Short-chain Dehydrogenase Research Articles

Insight into S-adenosylmethionine biosynthesis from the crystal structures of the human methionine adenosyltransferase catalytic and regulatory subunits

MAT (methionine adenosyltransferase) utilizes L-methionine and ATP to form SAM (S-adenosylmethionine), the principal methyl donor in biological methylation. Mammals encode a liver-specific isoenzyme, MAT1A, that is genetically linked with an inborn metabolic disorder of hypermethioninaemia, as well as a ubiquitously expressed isoenzyme, MAT2A, whose enzymatic activity is regulated by an associated subunit MAT2B. To understand the molecular mechanism of MAT functions and interactions, we have crystallized the ligand-bound complexes of human MAT1A, MAT2A and MAT2B. The structures of MAT1A and MAT2A in binary complexes with their product SAM allow for a comparison with the Escherichia coli and rat structures. This facilitates the understanding of the different substrate or product conformations, mediated by the neighbouring gating loop, which can be accommodated by the compact active site during catalysis. The structure of MAT2B reveals an SDR (short-chain dehydrogenase/reductase) core with specificity for the NADP/H cofactor, and harbours the SDR catalytic triad (YxxxKS). Extended from the MAT2B core is a second domain with homology with an SDR sub-family that binds nucleotide-sugar substrates, although the equivalent region in MAT2B presents a more open and extended surface which may endow a different ligand/protein-binding capability. Together, the results of the present study provide a framework to assign structural features to the functional and catalytic properties of the human MAT proteins, and facilitate future studies to probe new catalytic and binding functions.

Disruption of the histidine triad nucleotide-binding hint2 gene in mice affects glycemic control and mitochondrial function

The histidine triad nucleotide-binding (HINT2) protein is a mitochondrial adenosine phosphoramidase expressed in the liver and pancreas. Its physiological function is unknown. To elucidate the role of HINT2 in liver physiology, the mouse Hint2 gene was deleted. Hint2(-/-) and Hint2(+/+) mice were generated in a mixed C57Bl6/J × 129Sv background. At 20 weeks, the phenotypic changes in Hint2(-/-) relative to Hint2(+/+) mice were an accumulation of hepatic triglycerides, decreased tolerance to glucose, a defective counter-regulatory response to insulin-provoked hypoglycemia, and an increase in plasma interprandial insulin but a decrease in glucose-stimulated insulin secretion and defective thermoregulation upon fasting. Leptin messenger RNA (mRNA) in adipose tissue and plasma leptin were elevated. In mitochondria from Hint2(-/-) hepatocytes, state 3 respiration was decreased, a finding confirmed in HepG2 cells where HINT2 mRNA was silenced. The linked complex II-III electron transfer was decreased in Hint2(-/-) mitochondria, which was accompanied by a lower content of coenzyme Q. Hypoxia-inducible factor-2α expression and the generation of reactive oxygen species were increased. Electron microscopy of mitochondria in Hint2(-/-) mice aged 12 months revealed clustered, fused organelles. The hepatic activities of 3-hydroxyacyl-coenzyme A dehydrogenase short chain and glutamate dehydrogenase (GDH) were decreased by 68% and 60%, respectively, without a change in protein expression. GDH activity was similarly decreased in HINT2-silenced HepG2 cells. When measured in the presence of purified sirtuin 3, latent GDH activity was recovered (126% in Hint2(-/-) versus 83% in Hint2(+/+) ). This suggests a greater extent of acetylation in Hint2(-/-) than in Hint2(+/+) . Hint2/HINT2 positively regulates mitochondrial lipid metabolism and respiration and glucose homeostasis. The absence of Hint2 provokes mitochondrial deformities and a change in the pattern of acetylation of selected proteins.

