Porcine carbonyl reductase. structural basis for a functional monomer in short chain dehydrogenases/reductases

Porcine testicular carbonyl reductase (PTCR) belongs to the short chain dehydrogenases/reductases (SDR) superfamily and catalyzes the NADPH-dependent reduction of ketones on steroids and prostaglandins. The enzyme shares nearly 85% sequence identity with the NADPH-dependent human 15-hydroxyprostaglandin dehydrogenase/carbonyl reductase. The tertiary structure of the enzyme at 2.3 A reveals a fold characteristic of the SDR superfamily that uses a Tyr-Lys-Ser triad as catalytic residues, but exhibits neither the functional homotetramer nor the homodimer that distinguish all SDRs. It is the first known monomeric structure in the SDR superfamily. In PTCR, which is also active as a monomer, a 41-residue insertion immediately before the catalytic Tyr describes an all-helix subdomain that packs against interfacial helices, eliminating the four-helix bundle interface conserved in the superfamily. An additional anti-parallel strand in the PTCR structure also blocks the other strand-mediated interface. These novel structural features provide the basis for the scaffolding of one catalytic site within a single molecule of the enzyme.     

Comparative functional analysis of human medium-chain dehydrogenases, short-chain dehydrogenases/reductases and aldo-keto reductases with retinoids

Retinoic acid biosynthesis in vertebrates occurs in two consecutive steps: the oxidation of retinol to retinaldehyde followed by the oxidation of retinaldehyde to retinoic acid. Enzymes of the MDR (medium-chain dehydrogenase/reductase), SDR (short-chain dehydrogenase/reductase) and AKR (aldo-keto reductase) superfamilies have been reported to catalyse the conversion between retinol and retinaldehyde. Estimation of the relative contribution of enzymes of each type was difficult since kinetics were performed with different methodologies, but SDRs would supposedly play a major role because of their low K(m) values, and because they were found to be active with retinol bound to CRBPI (cellular retinol binding protein type I). In the present study we employed detergent-free assays and HPLC-based methodology to characterize side-by-side the retinoid-converting activities of human MDR [ADH (alcohol dehydrogenase) 1B2 and ADH4), SDR (RoDH (retinol dehydrogenase)-4 and RDH11] and AKR (AKR1B1 and AKR1B10) enzymes. Our results demonstrate that none of the enzymes, including the SDR members, are active with CRBPI-bound retinoids, which questions the previously suggested role of CRBPI as a retinol supplier in the retinoic acid synthesis pathway. The members of all three superfamilies exhibit similar and low K(m) values for retinoids (0.12-1.1 microM), whilst they strongly differ in their kcat values, which range from 0.35 min(-1) for AKR1B1 to 302 min(-1) for ADH4. ADHs appear to be more effective retinol dehydrogenases than SDRs because of their higher kcat values, whereas RDH11 and AKR1B10 are efficient retinaldehyde reductases. Cell culture studies support a role for RoDH-4 as a retinol dehydrogenase and for AKR1B1 as a retinaldehyde reductase in vivo.     

Molecular docking for substrate identification: the short-chain dehydrogenases/reductases

Protein ligand docking has recently been investigated as a tool for protein function identification, with some success in identifying both known and unknown substrates of proteins. However, identifying a protein's substrate when cross-docking a large number of enzymes and their cognate ligands remains a challenge. To explore a more limited yet practically important and timely problem in more detail, we have used docking for identifying the substrates of a single protein family with remarkable substrate diversity, the short-chain dehydrogenases/reductases.We examine different protocols for identifying candidate substrates for 27 short-chain dehydrogenase/reductase proteins of known catalytic function. We present the results of docking > 900 metabolites from the human metabolome to each of these proteins together with their known cognate substrates and products, and we investigate the ability of docking to (a) reproduce a viable binding mode for the substrate and (b) to rank the substrate highly amongst the dataset of other metabolites. In addition, we examine whether our docking results provide information about the nature of the substrate, based on the best-scoring metabolites in the dataset. We compare two different docking methods and two alternative scoring functions for one of the docking methods, and we attempt to rationalise both successes and failures.Finally, we introduce a new protocol, whereby we dock only a set of representative structures (medoids) to each of the proteins, in the hope of characterising each binding site in terms of its ligand preferences, with a reduced computational cost. We compare the results from this protocol with our original docking experiments, and we find that although the rank of the representatives correlates well with the mean rank of the clusters to which they belong, a simple structure-based clustering is too naïve for the purpose of substrate identification. Many clusters comprise ligands with widely varying affinities for the same protein; hence important candidates can be missed if a single representative is used.     

