Crystal structures of the archaeal UDP‐GlcNAc 2‐epimerase from Methanocaldococcus jannaschii reveal a conformational change induced by UDP‐GlcNAc

Uridine diphosphate N ‐ acetylglucosamine (UDP‐GlcNAc) 2‐epimerase catalyzes the interconversion of UDP‐GlcNAc to UDP‐N‐acetylmannosamine (UDP‐ManNAc), which is used in the biosynthesis of cell surface polysaccharides in bacteria. Biochemical experiments have demonstrated that mutation of this enzyme causes changes in cell morphology and the thermoresistance of the cell wall. Here, we present the crystal structures of Methanocaldococcus jannaschii UDP‐GlcNAc 2‐epimerase in open and closed conformations. A comparison of these crystal structures shows that upon UDP and UDP‐GlcNAc binding, the enzyme undergoes conformational changes involving a rigid‐body movement of the C‐terminal domain. We also present the crystal structure of Bacillus subtilis UDP‐GlcNAc 2‐epimerase in the closed conformation in the presence of UDP and UDP‐GlcNAc. Although a structural overlay of these two closed‐form structures reveals that the substrate‐binding site is evolutionarily conserved, some areas of the allosteric site are distinct between the archaeal and bacterial UDP‐GlcNAc 2‐epimerases. This is the first report on the crystal structure of archaeal UDP‐GlcNAc 2‐epimerase, and our results clearly demonstrate the changes between the open and closed conformations of this enzyme. Proteins 2014; 82:1519–1526. © 2014 Wiley Periodicals, Inc.     

Synthesis of Large Surface‐Area g‐C3N4 Comodified with MnOx and Au‐TiO2 as Efficient Visible‐Light Photocatalysts for Fuel Production

Herein, this study successfully fabricates porous g‐C₃N₄‐based nanocomposites by decorating sheet‐like nanostructured MnO_x and subsequently coupling Au‐modified nanocrystalline TiO₂. It is clearly demonstrated that the as‐prepared amount‐optimized nanocomposite exhibits exceptional visible‐light photocatalytic activities for CO₂ conversion to CH₄ and for H₂ evolution, respectively by ≈28‐time (140 µmol g^?1 h^?1) and ≈31‐time (313 µmol g^?1 h^?1) enhancement compared to the widely accepted outstanding g‐C₃N₄ prepared with urea as the raw material, along with the calculated quantum efficiencies of ≈4.92% and 2.78% at 420 nm wavelength. It is confirmed mainly based on the steady‐state surface photovoltage spectra, transient‐state surface photovoltage responses, fluorescence spectra related to the produced ?OH amount, and electrochemical reduction curves that the exceptional photoactivities are comprehensively attributed to the large surface area (85.5 m² g^?1) due to the porous structure, to the greatly enhanced charge separation and to the introduced catalytic functions to the carrier‐related redox reactions by decorating MnO_x and coupling Au‐TiO₂, respectively, to modulate holes and electrons. Moreover, it is suggested mainly based on the photocatalytic experiments of CO₂ reduction with isotope ¹³CO₂ and D₂O that the produced ?CO₂ and ?H as active radicals would be dominant to initiate the conversion of CO₂ to CH₄.     

Upregulation of Fas and FasL in Taiwan cobra phospholipase A2‐treated human neuroblastoma SK‐N‐SH cells through ROS‐ and Ca2+‐mediated p38 MAPK activation

The aim of the present study is to elucidate the signaling pathway involved in death of human neuroblastoma SK‐N‐SH cells induced by Naja naja atra phospholipase A₂ (PLA₂). Upon exposure to PLA₂, p38 MAPK activation, ERK inactivation, ROS generation, increase in intracellular Ca²⁺ concentration, and upregulation of Fas and FasL were found in SK‐N‐SH cells. SB202190 (p38MAPK inhibitor) suppressed upregulation of Fas and FasL. N‐Acetylcysteine (ROS scavenger) and BAPTA‐AM (Ca²⁺ chelator) abrogated p38 MAPK activation and upregulation of Fas and FasL expression, but restored phosphorylation of ERK. Activated ERK was found to attenuate p38 MAPK‐mediated upregulation of Fas and FasL. Deprivation of catalytic activity could not diminish PLA₂‐induced cell death and Fas/FasL upregulation. Moreover, the cytotoxicity of arachidonic acid and lysophosphatidylcholine was not related to the expression of Fas and FasL. Taken together, our results indicate that PLA₂‐induced cell death is, in part, elicited by upregulation of Fas and FasL, which is regulated by Ca²⁺‐ and ROS‐evoked p38 MAPK activation, and suggest that non‐catalytic PLA₂ plays a role for the signaling pathway. J. Cell. Biochem. 106: 93–102, 2009. © 2008 Wiley‐Liss, Inc.     