A Novel l-Xylulose Reductase Essential for l-Arabinose Catabolism in Trichoderma reesei

l-Xylulose reductases belong to the superfamily of short chain dehydrogenases and reductases (SDRs) and catalyze the NAD(P)H-dependent reduction of l-xylulose to xylitol in l-arabinose and glucuronic acid catabolism. Here we report the identification of a novel l-xylulose reductase LXR3 in the fungus Trichoderma reesei by a bioinformatic approach in combination with a functional analysis. LXR3, a 31 kDa protein, catalyzes the reduction of l-xylulose to xylitol via NADPH and is also able to convert d-xylulose, d-ribulose, l-sorbose, and d-fructose to their corresponding polyols. Transcription of lxr3 is specifically induced by l-arabinose and l-arabitol. Deletion of lxr3 affects growth on l-arabinose and l-arabitol and reduces total NADPH-dependent LXR activity in cell free extracts. A phylogenetic analysis of known l-xylulose reductases shows that LXR3 is phylogenetically different from the Aspergillus nigerl-xylulose reductase LxrA and, moreover, that all identified true l-xylulose reductases belong to different clades within the superfamily of SDRs. This indicates that the enzymes responsible for the reduction of l-xylulose in l-arabinose and glucuronic acid catabolic pathways have evolved independently and that even the fungal LXRs of the l-arabinose catabolic pathway have evolved in different clades of the superfamily of SDRs.

Structural and biochemical characterization of an atypical short‐chain dehydrogenase/reductase reveals an unusual cofactor preference

Short-chain dehydrogenases/reductases (SDRs) encompass a large and functionally diverse family of enzymes with representative members in all kingdoms of life. Despite the wealth of reactions catalyzed by SDRs, they operate through a well-conserved and efficient reaction mechanism centered in a conserved catalytic tetrad (Asn-Ser-Tyr-Lys) and the employment of an appropriate cofactor. In recent years, SDRs that lack the signature catalytic tetrad have been identified, thus adding a perplexing twist to SDR functionality. In the present study, we report the crystal structure of SDRvv, an atypical SDR from Vibrio vulnificus devoid of the catalytic tetrad, thereby defining the structural signature of this apparent SDR family outlier. Further structural analysis of SDRvv in complex with its putative cofactor NADPH, site-directed mutagenesis and binding studies via isothermal titration calorimetry, and further biochemical characterization have allowed us to dissect the cofactor preferences of SDRvv. The retained capacity to bind the NADPH cofactor, the conceivable existence of a proton relay and the conservation of the coordination distances between the key residues in the cofactor binding pocket define a first set of rules towards catalytic activity for SDRvv. The findings of the present study set the stage for deriving the identity of the natural substrate of SDRvv and add a new twist to the structure-function landscape for Rossmann-fold-dependent cofactor discrimination.

Engineered resistance and hypersusceptibility through functional metabolic studies of 100 genes in soybean to its major pathogen, the soybean cyst nematode

During pathogen attack, the host plant induces genes to ward off the pathogen while the pathogen often produces effector proteins to increase susceptibility of the host. Gene expression studies of syncytia formed in soybean root by soybean cyst nematode (Heterodera glycines) identified many genes altered in expression in resistant and susceptible roots. However, it is difficult to assess the role and impact of these genes on resistance using gene expression patterns alone. We selected 100 soybean genes from published microarray studies and individually overexpressed them in soybean roots to determine their impact on cyst nematode development. Nine genes reduced the number of mature females by more than 50 % when overexpressed, including genes encoding ascorbate peroxidase, β-1,4-endoglucanase, short chain dehydrogenase, lipase, DREPP membrane protein, calmodulin, and three proteins of unknown function. One gene encoding a serine hydroxymethyltransferase decreased the number of mature cyst nematode females by 45 % and is located at the Rhg4 locus. Four genes increased the number of mature cyst nematode females by more than 200 %, while thirteen others increased the number of mature cyst nematode females by more than 150 %. Our data support a role for auxin and ethylene in susceptibility of soybean to cyst nematodes. These studies highlight the contrasting gene sets induced by host and nematode during infection and provide new insights into the interactions between host and pathogen at the molecular level. Overexpression of some of these genes result in a greater decrease in the number of cysts formed than recognized soybean cyst nematode resistance loci.

