O-GlcNAcylation enhances CPS1 catalytic efficiency for ammonia and promotes ureagenesis.

Life-threatening hyperammonemia occurs in both inherited and acquired liver diseases affecting ureagenesis, the main pathway for detoxification of neurotoxic ammonia in mammals. Protein O-GlcNAcylation is a reversible and nutrient-sensitive post-translational modification using as substrate UDP-GlcNAc, the end-product of hexosamine biosynthesis pathway. Here we show that increased liver UDP-GlcNAc during hyperammonemia increases protein O-GlcNAcylation and enhances ureagenesis. Mechanistically, O-GlcNAcylation on specific threonine residues increased the catalytic efficiency for ammonia of carbamoyl phosphate synthetase 1 (CPS1), the rate-limiting enzyme in ureagenesis. Pharmacological inhibition of O-GlcNAcase, the enzyme removing O-GlcNAc from proteins, resulted in clinically relevant reductions of systemic ammonia in both genetic (hypomorphic mouse model of propionic acidemia) and acquired (thioacetamide-induced acute liver failure) mouse models of liver diseases. In conclusion, by fine-tuned control of ammonia entry into ureagenesis, hepatic O-GlcNAcylation of CPS1 increases ammonia detoxification and is a novel target for therapy of hyperammonemia in both genetic and acquired diseases.

The role of UDP-glycosyltransferases in xenobioticresistance.

Uridine diphosphate sugar-utilizing glycosyltransferases (UGTs) are an enzyme superfamily that catalyzes glycosyl residues transfer from activated nucleotide sugars to acceptor molecules. In addition to various endogenous compounds, numerous xenobiotics are substrates of UGTs. As the glycosides formed are generally less active/toxic and more hydrophilic than aglycones, UGTs effectively protect organisms from potentially harmful xenobiotics. Therefore, increased UGT expression and/or activity improve the protection of the organism and may contribute to the development of individuals that become more resistant to certain xenobiotics. While the function of UGTs in the resistance of human cancer cells to chemotherapy is now well known, other organisms and other xenobiotics have attracted much less attention. This review was designed to fill this knowledge gap by presenting complex information about the role of UGTs in xenobiotic-resistance in various organisms. This summarization and evaluation of the available information reveals that UGTs play an important role in defense against xenobiotics not only in humans, but in countless other organisms such as parasites, insects, and plants. Moreover, many recent studies clearly show the participation of UGTs in the resistance of nematodes to anthelmintics, insects to insecticides, weeds to herbicides as well as humans to various drugs (not only those used in cancer therapy but also in the treatment of epilepsy, psychiatric disorders, hypertension, hypercholesterolemia, and HIV infection). Nevertheless, although the contribution of UGTs to xenobiotic resistance in diverse organisms has become obvious, many pieces of information remain missing, for example with regard to the mechanisms of UGT regulation.

Genome-Wide Identification and Expression Analysis of Udp-Glucuronosyltransferases in the Whitefly Bemisia Tabaci (Gennadius) (HemipterA: Aleyrodidae).

Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is an important agricultural pest worldwide. Uridine diphosphate (UDP)-glucuronosyltransferases (UGTs) are one of the largest and most ubiquitous groups of proteins. Because of their role in detoxification, insect UGTs are attracting increasing attention. In this study, we identified and analyzed UGT genes in B. tabaci MEAM1 to investigate their potential roles in host adaptation and reproductive capacity. Based on phylogenetic and structural analyses, we identified 76 UGT genes in the B. tabaci MEAM1 genome. RNA-seq and real-time quantitative PCR (RT-qPCR) revealed differential expression patterns of these genes at different developmental stages and in association with four host plants (cabbage, cucumber, cotton and tomato). RNA interference results of selected UGTs showed that, when UGT352A1, UGT352B1, and UGT354A1 were respectively silenced by feeding on dsRNA, the fecundity of B. tabaci MEAM1 was reduced, suggesting that the expressions of these three UGT genes in this species may be associated with host-related fecundity. Together, our results provide detailed UGTs data in B.tabaci and help guide future studies on the mechanisms of host adaptation by B.tabaci.

Detection and validation of EST-SSR markers associated with sugar-related traits in sugarcane using linkage and association mapping.