Supramolecular Engineering for Formamidinium‐Based Layered 2D Perovskite Solar Cells: Structural Complexity and Dynamics Revealed by Solid‐State NMR Spectroscopy

Perovskite solar cells are one of the most promising photovoltaic technologies, although their molecular level design and stability toward environmental factors remain a challenge. Layered 2D Ruddlesden–Popper perovskite phases feature an organic spacer bilayer that enhances their environmental stability. Here, the concept of supramolecular engineering of 2D perovskite materials is demonstrated in the case of formamidinium (FA) containing A₂FA_n?1Pb_nI_3n+1 formulations by employing (adamantan‐1‐yl)methanammonium (A) spacers exhibiting propensity for strong Van der Waals interactions complemented by structural adaptability. The molecular design translates into desirable structural features and phases with different compositions and dimensionalities, identified uniquely at the atomic level by solid‐state NMR spectroscopy. For A₂FA₂Pb₃I₁₀, efficiencies exceeding 7% in mesoscopic device architectures without any additional treatment or use of antisolvents for ambient temperature film deposition are achieved. This performance improvement over the state‐of‐the‐art FA‐based 2D perovskites is accompanied by high operational stability under humid ambient conditions, which illustrates the utility of the approach in perovskite solar cells and sets the basis for advanced supramolecular design in the future.     

Antagonist binding and induced conformational dynamics of GPCR A2A adenosine receptor

The A_2A adenosine receptor (A_2AAR) is a unique G‐protein coupled receptor (GPCR), because besides agonist, its antagonist could also lead to therapeutic relevance. Based on A_2AAR‐antagonist crystal structure, we have studied the binding mechanism of two distinct antagonists, ZM241385 and KW6002, and dynamic behaviors of A_2AAR induced by antagonist binding. Key residues interacting with both antagonists and residues specifically binding to one of them are identified. ZM241385 specifically bound to S67^2.65, M177^5.38, and N253^6.55, while KW6002 binds to F62^2.60, A81^3.29, and H264^7.29. Moreover, interactions with L167^5.28 are found for both antagonists, which were not reported in agonist binding. The dynamic behaviors of antagonist bound holo‐A_2AARs were found to be different from the apo‐A_2AAR in three typical functional switches, (i) the “ionic lock” was in equilibrium between formation and breakage in apo‐A_2AAR, but stayed broken in holo‐A_2AARs; (ii) the “rotamer toggle switch,” T88^3.36/F242^6.44/W246^6.48, adopted different rotameric conformations in apo‐A_2AAR and holo‐A_2AARs; (iii) apo‐A_2AAR preferred α‐helical intracellular loop (IC)2 and flexible IC3, while holo‐A_2AARs had a flexible IC2 and α‐helical IC3. Our results indicated that antagonist binding induced different conformational rearrangements of these characteristic functional switches in apo‐A_2AAR and holo‐A_2AARs. Proteins 2013; 81:1399–1410. © 2013 Wiley Periodicals, Inc.     

5‐HT2A Receptor Gene Promoter Polymorphism in Relation to Abdominal Obesity and Cortisol

Objective: There is considerable evidence that cortisol secretion is associated with obesity. The regulation of the 5‐hydroxytryptamine receptor 2A (5‐HT_2A) gene might play an essential role because it is involved in the control of cortisol secretion. Therefore, we examined the potential impact of the 5‐HT_2A ?1438G/A promoter polymorphism on obesity and estimates of insulin, glucose, and lipid metabolism as well as circulating hormones, including salivary cortisol, in 284 unrelated Swedish men born in 1944. Research Methods and Procedures: The subjects were genotyped by using polymerase chain reaction amplification of the promoter region of the gene for 5‐HT_2A followed by digestion of the reaction product with the restriction enzyme MspI. Results: The frequencies were 0.39 for allele ?1438A and 0.61 for allele ?1438G. Homozygotes for the ?1438G allele had, in comparison with ?1438A/A subjects, higher body mass index, waist‐to‐hip ratio, and abdominal sagittal diameter. Moreover, cortisol escape from 0.25‐mg dexamethasone suppression was found in subjects with the ?1438A/G genotype. Serum leptin, fasting insulin, and glucose, as well as serum lipids, were not different across the ?1438G/A genotype groups. Discussion: From these results, we suggest the possibility that an abnormal production rate of the 5‐HT_2A gene product might lead to the development of abdominal obesity. The pathophysiology could involve stress factors that destabilize the serotonin‐hypothalamic‐pituitary‐adrenal system in those with genetic vulnerability in the serotonin receptor gene.     