A novel and rapid long-chain hydroxyacyl-CoA dehydrogenase assay

INTRODUCTION Mitochondrial beta-oxidation is key physiological process in higher eukaryotes and consists of a four step reaction involving the concerted action of several chain length specifi c enzymes. Mitochondrial trifunctional protein (MTP) harbours three enzymatic activities required for the oxidation of long-chain fatty acids. Defi ciency of the second enzymatic activity of MTP, i.e. long-chain hydroxyacyl-CoA dehydrogenase activity (LCHAD), is a severe multiorgan disorder that has been included in the newborn screening programme. Identifi cation of LCHAD defi ciency occurs via the rapid and sensitive quantifi cation of acylcarnitines in plasma or serum. Although bloodspot acylcarnitine profi ling is highly indicative for this disorder, a conclusive diagnosis comes from a specifi c enzyme assay and/ or mutation analysis. However, mutation analysis is more time consuming. Measurement of LCHAD enzymatic activity is hampered by the presence of short-chain hydroxyacyl-CoA dehydrogenase (SCHAD), which also contributes to the enzymatic reaction. Therefore, we have developed and applied several means to eliminate the contribution of SCHAD. Here we report a novel, more direct, less time-consuming and less costly method.

A novel transcriptional repressor PhaR for the steroid-inducible expression of the 3,17β-hydroxysteroid dehydrogenase gene in Comamonas testosteroni ATCC11996

Comamonas testosteroni is able to catabolize a variety of steroids and polycyclic aromatic hydrocarbons and might be used in the bioremediation of contaminated environments. 3,17β-Hydroxysteroid dehydrogenase (3,17β-HSD) from C. testosteroni is a member of the short-chain dehydrogenase/reductase (SDR) superfamily and a key enzyme in steroid degradation. The genome of C. testosteroni ATCC11996 was sequenced in our previous work. In addition to the gene coding for 3,17β-HSD (βhsd), a novel transcriptional repressor phaR gene (phaR) which locates 2290bp upstream of the βhsd gene was found. PhaR knock-out mutants of C. testosteroni were prepared and shown to grow better than wild-type C. testosteroni in the presence of 1mM testosterone, 0.5mM estradiol or 0.5mM cholesterol in both Standard 1 Nutrient (SIN) medium and 1:10 diluted SIN medium. After 1mM testosterone induction, 3,17β-HSD expression in the mutant was 2.5 times higher than in wild type C. testosteroni. Accordingly, PhaR is a repressor that controls 3,17β-HSD expression. Moreover, phaR knock-out mutants grow at higher rates and produce more protein in the presence of steroids as carbon source. However, ELISA results showed that 0.5mM estradiol and cholesterol could not induce βhsd gene expression in both wild-type and mutant C. testosteroni. Probably, in addition to the βhsd gene, PhaR regulates some other genes that relate to steroid degradation. The genes coding for PhaR and 3,17β-HSD together with their promoter domains were cloned into plasmids pK18 and pUC19. Escherichia coli HB101 was co-transformed with these plasmids. The results suggest that PhaR is a repressor, which might bind on a special βhsd promoter domain (214bp). A 2509bp DNA fragment that contained a putative promoter for the βhsd gene (without the phaR gene) was cloned into plasmid pUC2.5-3. The plasmid was transformed into HB101 (E. coli) and induced with testosterone. As a result, 3,17β-HSD expression was at a high level, but could not be further enhanced by testosterone. Taken together, phaR knock-out mutants have better ability to degrade steroids than wild-type C. testosteroni ATCC11996 and might therefore be used in bioremediation.