Molecular aspects of functional differences between alcohol and sorbitol dehydrogenases

The amino acid sequence of sheep liver sorbitol dehydrogenase has been fitted to the high-resolution model of the homologous horse liver alcohol dehydrogenase by computer graphics. This has allowed construction of a model of sorbitol dehydrogenase that provides explanations why sorbitol is not a substrate for alcohol dehydrogenase, why ethanol is not a substrate for sorbitol dehydrogenase, and what determines its specificity for polyols. An important feature of the model is that one of the ligands to the active site zinc atom is a glutamic acid residue instead of a cysteine residue, which is the corresponding ligand in the homologous alcohol dehydrogenases. This is one component of the structural change that can be related to the different substrate specificities, showing how altered enzymic activity might be brought about by structural changes of the kind that it is now possible to introduce by site-directed mutagenesis and recombinant DNA techniques.     

Critical residues for structure and catalysis in short-chain dehydrogenases/reductases

Short-chain dehydrogenases/reductases form a large, evolutionarily old family of NAD(P)(H)-dependent enzymes with over 60 genes found in the human genome. Despite low levels of sequence identity (often 10-30%), the three-dimensional structures display a highly similar alpha/beta folding pattern. We have analyzed the role of several conserved residues regarding folding, stability, steady-state kinetics, and coenzyme binding using bacterial 3beta/17beta-hydroxysteroid dehydrogenase and selected mutants. Structure determination of the wild-type enzyme at 1.2-A resolution by x-ray crystallography and docking analysis was used to interpret the biochemical data. Enzyme kinetic data from mutagenetic replacements emphasize the critical role of residues Thr-12, Asp-60, Asn-86, Asn-87, and Ala-88 in coenzyme binding and catalysis. The data also demonstrate essential interactions of Asn-111 with active site residues. A general role of its side chain interactions for maintenance of the active site configuration to build up a proton relay system is proposed. This extends the previously recognized catalytic triad of Ser-Tyr-Lys residues to form a tetrad of Asn-Ser-Tyr-Lys in the majority of characterized short-chain dehydrogenases/reductase enzymes.     

A structural account of substrate and inhibitor specificity differences between two naphthol reductases

Two short chain dehydrogenase/reductases mediate naphthol reduction reactions in fungal melanin biosynthesis. An X-ray structure of 1,3,6,8-tetrahydroxynaphthalene reductase (4HNR) complexed with NADPH and pyroquilon was determined for examining substrate and inhibitor specificities that differ from those of 1,3,8-trihydroxynaphthalene reductase (3HNR). The 1.5 A resolution structure allows for comparisons with the 1.7 A resolution structure of 3HNR complexed with the same ligands. The sequences of the two proteins are 46% identical, and they have the same fold. The 30-fold lower affinity of the 4HNR-NADPH complex for pyroquilon (a commercial fungicide that targets 3HNR) in comparison to that of the 3HNR-NADPH complex can be explained by unfavorable interactions between the anionic carboxyl group of the C-terminal Ile282 of 4HNR and CH and CH(2) groups of the inhibitor that are countered by favorable inhibitor interactions with 3HNR. 1,3,8-Trihydroxynaphthalene (3HN) and 1,3,6,8-tetrahydroxynaphthalene (4HN) were modeled onto the cyclic structure of pyroquilon in the 4HNR-NADPH-pyroquilon complex to examine the 300-fold preference of the enzyme for 4HN over 3HN. The models suggest that the C-terminal carboxyl group of Ile282 has a favorable hydrogen bonding interaction with the C6 hydroxyl group of 4HN and an unfavorable interaction with the C6 CH group of 3HN. Models of 3HN and 4HN in the 3HNR active site suggest a favorable interaction of the sulfur atom of the C-terminal Met283 with the C6 CH group of 3HN and an unfavorable one with the C6 hydroxyl group of 4HN, accounting for the 4-fold difference in substrate specificities. Thus, the C-terminal residues of the two naphthol reductase are determinants of inhibitor and substrate specificities.     