Structural, functional, and bioinformatics studies reveal a new snake venom homologue phospholipase A₂ class

Phospholipases A₂ (PLA₂s) are enzymes responsible for membrane disruption through Ca²⁺‐dependent hydrolysis of phospholipids. Lys49‐PLA₂s are well‐characterized homologue PLA₂s that do not show catalytic activity but can exert a pronounced local myotoxic effect. These homologue PLA₂s were first believed to present residual catalytic activity but experiments with a recombinant toxin show they are incapable of catalysis. Herein, we present a new homologue Asp49‐PLA₂ (BthTX‐II) that is also able to exert muscle damage. This toxin was isolated in 1992 and characterized as presenting very low catalytic activity. Interestingly, this myotoxic homologue Asp49‐PLA₂ conserves all the residues responsible for Ca²⁺ coordination and of the catalytic network, features thought to be fundamental for PLA₂ enzymatic activity. Previous crystallographic studies of apo BthTX‐II suggested this toxin could be catalytically inactive since a distortion in the calcium binding loop was observed. In this article, we show BthTX‐II is not catalytic based on an in vitro cell viability assay and time‐lapse experiments on C2C12 myotube cell cultures, X‐ray crystallography and phylogenetic studies. Cell culture experiments show that BthTX‐II is devoid of catalytic activity, as already observed for Lys49‐PLA₂s. Crystallographic studies of the complex BthTX‐II/Ca²⁺ show that the distortion of the calcium binding loop is still present and impairs ion coordination even though Ca²⁺ are found interacting with other regions of the protein. Phylogenetic studies demonstrate that BthTX‐II is more phylogenetically related to Lys49‐PLA₂s than to other Asp49‐PLA₂s, thus allowing Crotalinae subfamily PLA₂s to be classified into two main branches: a catalytic and a myotoxic one. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

High homocysteine levels prevent via H2S the CoCl2‐induced alteration of lymphocyte viability

High homocysteine (HCy) levels are associated with lymphocyte‐mediated inflammatory responses that are sometimes in turn related to hypoxia. Because adenosine is a potent lymphocyte suppressor produced in hypoxic conditions and shares metabolic pathways with HCy, we addressed the influence of high HCy levels on the hypoxia‐induced, adenosine‐mediated, alteration of lymphocyte viability. We treated mitogen‐stimulated human lymphocytes isolated from healthy individuals and the human lymphoma T‐cell line CEM with cobalt chloride (CoCl₂)to reproduce hypoxia. We found that CoCl₂‐altered cell viability was dose‐dependently reversed using HCy. In turn, the HCy effect was inhibited using DL‐propargylglycine, a specific inhibitor of the hydrogen sulphide (H₂S)‐synthesizing enzyme cystathionine‐γ‐lyase involved in HCy catabolism. We then addressed the intracellular metabolic pathway of adenosine and HCy, and the role of the adenosine A_2A receptor (A_2AR). We observed that: (i) hypoxic conditions lowered the intracellular concentration of HCy by increasing adenosine production, which resulted in high A_2AR expression and 3′, 5′‐cyclic adenosine monophosphate production; (ii) increasing intracellular HCy concentration reversed the hypoxia‐induced adenosinergic signalling despite high adenosine concentration by promoting both S‐adenosylhomocysteine and H₂S production; (iii) DL‐propargylglycine that inhibits H₂S production abolished the HCy effect. Together, these data suggest that high HCy levels prevent, via H₂S production and the resulting down‐regulation of A_2AR expression, the hypoxia‐induced adenosinergic alteration of lymphocyte viability. We point out the relevance of these mechanisms in the pathophysiology of cardiovascular diseases.     