Potential Immunogenic Polypeptides of Burkholderia pseudomallei Identified by Shotgun Expression Library and Evaluation of Their Efficacy for Serodiagnosis of Melioidosis

The search for novel immunogenic polypeptides to improve the accuracy and reliability of serologic diagnostic methods for Burkholderia pseudomallei infection is ongoing. We employed a rapid and efficient approach to identify such polypeptides with sera from melioidosis patients using a small insert genomic expression library created from clinically confirmed local virulent isolates of B. pseudomallei. After 2 rounds of immunoscreening, 6 sero-positive clones expressing immunogenic peptides were sequenced and their identities were: benzoate 1,2-dioxygenase beta subunit, a putative 200 kDa antigen p200, phosphotransferase enzyme family protein, short chain dehydrogenase and 2 hypothetical proteins. These immunogens were then transferred to an ELISA platform for further large scale screening. By combining shotgun expression library and ELISA assays, we identified 2 polypeptides BPSS1904 (benzoate 1,2-dioxygenase beta subunit) and BPSL3130 (hypothetical protein), which had sensitivities of 78.9% and 79.4% and specificities of 88.1% and 94.8%, respectively in ELISA test, thus suggesting that both are potential candidate antigens for the serodiagnosis of infections caused by B. pseudomallei.

Transcriptional analysis of Clostridium beijerinckii NCIMB 8052 to elucidate role of furfural stress during acetone butanol ethanol fermentation

BackgroundFurfural is the prevalent microbial inhibitor generated during pretreatment and hydrolysis of lignocellulose biomass to monomeric sugars, but the response of acetone butanol ethanol (ABE) producing Clostridium beijerinckii NCIMB 8052 to this compound at the molecular level is unknown. To discern the effect of furfural on C. beijerinckii and to gain insight into molecular mechanisms of action and detoxification, physiological changes of furfural-stressed cultures during acetone butanol ethanol (ABE) fermentation were studied, and differentially expressed genes were profiled by genome-wide transcriptional analysis.ResultsA total of 5,003 C. beijerinckii NCIMB 8052 genes capturing about 99.7% of the genome were examined. About 111 genes were differentially expressed (up- or down-regulated) by C. beijerinckii when it was challenged with furfural at acidogenic growth phase compared with 721 genes that were differentially expressed (up- or down-regulated) when C. beijerinckii was challenged with furfural at solventogenic growth phase. The differentially expressed genes include genes related to redox and cofactors, membrane transporters, carbohydrate, amino sugar and nucleotide sugar metabolisms, heat shock proteins, DNA repair, and two-component signal transduction system. While C. beijerinckii exposed to furfural stress during the acidogenic growth phase produced 13% more ABE than the unstressed control, ABE production by C. beijerinckii ceased following exposure to furfural stress during the solventogenic growth phase.ConclusionGenome-wide transcriptional response of C. beijerinckii to furfural stress was investigated for the first time using microarray analysis. Stresses emanating from ABE accumulation in the fermentation medium; redox balance perturbations; and repression of genes that code for the phosphotransferase system, cell motility and flagellar proteins (and combinations thereof) may have caused the premature termination of C. beijerinckii 8052 growth and ABE production following furfural challenge at the solventogenic phase.This study provides insights into basis for metabolic engineering of C. beijerinckii NCIMB 8052 for enhanced tolerance of lignocellulose-derived microbial inhibitory compounds, thereby improving bioconversion of lignocellulose biomass hydrolysates to biofuels and chemicals. Indeed, two enzymes encoded by Cbei_3974 and Cbei_3904 belonging to aldo/keto reductase (AKR) and short-chain dehydrogenase/reductase (SDR) families have been identified to be involved in furfural detoxification and tolerance.

