients, 11 cluster in two distinct groups around the Fe(II) (Pro(173), His(175), Gln(176), Asp(177), and His(220)) and 2OG binding sites (Trp(193), Glu(197), Ile(199), Gly(204), Asn(269), and Arg(275)). PAHX may be the first of a new subfamily of coenzyme A-binding 2OG oxygenases.

The coenzyme specificity of Candida tenuis xylose reductase (AKR2B5) explored by site-directed mutagenesis and X-ray crystallography.

CtXR (xylose reductase from the yeast Candida tenuis; AKR2B5) can utilize NADPH or NADH as co-substrate for the reduction of D-xylose into xylitol, NADPH being preferred approx. 33-fold. X-ray structures of CtXR bound to NADP+ and NAD+ have revealed two different protein conformations capable of accommodating the presence or absence of the coenzyme 2'-phosphate group. Here we have used site-directed mutagenesis to replace interactions specific to the enzyme-NADP+ complex with the aim of engineering the co-substrate-dependent conformational switch towards improved NADH selectivity. Purified single-site mutants K274R (Lys274-->Arg), K274M, K274G, S275A, N276D, R280H and the double mutant K274R-N276D were characterized by steady-state kinetic analysis of enzymic D-xylose reductions with NADH and NADPH at 25 degrees C (pH 7.0). The results reveal between 2- and 193-fold increases in NADH versus NADPH selectivity in the mutants, compared with the wild-type, with only modest alterations of the original NADH-linked xylose specificity and catalytic-centre activity. Catalytic reaction profile analysis demonstrated that all mutations produced parallel effects of similar magnitude on ground-state binding of coenzyme and transition state stabilization. The crystal structure of the double mutant showing the best improvement of coenzyme selectivity versus wild-type and exhibiting a 5-fold preference for NADH over NADPH was determined in a binary complex with NAD+ at 2.2 A resolution.

Structure of xylose reductase bound to NAD+ and the basis for single and dual co-substrate specificity in family 2 aldo-keto reductases.

The co-ordinates reported have been submitted to the Protein Data Bank under accession number 1MI3. Xylose reductase (XR; AKR2B5) is an unusual member of aldo-keto reductase superfamily, because it is one of the few able to efficiently utilize both NADPH and NADH as co-substrates in converting xylose into xylitol. In order to better understand the basis for this dual specificity, we have determined the crystal structure of XR from the yeast Candida tenuis in complex with NAD(+) to 1.80 A resolution (where 1 A=0.1 nm) with a crystallographic R -factor of 18.3%. A comparison of the NAD(+)- and the previously determined NADP(+)-bound forms of XR reveals that XR has the ability to change the conformation of two loops. To accommodate both the presence and absence of the 2'-phosphate, the enzyme is able to adopt different conformations for several different side chains on these loops, including Asn(276), which makes alternative hydrogen-bonding interactions with the adenosine ribose. Also critical is the presence of Glu(227) on a short rigid helix, which makes hydrogen bonds to both the 2'- and 3'-hydroxy groups of the adenosine ribose. In addition to changes in hydrogen-bonding of the adenosine, the ribose unmistakably adopts a 3'- endo conformation rather than the 2'- endo conformation seen in the NADP(+)-bound form. These results underscore the importance of tight adenosine binding for efficient use of either NADH or NADPH as a co-substrate in aldo-keto reductases. The dual specificity found in XR is also an important consideration in designing a high-flux xylose metabolic pathway, which may be improved with an enzyme specific for NADH.