Sequence‐dependent folding and unfolding of ligand‐bound purine riboswitches

Riboswitch regulation of gene expression requires ligand‐mediated RNA folding. From the fluorescence lifetime distribution of bound 2‐aminopurine ligand, we resolve three RNA conformers (C_o, C_i, C_c) of the liganded G‐ and A‐sensing riboswitches from Bacillus subtilis. The ligand binding affinities, and sensitivity to Mg²⁺, together with results from mutagenesis, suggest that C_o and C_i are partially unfolded species compromised in key loop‐loop interactions present in the fully folded C_c. These data verify that the ligand‐bound riboswitches may dynamically fold and unfold in solution, and reveal differences in the distribution of folded states between two structurally homologous purine riboswitches: Ligand‐mediated folding of the G‐sensing riboswitch is more effective, less dependent on Mg²⁺, and less debilitated by mutation, than the A‐sensing riboswitch, which remains more unfolded in its liganded state. We propose that these sequence‐dependent RNA dynamics, which adjust the balance of ligand‐mediated folding and unfolding, enable different degrees of kinetic discrimination in ligand binding, and fine‐tuning of gene regulatory mechanisms. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 953–965, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Family 34 glycosyltransferase (GT34) genes and proteins in Pinus radiata (radiata pine) and Pinus taeda (loblolly pine)

Using a functional genomics approach, four candidate genes (PtGT34A, PtGT34B, PtGT34C and PtGT34D) were identified in Pinus taeda. These genes encode CAZy family GT34 glycosyltransferases that are involved in the synthesis of cell‐wall xyloglucans and heteromannans. The full‐length coding sequences of three orthologs (PrGT34A, B and C) were isolated from a xylem‐specific cDNA library from the closely related Pinus radiata. PrGT34B is the ortholog of XXT1 and XXT2, the two main xyloglucan (1→6)‐α‐xylosyltransferases in Arabidopsis thaliana. PrGT34C is the ortholog of XXT5 in A. thaliana, which is also involved in the xylosylation of xyloglucans. PrGT34A is an ortholog of a galactosyltransferase from fenugreek (Trigonella foenum‐graecum) that is involved in galactomannan synthesis. Truncated coding sequences of the genes were cloned into plasmid vectors and expressed in a Sf9 insect cell‐culture system. The heterologous proteins were purified, and in vitro assays showed that, when incubated with UDP‐xylose and cellotetraose, cellopentaose or cellohexaose, PrGT34B showed xylosyltransferase activity, and, when incubated with UDP‐galactose and the same cello‐oligosaccharides, PrGT34B showed some galactosyltransferase activity. The ratio of xylosyltransferase to galactosyltransferase activity was 434:1. Hydrolysis of the galactosyltransferase reaction products using galactosidases showed the linkages formed were α‐linkages. Analysis of the products of PrGT34B by MALDI‐TOF MS showed that up to three xylosyl residues were transferred from UDP‐xylose to cellohexaose. The heterologous proteins PrGT34A and PrGT34C showed no detectable enzymatic activity.     

Fungal community composition and function after long‐term exposure of northern forests to elevated atmospheric CO2 and tropospheric O3

The long‐term effects of rising atmospheric carbon dioxide (CO₂) and tropospheric O₃ concentrations on fungal communities in soil are not well understood. Here, we examine fungal community composition and the activities of cellobiohydrolase and N‐acetylglucosaminidase (NAG) after 10 years of exposure to 1.5 times ambient levels of CO₂ and O₃ in aspen and aspen–birch forest ecosystems, and compare these results to earlier studies in the same long‐term experiment. The forest floor community was dominated by saprotrophic fungi, and differed slightly between plant community types, as did NAG activity. Elevated CO₂ and O₃ had small but significant effects on the distribution of fungal genotypes in this horizon, and elevated CO₂ also lead to an increase in the proportion of Sistotrema spp. within the community. Yet, although cellobiohydrolase activity was lower in the forest floor under elevated O₃, it was not affected by elevated CO₂. NAG was also unaffected. The soil community was dominated by ectomycorrhizal species. Both CO₂ and O₃ had a minor effect on the distribution of genotypes; however, phylogenetic analysis indicated that under elevated O₃Cortinarius and Inocybe spp. increased in abundance and Laccaria and Tomentella spp. declined. Although cellobiohydrolase activity in soil was unaffected by either CO₂ or O₃, NAG was higher (～29%) under CO₂ in aspen–birch, but lower (～18%) under aspen. Time series analysis indicated that CO₂ increased cellulolytic enzyme activity during the first 5 years of the experiment, but that the magnitude of this effect diminished over time. NAG activity also showed strong early stimulation by elevated CO₂, but after 10 years this effect is no longer evident. Elevated O₃ appears to have variable stimulatory and repressive effects depending on the soil horizon and time point examined.     