Molecular cloning and catalytic characterization of a recombinant tropine biosynthetic tropinone reductase from Withania coagulans leaf

Tropinone reductases (TRs) are small proteins belonging to the SDR (short chain dehydrogenase/reductase) family of enzymes. TR-I and TR-II catalyze the conversion of tropinone into tropane alcohols (tropine and pseudotropine, respectively). The steps are intermediary enroute to biosynthesis of tropane esters of medicinal importance, hyoscyamine/scopolamine, and calystegins, respectively. Biosynthesis of tropane alkaloids has been proposed to occur in roots. However, in the present report, a tropine forming tropinone reductase (TR-I) cDNA was isolated from the aerial tissue (leaf) of a medicinal plant, Withania coagulans. The ORF was deduced to encode a polypeptide of 29.34kDa. The complete cDNA (WcTRI) was expressed in E. coli and the recombinant His-tagged protein was purified for functional characterization. The enzyme had a narrow pH range of substantial activity with maxima at 6.6. Relatively superior thermostability of the enzyme (30% retention of activity at 60°C) was catalytic novelty in consonance with the desert area restricted habitat of the plant. The in vitro reaction kinetics predominantly favoured the forward reaction. The enzyme had wide substrate specificity but did not cover the substrates of other well-known plant SDR related to menthol metabolism. To our knowledge, this pertains to be the first report on any gene and enzyme of secondary metabolism from the commercially and medicinally important vegetable rennet species.

Correlation between sequence, structure and function for trisporoid processing proteins in the model zygomycete Mucor mucedo

Terpenoids, steroids, carotenoids, phytoenes and other chemically related substance groups fulfill multiple functions in all realms of the organismic world. This analysis focuses on trisporoids that operate as pheromones in the phylogenetically ancient fungal group of mucoralean zygomycetes. Trisporoids serve as pheromones for recognizing complementary mating partners and for inducing the differentiation program towards sexual spore formation. Trisporoids are synthesized by oxidative degradation of β-carotene. Structurally, they are related to retinoids in mammals and abscisic acid in vascular plants. In order to evaluate evolutionary relationships between proteins involved in trisporoid binding and also for checking possibilities to recognize functionally related proteins by sequence and structure comparisons, we compared representative proteins of different origins. Towards this goal, we calculated three-dimensional structures for 4-dihydromethyltrisporate dehydrogenase (TSP1) and 4-dihydrotrisporin dehydrogenase (TSP2), the two proteins involved in trisporic acid synthesis that have unequivocally been correlated with their catalytic function for the model zygomycete Mucor mucedo. TSP1 is an aldo–keto reductase with a TIM-barrel structure, TSP2 belongs to short-chain dehydrogenases, characterized by a Rossmann fold. Evidently, functional conservation, even implying very similar substrates and identical cosubstrates of enzymes in a single organism, turns out to be essentially independent of basic protein structure. The binding sites for NADP and trisporoid ligands in the proteins were determined by docking studies, revealing those regions affecting substrate specificity. Despite the pronounced differences in amino acid sequence and tertiary structure, the surfaces around the active sites are comparable between TSP1 and TSP2. Two binding regions were identified, one sterically open and a second closed one. In contrast to TSP1, all docking models for TSP2 place the trisporoid into the second, channel-like region.

FUNCTIONAL ANALYSIS OF A MOSQUITO SHORT‐CHAIN DEHYDROGENASE CLUSTER

The short-chain dehydrogenases (SDR) constitute one of the oldest and largest families of enzymes with over 46,000 members in sequence databases. About 25% of all known dehydrogenases belong to the SDR family. SDR enzymes have critical roles in lipid, amino acid, carbohydrate, hormone, and xenobiotic metabolism as well as in redox sensor mechanisms. This family is present in archaea, bacteria, and eukaryota, emphasizing their versatility and fundamental importance for metabolic processes. We identified a cluster of eight SDRs in the mosquito Aedes aegypti (AaSDRs). Members of the cluster differ in tissue specificity and developmental expression. Heterologous expression produced recombinant proteins that had diverse substrate specificities, but distinct from the conventional insect alcohol (ethanol) dehydrogenases. They are all NADP⁺-dependent and they have S-enantioselectivity and preference for secondary alcohols with 8-15 carbons. Homology modeling was used to build the structure of AaSDR1 and two additional cluster members. The computational study helped explain the selectivity toward the (10S)-isomers as well as the reduced activity of AaSDR4 and AaSDR9 for longer isoprenoid substrates. Similar clusters of SDRs are present in other species of insects, suggesting similar selection mechanisms causing duplication and diversification of this family of enzymes.