Chicken muscle aldose reductase: purification, properties and relationship to other chicken aldo/keto reductases

An enzyme that catalyzes the NADPH-dependent reduction of a wide range of aromatic and hydroxy-aliphatic aldehydes was purified from chicken breast muscle. This enzyme shares many properties with mammalian aldose reductases including molecular weight, relative substrate specificity, Michaelis constants, an inhibitor specificity. Therefore, it seems appropriate to call this enzyme an aldose reductase (EC 1.1.1.21). Chicken muscle aldose reductase appears to be kinetically identical to an aldose reductase that has been purified from chicken kidney (Hara et al., Eur. J. Biochem. 133, 207-214) and to hen muscle L-glycol dehydrogenase (Bernado et al., Biochim. biophys. Acta 659, 189-198). The association of this aldose reductase with muscular dystrophy in the chick is discussed.     

Structure of Toxoplasma gondii LDH1: active-site differences from human lactate dehydrogenases and the structural basis for efficient APAD+ use

While within a human host the opportunistic pathogen Toxoplasma gondii relies heavily on glycolysis for its energy needs. Lactate dehydrogenase (LDH), the terminal enzyme in anaerobic glycolysis necessary for NAD(+) regeneration, therefore represents an attractive therapeutic target. The tachyzoite stage lactate dehydrogenase (LDH1) from the parasite T. gondii has been crystallized in apo form and in ternary complexes containing NAD(+) or the NAD(+)-analogue 3-acetylpyridine adenine dinucleotide (APAD(+)) and sulfate or the inhibitor oxalate. Comparison of the apo and ternary models shows an active-site loop that becomes ordered upon substrate binding. This active-site loop is five residues longer than in most LDHs and necessarily adopts a different conformation. While loop isomerization is fully rate-limiting in prototypical LDHs, kinetic data suggest that LDH1's rate is limited by chemical steps. The importance of charge neutralization in ligand binding is supported by the complexes that have been crystallized as well as fluorescence quenching experiments performed with ligands at low and high pH. A methionine that replaces a serine residue and displaces an ordered water molecule often seen in LDH structures provides a structural explanation for reduced substrate inhibition. Superimposition of LDH1 with human muscle- and heart-specific LDH isoforms reveals differences in residues that line the active site that increase LDH1's hydrophobicity. These differences will aid in designing inhibitors specific for LDH1 that may be useful in treating toxoplasmic encephalitis and other complications that arise in immune-compromised individuals.     

Identification and characterization of retinoid-active short-chain dehydrogenases/reductases in Drosophila melanogaster

Background

In chordates, retinoid metabolism is an important target of short-chain dehydrogenases/reductases (SDRs). It is not known whether SDRs play a role in retinoid metabolism of protostomes, such as Drosophila melanogaster.

Methods

Drosophila genome was searched for genes encoding proteins with ∼ 50% identity to human retinol dehydrogenase 12 (RDH12). The corresponding proteins were expressed in Sf9 cells and biochemically characterized. Their phylogenetic relationships were analyzed using PHYLIP software.

Results

A total of six Drosophila SDR genes were identified. Five of these genes are clustered on chromosome 2 and one is located on chromosome X. The deduced proteins are 300 to 406 amino acids long and are associated with microsomal membranes. They recognize all-trans-retinaldehyde and all-trans-3-hydroxyretinaldehyde as substrates and prefer NADPH as a cofactor. Phylogenetically, Drosophila SDRs belong to the same branch of the SDR superfamily as human RDH12, indicating a common ancestry early in bilaterian evolution, before a protostome–deuterostome split.

Conclusions

Similarities in the substrate and cofactor specificities of Drosophila versus human SDRs suggest conservation of their function in retinoid metabolism throughout protostome and deuterostome phyla.

General significance

The discovery of Drosophila retinaldehyde reductases sheds new light on the conversion of β-carotene and zeaxantine to visual pigment and provides a better understanding of the evolutionary roots of retinoid-active SDRs.     