Pyrimidine Nucleotide-Stimulated Thromboxane A2 Release from Cultured GLIA

1. Uridine triphosphate (UTP), uridine diphosphate (UDP), cytidine triphosphate (CTP), and deoxythymidine triphosphate (TTP) caused concentration-dependent increases in the release of thromboxane A₂ (TXA₂) from cultured glia prepared from the newborn rat cerebral cortex. Although each of the pyrimidine nucleotides displayed similar potencies, CTP and TTP were considerably less effective than either UTP or UDP. The purine nucleotide ATP was equally as potent as the pyrimidine nucleotides but was marginally less effective than either UTP or UDP.2. The ability of UTP, UDP, TTP, and CTP to promote TXA₂ release from cultured glia was inhibited in a concentration-dependent manner by suramin and was markedly reduced when incubations were performed either in Ca²⁺-free medium or on cultures which had been maintained in serum-free growth medium for 4 days prior to experimentation.3. Challenges with UTP and UDP in combination were found to elicit a response which was no different from the effects of these nucleotides alone; in addition, their effects were reversed by the phospholipase A₂ inhibitor ONO-RS-082. A slight reduction in UTP-and UDP-stimulated TXA₂ release was observed in cultures grown in the presence of leucine methyl ester, a treatment reported to limit microglial survival.4. These results suggest that glia are targets for extracellular pyrimidine nucleotides and that their ability to release eicosanoids from these cells may be important in the brain's response to damage.     

The CysB motif of Rev3p involved in the formation of the four‐subunit DNA polymerase ζ is required for defective‐replisome‐induced mutagenesis

Eukaryotic DNA replication is performed by high‐fidelity multi‐subunit replicative B‐family DNA polymerases (Pols) α, δ and ?. Those complexes are composed of catalytic and accessory subunits and organized in multicomplex machinery: the replisome. The fourth B‐family member, DNA polymerase zeta (Pol ζ), is responsible for a large portion of mutagenesis in eukaryotic cells. Two forms of Pol ζ have been identified, a hetero‐dimeric (Pol ζ₂) and a hetero‐tetrameric (Pol ζ₄) ones and recent data have demonstrated that Pol ζ₄ is responsible for damage‐induced mutagenesis. Here, using yeast Pol ζ mutant defective in the assembly of the Pol ζ four‐subunit form, we show in vivo that [4Fe‐4S] cluster in Pol ζ catalytic subunit (Rev3p) is also required for spontaneous (wild‐type cells) and defective‐replisome‐induced mutagenesis – DRIM (pol3‐Y708A, pol2‐1 or psf1‐100 cells), when cells are not treated with any external damaging agents.     

Three metabolites from an entomopathogenic bacterium,Xenorhabdus nematophila,inhibit larval development of Spodoptera exigua (Lepidoptera: Noctuidae) by inhibiting a digestive enzyme,phospholipase A2

Abstract An entomopathogenic bacterium, Xenorhabdus nematophila, has been known to induce significant immunosuppression of target insects by inhibiting immune‐associated phospholipase A₂ (PLA₂), which subsequently shuts down biosynthesis of eicosanoids that are critical in immune mediation in insects. Some metabolites originated from the bacterial culture broth have been identified and include benzylideneacetone, proline‐tyrosine and acetylated phenylalanine‐glycine‐valine, which are known to inhibit enzyme activity of PLA₂ extracted from hemocyte and fat body. This study tested their effects on digestive PLA₂ of the beet armyworm, Spodoptera exigua. Young larvae fed different concentrations of the three metabolites resulted in significant adverse effects on larval development even at doses below 100 μg/mL. In particular, they induced significant reduction in digestive efficiency of ingested food. All three metabolites significantly inhibited catalytic activity of digestive PLA₂ extracted from midgut lumen of the fifth instar larvae at a low micromolar range. These results suggest that the inhibitory activities of the three bacterial metabolites on digestive PLA₂ of S. exigua midgut may explain some of their oral toxic effects.     