Dothistromin genes at multiple separate loci are regulated by AflR

In fungi, genes involved in the production of secondary metabolites are generally clustered at one location. There are some exceptions, such as genes required for synthesis of dothistromin, a toxin that is a chemical analog of the aflatoxin precursor versicolorin A and made by the pine needle pathogen Dothistroma septosporum. The availability of the D. septosporum genome sequence enabled identification of putative dothistromin genes, including an ortholog of the aflatoxin regulatory gene AflR, and revealed that most of the genes are spread over six separate regions (loci) on chromosome 12 (1.3Mb). Here we show that levels of expression of the widely dispersed genes in D. septosporum are not correlated with gene location with respect to their distance from a telomere, but that AflR regulates them. The production of dothistromin by D. septosporum in which the AflR gene was knocked out (ΔDsAflR) was drastically reduced, but still detectable. This is in contrast to orthologous ΔAflR mutants in Aspergillus species that lack any aflatoxin production. Expression patterns in ΔDsAflR mutants helped to predict the complete set of genes involved in dothistromin production. This included a short-chain aryl alcohol dehydrogenase (NorB), which is located on chromosome 11 rather than chromosome 12, but was 24-fold down regulated in ΔDsAflR. An orthologous set of dothistromin genes, organized in a similar fragmented cluster arrangement to that seen in D. septosporum, was found in the closely related tomato pathogen Cladosporium fulvum even though this species does not produce dothistromin. In C. fulvum, pseudogenization of key biosynthetic genes explains the lack of dothistromin production. The fragmented arrangement of dothistromin genes provides an example of coordinated control of a dispersed set of secondary metabolite genes; it also provides an example where loss of dothistromin production might have allowed adaptation to a new pathogenic lifestyle.

Classification and nomenclature of the superfamily of short-chain dehydrogenases/reductases (SDRs)

The short-chain dehydrogenases/reductases (SDRs) constitute one of the largest protein superfamilies known today. The members are distantly related with typically 20–30% residue identity in pair-wise comparisons. Still, all hitherto structurally known SDRs present a common three-dimensional structure consisting of a Rossmann fold with a parallel beta sheet flanked by three helices on each side. Using hidden Markov models (HMMs), we have developed a semi-automated subclassification system for this huge family. Currently, 75% of all SDR forms have been assigned to one of the 464 families totalling 122,940 proteins. There are 47 human SDR families, corresponding to 75 genes. Most human SDR families (35 families) have only one gene, while 12 have between 2 and 8 genes. For more than half of the human SDR families, the three-dimensional fold is known. The number of SDR members increases considerably every year, but the number of SDR families now starts to converge. The classification method has paved the ground for a sustainable and expandable nomenclature system. Information on the SDR superfamily is continuously updated at http://sdr-enzymes.org/.

The Plant Short-Chain Dehydrogenase (SDR) superfamily: genome-wide inventory and diversification patterns