Molecular differences between rat-liver and rat-kidney biliverdin reductase. Implications for their in vivo regulation

Rat-liver biliverdin reductase exists in two molecular forms. The major form 1 has a molecular mass of 34 kDa, while the minor form 2 has a molecular mass of 56 kDa. Form 1 was converted into a second major form (form 3) with a molecular mass of 68 kDa by a NAD+-dependent peroxisomal dehydrogenase which was induced under conditions of oxidative stress [Frydman, R. B., Tomaro, M. L., Awruch, J. & Frydman, B. (1984) Biochem. Biophys. Res. Commun. 121, 249]. Molecular form 1 from rat kidney was not affected by the dehydrogenase, and a structural explanation for this difference was therefore sought. Both form 1 biliverdin reductases, isolated from rat liver and kidney, were purified to homogeneity using affinity chromatography, FPLC and HPLC techniques. The homogeneous enzymes were found to be identical when compared by their HPLC retention times, amino acid compositions and electrophoretic behaviour on polyacrylamide gels under non-denaturing conditions and on SDS/polyacrylamide gels. On HPLC analysis the peptides resulting from the CNBr cleavage were found to be the same for both enzymes, when either the native enzymes or their thioethylpyridine derivatives were compared. When the HPLC fingerprints of the tryptic digests were compared, they were found to be very similar, except for a peptide eluting at 31.60 min in the liver digest and at 23.60 min in the kidney digest. When the enzyme from both origins was alkylated with 4-dimethylaminoazobenzene-4'-iodoacetamide and then digested with trypsin, the HPLC fingerprints of the alkylated cysteine-carrying peptides were almost identical, except for a peptide with a retention time of 19.03 min in the liver digest and of 18.19 min in the kidney digest. The liver reductase was not amenable to Edman degradation suggesting a block at the NH2-terminus; in the kidney enzyme, however, it was free and an NH2-terminal sequence of 12 amino acids could be determined. The liver enzyme was found to be more sensitive toward p-hydroxymercuriphenyl sulfonate than the kidney enzyme.     

Extended superfamily of short alcohol-polyol-sugar dehydrogenases: structural similarities between glucose and ribitol dehydrogenases

The recently determined primary structure of glucose dehydrogenase from Bacillus megaterium was scanned by computerized comparisons for similarities with known polyol and alcohol dehydrogenases. The results revealed a highly significant similarity between this glucose dehydrogenase and ribitol dehydrogenase from Klebsiella aerogenes. Sixty-one positions of the 262 in glucose dehydrogenase are identical between these two proteins (23% identity), fitting into a homology alignment for the complete polypeptide chains. The extent of similarity is equivalent to that between other highly divergent but clearly related dehydrogenases (two zinc-containing alcohol dehydrogenases, 25% sorbitol and zinc-containing alcohol dehydrogenases, 25%; ribitol and non-zinc-containing alcohol dehydrogenases, 20%), and suggests an ancestral relationship between glucose and ribitol dehydrogenases from different bactera. The similarities fit into a previously suggested evolutionary scheme comprising short and long alcohol and polyol dehydrogenases, and greatly extend the former group to one composed of non-zinc-containing alcohol-polyol-glucose dehydrogenases.     

Molecular structural differences between type-2-diabetic and healthy glycogen

Glycogen is a highly branched glucose polymer functioning as a glucose buffer in animals. Multiple-detector size exclusion chromatography and fluorophore-assisted carbohydrate electrophoresis were used to examine the structure of undegraded native liver glycogen (both whole and enzymatically debranched) as a function of molecular size, isolated from the livers of healthy and db/db mice (the latter a type 2 diabetic model). Both the fully branched and debranched levels of glycogen structure showed fundamental differences between glycogen from healthy and db/db mice. Healthy glycogen had a greater population of large particles, with more α particles (tightly linked assemblages of smaller β particles) than glycogen from db/db mice. These structural differences suggest a new understanding of type 2 diabetes.     

Acyl-CoA dehydrogenases and acyl-CoA oxidases. Structural basis for mechanistic similarities and differences.