ZmGA3ox2, a candidate gene for a major QTL,qPH3.1, for plant height in maize

Maize plant height is closely associated with biomass, lodging resistance and grain yield. Determining the genetic basis of plant height by characterizing and cloning plant height genes will guide the genetic improvement of crops. In this study, a quantitative trait locus (QTL) for plant height, qPH3.1, was identified on chromosome 3 using populations derived from a cross between Zong3 and its chromosome segment substitution line, SL15. The plant height of the two lines was obviously different, and application of exogenous gibberellin A₃ removed this difference. QTL mapping placed qPH3.1 within a 4.0 cM interval, explaining 32.3% of the phenotypic variance. Furthermore, eight homozygous segmental isolines (SILs) developed from two larger F₂ populations further narrowed down qPH3.1 to within a 12.6 kb interval. ZmGA3ox2, an ortholog of OsGA3ox2, which encodes a GA3 β‐hydroxylase, was positionally cloned. Association mapping identified two polymorphisms in ZmGA3ox2 that were significantly associated with plant height across two experiments. Quantitative RT‐PCR showed that SL15 had higher ZmGA3ox2 expression relative to Zong3. The resultant higher GA₁ accumulation led to longer internodes in SL15 because of increased cell lengths. Moreover, a large deletion in the coding region of ZmGA3ox2 is responsible for the dwarf mutant d1‐6016. The successfully isolated qPH3.1 enriches our knowledge on the genetic basis of plant height in maize, and provides an opportunity for improvement of plant architecture in maize breeding.     

Crystal Structures of Bacillus cereus NCTU2 Chitinase Complexes with Chitooligomers Reveal Novel Substrate Binding for Catalysis: A CHITINASE WITHOUT CHITIN BINDING AND INSERTION DOMAINS*

Chitinases hydrolyze chitin, an insoluble linear polymer of N-acetyl-d-glucosamine (NAG)_n, into nutrient sources. Bacillus cereus NCTU2 chitinase (ChiNCTU2) predominantly produces chitobioses and belongs to glycoside hydrolase family 18. The crystal structure of wild-type ChiNCTU2 comprises only a catalytic domain, unlike other chitinases that are equipped with additional chitin binding and insertion domains to bind substrates into the active site. Lacking chitin binding and chitin insertion domains, ChiNCTU2 utilizes two dynamic loops (Gly-67—Thr-69 and Ile-106–Val-112) to interact with (NAG)_n, generating novel substrate binding and distortion for catalysis. Gln-109 is crucial for direct binding with substrates, leading to conformational changes of two loops with a maximum shift of ∼4.6 Å along the binding cleft. The structures of E145Q, E145Q/Y227F, and E145G/Y227F mutants complexed with (NAG)_n reveal (NAG)₂, (NAG)₂, and (NAG)₄ in the active site, respectively, implying various stages of reaction: before hydrolysis, E145G/Y227F with (NAG)₄; in an intermediate state, E145Q/Y227F with a boat-form NAG at the −1 subsite, −1-(NAG); after hydrolysis, E145Q with a chair form −1-(NAG). Several residues were confirmed to play catalytic roles: Glu-145 in cleavage of the glycosidic bond between −1-(NAG) and +1-(NAG); Tyr-227 in the conformational change of −1-(NAG); Asp-143 and Gln-225 in stabilizing the conformation of −1-(NAG). Additionally, Glu-190 acts in the process of product release, and Tyr-193 coordinates with water for catalysis. Residues Asp-143, E145Q, Glu-190, and Tyr-193 exhibit multiple conformations for functions. The inhibitors zinc ions and cyclo-(l-His-l-Pro) are located at various positions and confirm the catalytic-site topology. Together with kinetics analyses of related mutants, the structures of ChiNCTU2 and its mutant complexes with (NAG)_n provide new insights into its substrate binding and the mechanistic action.     