BackgroundShort-chain dehydrogenases/reductases (SDRs) form one of the largest and oldest NAD(P)(H) dependent oxidoreductase families. Despite a conserved ‘Rossmann-fold’ structure, members of the SDR superfamily exhibit low sequence similarities, which constituted a bottleneck in terms of identification. Recent classification methods, relying on hidden-Markov models (HMMs), improved identification and enabled the construction of a nomenclature. However, functional annotations of plant SDRs remain scarce.ResultsWide-scale analyses were performed on ten plant genomes. The combination of hidden Markov model (HMM) based analyses and similarity searches led to the construction of an exhaustive inventory of plant SDR. With 68 to 315 members found in each analysed genome, the inventory confirmed the over-representation of SDRs in plants compared to animals, fungi and prokaryotes. The plant SDRs were first classified into three major types — ‘classical’, ‘extended’ and ‘divergent’ — but a minority (10% of the predicted SDRs) could not be classified into these general types (‘unknown’ or ‘atypical’ types). In a second step, we could categorize the vast majority of land plant SDRs into a set of 49 families. Out of these 49 families, 35 appeared early during evolution since they are commonly found through all the Green Lineage. Yet, some SDR families — tropinone reductase-like proteins (SDR65C), ‘ABA2-like’-NAD dehydrogenase (SDR110C), ‘salutaridine/menthone-reductase-like’ proteins (SDR114C), ‘dihydroflavonol 4-reductase’-like proteins (SDR108E) and ‘isoflavone-reductase-like’ (SDR460A) proteins — have undergone significant functional diversification within vascular plants since they diverged from Bryophytes. Interestingly, these diversified families are either involved in the secondary metabolism routes (terpenoids, alkaloids, phenolics) or participate in developmental processes (hormone biosynthesis or catabolism, flower development), in opposition to SDR families involved in primary metabolism which are poorly diversified.ConclusionThe application of HMMs to plant genomes enabled us to identify 49 families that encompass all Angiosperms (‘higher plants’) SDRs, each family being sufficiently conserved to enable simpler analyses based only on overall sequence similarity. The multiplicity of SDRs in plant kingdom is mainly explained by the diversification of large families involved in different secondary metabolism pathways, suggesting that the chemical diversification that accompanied the emergence of vascular plants acted as a driving force for SDR evolution.

Biocatalytic characterization of a short-chain alcohol dehydrogenase with broad substrate specificity from thermophilic Carboxydothermus hydrogenoformans

The gene encoding a novel short-chain alcohol dehydrogenase in the thermophilic bacterium, Carboxydothermus hydrogenoformans, was identified and overexpressed in Escherichia coli. The enzyme was thermally stable and displayed the highest activity at 70°C and pH 6.0. It preferred NAD(H) over NADP(H) as a cofactor and exhibited broad substrate specificity towards aliphatic ketones, cycloalkanones, aromatic ketones, and ketoesters. Furthermore, ethyl benzoylformate was asymmetrically reduced by the purified enzyme, using an additional coupled NADH regeneration system, with 95% conversion and in an enantiomeric excess of (99.9%). The results of this study may lead to the discovery of a novel method for asymmetric reduction of alcohols, which is an important tool in organic synthesis.

A subcomplex of human mitochondrial RNase P is a bifunctional methyltransferase—extensive moonlighting in mitochondrial tRNA biogenesis

Transfer RNAs (tRNAs) reach their mature functional form through several steps of processing and modification. Some nucleotide modifications affect the proper folding of tRNAs, and they are crucial in case of the non-canonically structured animal mitochondrial tRNAs, as exemplified by the apparently ubiquitous methylation of purines at position 9. Here, we show that a subcomplex of human mitochondrial RNase P, the endonuclease removing tRNA 5′ extensions, is the methyltransferase responsible for m1G9 and m1A9 formation. The ability of the mitochondrial tRNA:m1R9 methyltransferase to modify both purines is uncommon among nucleic acid modification enzymes. In contrast to all the related methyltransferases, the human mitochondrial enzyme, moreover, requires a short-chain dehydrogenase as a partner protein. Human mitochondrial RNase P, thus, constitutes a multifunctional complex, whose subunits moonlight in cascade: a fatty and amino acid degradation enzyme in tRNA methylation and the methyltransferase, in turn, in tRNA 5′ end processing.