Acyl-CoA dehydrogenases and acyl-CoA oxidases are two closely related FAD-containing enzyme families that are present in mitochondria and peroxisomes, respectively. They catalyze the dehydrogenation of acyl-CoA thioesters to the corresponding trans-2-enoyl-CoA. This review examines the structure of medium chain acyl-CoA dehydrogenase, as a representative of the dehydrogenase family, with respect to the catalytic mechanism and its broad chain length specificity. Comparing the structures of four other acyl-CoA dehydrogenases provides further insights into the structural basis for the substrate specificity of each of these enzymes. In addition, the structure of peroxisomal acyl-CoA oxidase II from rat liver is compared to that of medium chain acyl-CoA dehydrogenase, and the structural basis for their different oxidative half reactions is discussed.     

Molecular and structural basis for N-glycan-dependent determination of glycoprotein fates in cells

Background

N-linked oligosaccharides operate as tags for protein quality control, consigning glycoproteins to different fates, i.e. folding in the endoplasmic reticulum (ER), vesicular transport between the ER and the Golgi complex, and ER-associated degradation of glycoproteins, by interacting with a panel of intracellular lectins in the early secretory pathway.

Scope of review

This review summarizes the current state of knowledge regarding the molecular and structural basis for glycoprotein-fate determination in cells that is achieved through the actions of the intracellular lectins and its partner proteins.

Major conclusions

Cumulative frontal affinity chromatography (FAC) data demonstrated that the intracellular lectins exhibit distinct sugar-binding specificity profiles. The glycotopes recognized by these lectins as fate determinants are embedded in the triantennary structures of the high-mannose-type oligosaccharides and are exposed upon trimming of the outer glucose and mannose residues during the N-glycan processing pathway. Furthermore, recently emerged 3D structural data offer mechanistic insights into functional interplay between an intracellular lectin and its binding partner in the early secretory pathway.

General significance

Structural biology approaches in conjunction with FAC methods provide atomic pictures of the mechanisms behind the glycoprotein-fate determination in cells. This article is a part of a Special issue entitled: Glycoproteomics.     

Molecular and structural basis for redox regulation of beta-actin

An essential consequence of growth factor-mediated signal transduction is the generation of intracellular H₂O₂. It operates as a second messenger in the control of actin microfilament dynamics, causing rapid and dramatic changes in the morphology and motile activity of stimulated cells. Little is understood about the molecular mechanisms causing these changes in the actin system. Here, it is shown that H₂O₂ acts directly upon several levels of this system, and some of the mechanistic effects are detailed. We describe the impact of oxidation on the polymerizability of non-muscle β/γ-actin and compare with that of muscle α-actin. Oxidation of β/γ-actin can cause a complete loss of polymerizability, crucially, reversible by the thioredoxin system. Further, oxidation of the actin impedes its interaction with profilin and causes depolymerization of filamentous actin. The effects of oxidation are critically dependent on the nucleotide state and the concentration of Ca²⁺. We have determined the crystal structure of oxidized β-actin to a resolution of 2.6 Å. The arrangement in the crystal implies an antiparallel homodimer connected by an intermolecular disulfide bond involving cysteine 374. Our data indicate that this dimer forms under non-polymerizing and oxidizing conditions. We identify oxidation of cysteine 272 in the crystallized actin dimer, likely to a cysteine sulfinic acid. In β/γ-actin, this is the cysteine residue most reactive towards H₂O₂ in solution, and we suggest plausible structural determinants for its reactivity. No other oxidative modification was obvious in the structure, highlighting the specificity of the oxidation by H₂O₂. Possible consequences of the observed effects in a cellular context and their potential relevance are discussed.     

Detection of structural differences between nuclear and mitochondrial glutamate dehydrogenases by the use of immunoadsorbents.