Simultaneous inhibition of anti‐coagulation and inflammation: crystal structure of phospholipase A2 complexed with indomethacin at 1.4 Å resolution reveals the presence of the new common ligand‐binding site

A novel ligand‐binding site with functional implications has been identified in phospholipase A₂ (PLA₂). The binding of non‐steroidal anti‐inflammatory agent indomethacin at this site blocks both catalytic and anti‐coagulant actions of PLA₂. A group IIA PLA₂ has been isolated from Daboia russelli pulchella (Russell's viper) which is enzymatically active as well as induces a strong anti‐coagulant action. The binding studies have shown that indomethacin reduces the effects of both anti‐coagulant and pro‐inflammatory actions of PLA₂. A group IIA PLA₂ was co‐crystallized with indomethacin and the structure of the complex has been determined at 1.4 Å resolution. The structure determination has revealed the presence of an indomethacin molecule in the structure of PLA₂ at a site which is distinct from the conventional substrate‐binding site. One of the carboxylic group oxygen atoms of indomethacin interacts with Asp 49 and His 48 through the catalytically important water molecule OW 18 while the second carboxylic oxygen atom forms an ionic interaction with the side chain of Lys 69. It is well known that the residues, His 48 and Asp 49 are essential for catalysis while Lys 69 is a part of the anti‐coagulant loop (residues, 54–77). Indomethacin binds in such a manner that it blocks the access to both, it works as a dual inhibitor for catalytic and anti‐coagulant actions of PLA₂. This new binding site in PLA₂ has been observed for the first time and indomethacin is the first compound that has been shown to bind at this novel site resulting in the prevention of anti‐coagulation and inflammation. Copyright © 2009 John Wiley & Sons, Ltd.     

The disulfide oxidoreductase SdbA is active in Streptococcus gordonii using a single C‐terminal cysteine of the CXXC motif

Recently, we identified a novel disulfide oxidoreductase, SdbA, in the oral bacterium Streptococcus gordonii. Disulfide oxidoreductases form disulfide bonds in nascent proteins using a CXXC catalytic motif. Typically, the N‐terminal cysteine interacts with substrates, whereas the C‐terminal cysteine is buried and only reacts with the first cysteine of the motif. In this study, we investigated the SdbA C⁸⁶P⁸⁷D⁸⁸C⁸⁹ catalytic motif. In vitro, SdbA single cysteine variants at the N or C‐terminal position (SdbA_C86P and SdbA_C89A) were active but displayed different susceptibility to oxidation, and N‐terminal cysteine was prone to sulfenylation. In S. gordonii, mutants with a single N‐terminal cysteine were inactive and formed unstable disulfide adducts with other proteins. Activity was partially restored by inactivation of pyruvate oxidase, a hydrogen peroxide generator. Presence of the C‐terminal cysteine alone (in the SdbA_C86P variant) could complement the ΔsdbA mutant and restore disulfide bond formation in recombinant and natural protein substrates. These results provide evidence that certain disulfide oxidoreductases can catalyze disulfide bond formation using a single cysteine of the CXXC motif, including the buried C‐terminal cysteine.     

The functional vanGCd cluster of Clostridium difficile does not confer vancomycin resistance

vanG_Cd, a cryptic gene cluster highly homologous to the vanG gene cluster of Enterococcus faecalis is largely spread in Clostridium difficile. Since emergence of vancomycin resistance would have dramatic clinical consequences, we have evaluated the capacity of the vanG_Cd cluster to confer resistance. We showed that expression of vanG_Cd is inducible by vancomycin and that VanG_Cd, VanXY_Cd and VanT_Cd are functional, exhibiting D‐Ala : D‐Ser ligase, D,D‐dipeptidase and D‐Ser racemase activities respectively. In other bacteria, these enzymes are sufficient to promote vancomycin resistance. Trans‐complementation of C. difficile with the vanC resistance operon of Enterococcus gallinarum faintly impacted the MIC of vancomycin, but did not promote vancomycin resistance in C. difficile. Sublethal concentration of vancomycin led to production of UDP‐MurNAc‐pentapeptide[D‐Ser], suggesting that the vanG_Cd gene cluster is able to modify the peptidoglycan precursors. Our results indicated amidation of UDP‐MurNAc‐tetrapeptide, UDP‐MurNAc‐pentapeptide[D‐Ala] and UDP‐MurNAc‐pentapeptide[D‐Ser]. This modification is passed on the mature peptidoglycan where a muropeptide Tetra‐Tetra is amidated on the meso‐diaminopimelic acid. Taken together, our results suggest that the vanG_Cd gene cluster is functional and is prevented from promoting vancomycin resistance in C. difficile.