The Atypical Short-Chain Dehydrogenases HCF173 and HCF244 Are Jointly Involved in Translational Initiation of the psbA mRNA of Arabidopsis

The related proteins D1 and D2 together build up the photosystem II reaction center. Synthesis of D1 (PsbA) is highly regulated in all photosynthetic organisms. The mechanisms and specific protein factors involved in controlled expression of the psbA gene in higher plants are highly elusive. Here, we report on the identification of a chloroplast-located protein, HCF244 (for high chlorophyll fluorescence244), which is essentially required for translational initiation of the psbA messenger RNA in Arabidopsis (Arabidopsis thaliana). The factor is highly conserved between land plants, algae, and cyanobacteria. HCF244 was identified by coexpression analysis of HCF173, which encodes a protein that is also necessary for psbA translational initiation and in addition for stabilization of this messenger RNA. Phenotypic characterization of the mutants hcf244 and hcf173 suggests that the corresponding proteins operate cooperatively during psbA translation. Immunolocalization studies detected the majority of the two proteins at the thylakoid membrane. Both HCF244 and HCF173 are members of the atypical short-chain dehydrogenase/reductase superfamily, a modified group, which has lost enzyme activity but acquires new functions in the metabolism of the cell.

Structure and Mechanism of Human UDP-xylose Synthase: EVIDENCE FOR A PROMOTING ROLE OF SUGAR RING DISTORTION IN A THREE-STEP CATALYTIC CONVERSION OF UDP-GLUCURONIC ACID

UDP-xylose synthase (UXS) catalyzes decarboxylation of UDP-D-glucuronic acid to UDP-xylose. In mammals, UDP-xylose serves to initiate glycosaminoglycan synthesis on the protein core of extracellular matrix proteoglycans. Lack of UXS activity leads to a defective extracellular matrix, resulting in strong interference with cell signaling pathways. We present comprehensive structural and mechanistic characterization of the human form of UXS. The 1.26-Å crystal structure of the enzyme bound with NAD(+) and UDP reveals a homodimeric short-chain dehydrogenase/reductase (SDR), belonging to the NDP-sugar epimerases/dehydratases subclass. We show that enzymatic reaction proceeds in three chemical steps via UDP-4-keto-D-glucuronic acid and UDP-4-keto-pentose intermediates. Molecular dynamics simulations reveal that the D-glucuronyl ring accommodated by UXS features a marked (4)C(1) chair to B(O,3) boat distortion that facilitates catalysis in two different ways. It promotes oxidation at C(4) (step 1) by aligning the enzymatic base Tyr(147) with the reactive substrate hydroxyl and it brings the carboxylate group at C(5) into an almost fully axial position, ideal for decarboxylation of UDP-4-keto-D-glucuronic acid in the second chemical step. The protonated side chain of Tyr(147) stabilizes the enolate of decarboxylated C(4) keto species ((2)H(1) half-chair) that is then protonated from the Si face at C(5), involving water coordinated by Glu(120). Arg(277), which is positioned by a salt-link interaction with Glu(120), closes up the catalytic site and prevents release of the UDP-4-keto-pentose and NADH intermediates. Hydrogenation of the C(4) keto group by NADH, assisted by Tyr(147) as catalytic proton donor, yields UDP-xylose adopting the relaxed (4)C(1) chair conformation (step 3).

Tautomers of Anthrahydroquinones: Enzymatic Reduction and Implications for Chrysophanol, Monodictyphenone, and Related Xanthone Biosyntheses

Reduction of emodin by sodium dithionite resulted in the formation of two tautomeric forms of emodin hydroquinone. Subsequent conversion by the short-chain dehydrogenase/reductase (SDR) MdpC into the corresponding 3-hydroxy-3,4-dihydroanthracen-1(2H)-one implies that deoxygenation is the first step in monodictyphenone biosynthesis. Implications for chrysophanol formation as well as reaction sequences in the related xanthone, ergochrome, and bianthraquinone biosyntheses are discussed.