Structural differences between crystalline mitochondrial and nuclear glutamate dehydrogenases from ox liver have been detected by immunological techniques. Antisera prepared against each enzyme precipitate both glutamate dehydrogenases; upon immunodiffusion, the antiserum against the nuclear enzyme gives a line of incomplete identity with the two antigens, whereas the antiserum against the mitochondrial enzyme gives a line of complete identity. Fractionation of the antibodies contained in each antiserum by means of an immunoadsorbent, to which the nuclear or the mitochondrial enzyme has been covalently linked, shows that nuclear glutamate dehydrogenase (GDH) contains specific antigenic determinants as well as determinants common to the mitochondrial enzyme, whereas the latter appears to have no antigenic portions which are not present in the nuclear antigen, in accord with the results of immunodiffusion. The antibodies against determinants common to both enzymes precipitate and inhibit them, whereas the specific anti-nuclear GDH antibodies precipitate but do not inhibit the nuclear antigen.     

A new member of the short-chain dehydrogenases/reductases superfamily: Purification, characterization and substrate specificity of a recombinant carbonyl reductase from Pichia stipitis

A novel short-chain dehydrogenases/reductases superfamily (SDRs) reductase (PsCR) from Pichia stipitis that produced ethyl (S)-4-chloro-3-hydroxybutanoate with greater than 99% enantiomeric excess, was purified to homogeneity using fractional ammonium sulfate precipitation followed by DEAE-Sepharose chromatography. The enzyme purified from recombinant Escherichia coli had a molecular mass of about 35 kDa on SDS–PAGE and only required NADPH as an electron donor. The K_m value of PsCR for ethyl 4-chloro-3-oxobutanoate was 4.9 mg/mL and the corresponding V_max was 337 μmol/mg protein/min. The catalytic efficiency value was the highest ever reported for reductases from yeasts. Moreover, PsCR exhibited a medium-range substrate spectrum toward various keto and aldehyde compounds, i.e., ethyl-3-oxobutanoate with a chlorine substitution at the 2 or 4-position, or α,β-diketones. In addition, the activity of the enzyme was strongly inhibited by SDS and β-mercaptoethanol, but not by ethylene diamine tetra acetic acid.     

Molecular basis of the differences between normal and tumor tissues of gastric cancer

To be able to describe the differences between the normal and tumor tissues of gastric cancer at a molecular level would be essential in the study of the disease. We investigated the gene expression pattern in the two types of tissues from gastric cancer by performing expression profiling of 86 tissues on 17K complementary DNA microarrays. To select for the differentially expressed genes, class prediction algorithm was employed. For predictor selection, samples were first divided into a training (n=58), and a test set (n=28). A group of 894 genes was selected by a t-test in a training set, which was used for cross-validation in the training set and class (normal or tumor) prediction in the test set. Smaller groups of 894 genes were individually tested for their ability to correctly predict the normal or tumor samples based on gene expression pattern. The expression ratios of the 5 genes chosen from microarray data can be validated by real time RT-PCR over 6 tissue samples, resulting in a high level of correlation, individually or combined. When a representative predictor set of 92 genes was examined, pathways of 'focal adhesion' (with gene components of THBS2, PDGFD, MAPK1, COL1A2, COL6A3), 'ECM-receptor interaction' pathway (THBS2, COL1A2, COL6A3, FN1) and 'TGF-beta signaling' (THBS2, MAPK1, INHBA) represent some of the main differences between normal and tumor of gastric cancer at a molecular level.     

Mating interactions between two biotypes of the whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae) in Australia

The biological consequences of mating interactions between indigenous and exotic biotypes of Bemisia tabaci (Gennadius) in Australia were studied using a combination of field and laboratory experiments. The key results of the interaction between the B and eastern Australian biotypes were reduced population increase, a marked increase in the proportion of male progeny, fewer eggs produced by females paired with males of different biotype and no difference in the numbers of eggs per unmated female and females paired with males of the same biotype. In addition, there was no change in the proportion of eggs hatching, mixed biotype pairs spent more time courting than single biotype pairs and a low level of hybridization in field cages and small containers was observed. These observations suggest three possibilities. The first is the 'distracting male hypothesis' in which mating pairs made up of different biotypes apportion more time to courtship and less time to egg laying than single biotype pairs. The second invokes the 'single-locus complementary sex determination model' in which the production of non-viable diploid male zygotes may explain the reduction in eggs laid. The third is cytoplasmic incompatibility between biotypes caused by Wolbachia. The results also suggest that the geographical distribution of clusters of related biotypes both overseas and in Australia may be explained by between-biotype interactions leading to the formation of parapatric populations.