Sugar-related traits are of great importance in sugarcane breeding. In the present study, quantitative trait loci (QTL) mapping validated with association mapping was used to identify expressed sequence tag-simple sequence repeats (EST-SSRs) associated with sugar-related traits. For linkage mapping, 524 EST-SSRs, 241 Amplified Fragment Length Polymorphisms, and 10 genomic SSR markers were mapped using 283 F1 progenies derived from an interspecific cross. Six regions were identified using Multiple QTL Mapping, and 14 unlinked markers using single marker analysis. Association analysis was performed on a set of 200 accessions, based on the mixed linear model. Validation of the EST-SSR markers using association mapping within the target QTL genomic regions identified two EST-SSR markers showing a putative relationship with uridine diphosphate (UDP) glycosyltransferase, and beta-amylase, which are associated with pol and sugar yield. These functional markers can be used for marker-assisted selection of sugarcane.

Overexpression of SrUGT76G1 in Stevia alters major steviol glycosides composition towards improved quality.

Steviol glycosides (SGs) are extracted from Stevia leaves for use as a natural sweetener. Among SGs, stevioside is most abundant in leaf extracts followed by rebaudioside A (Reb A). However, Reb A is of particular interest because of its sweeter and more pleasant taste compared to stevioside. Therefore, the development of new Stevia varieties with a higher Reb A to stevioside ratio would be desirable for the production of higher quality natural sweeteners. Here, we generated transgenic Stevia plants overexpressing Stevia UDP-glycosyltransferase 76G1 (SrUGT76G1) that is known to convert stevioside to Reb A through 1,3-ß-d-glucosylation in vitro. Interestingly, by overexpressing SrUGT76G1, the Reb A to stevioside ratio was drastically increased from 0.30 in wild-type (WT) plants up to 1.55 in transgenic lines without any significant changes in total SGs content. This was contributed by a concurrent increase in Reb A content and a decrease in stevioside content. Additionally, we were able to find an increase in the Reb C to dulcoside A ratio in transgenic lines. Using the glutathione S-transferase-tagged SrUGT76G1 recombinant protein for an in vitro glucosyltransferase assay, we further demonstrated that Reb C can be produced from the glucosylation of dulcoside A by SrUGT76G1. Transgenic Stevia plants having higher Reb A to stevioside ratio were visually indistinguishable from WT plants. Taken together, our results demonstrate that the overexpression of SrUGT76G1 in Stevia is an effective way to generate new Stevia varieties with higher proportion of the more preferred Reb A without compromising on plant development.

UDP-glycosyltransferase family in Haemonchus contortus: Phylogenetic analysis, constitutive expression, sex-differences and resistance-related differences.

UDP-glycosyltransferases (UGT), catalysing conjugation of UDP-activated sugar donors to small lipophilic chemicals, are widespread in living organisms from bacteria to fungi, plant, or animals. The progress of genome sequencing has enabled an assessment of the UGT multigene family in Haemonchus contortus (family Trichostrongylidae, Nematoda), a hematophagous gastrointestinal parasite of small ruminants. Here we report 32 putative UGT genes divided into 15 UGT families. Phylogenetic analysis in comparison with UGTs from Caenorhabditis elegans, a free-living model nematode, revealed several single member homologues, a lack of the dramatic gene expansion seen in C. elegans, but also several families (UGT365, UGT366, UGT368) expanded in H. contortus only. The assessment of constitutive UGT mRNA expression in H. contortus adults identified significant differences between females and males. In addition, we compared the expression of selected UGTs in the drug-sensitive ISE strain to two benzimidazole-resistant strains, IRE and WR, with different genetic backgrounds. Constitutive expression of UGT368B2 was significantly higher in both resistant strains than in the sensitive strain. As resistant strains were able to deactivate benzimidazole anthelmintics via glycosylation more effectively then the sensitive strain, UGT368B2 enhanced constitutive expression might contribute to drug resistance in H. contortus.

UDP and NTF2 are the most consistently expressed genes in Panax ginseng roots at different growth stages.

Reverse transcription­quantitative polymerase chain reaction (RT­qPCR) analysis relies on normalization against a consistently expressed reference gene. However, it has been reported that reference gene expression levels often vary markedly between samples as they are usually selected based solely on convention. The advent of RNA sequencing technology offers the opportunity to select reference genes with the least variability in steady­state transcript levels. To identify the most consistently stable genes, which are a prerequisite for obtaining reliable gene expression data, the present study analyzed transcriptomes from six Panax ginseng transcriptome data sets, representing six growth stages, and selected 21 candidate reference genes for screening using RT­qPCR. Of the 21 candidate genes, 13 had not been reported previously. The geNorm, NormFinder and BestKeeper programs were used to analyze the stability of the 21 candidate reference genes. The results showed that UDP­N­acetylgalactosamine transporter and nuclear transport factor 2 were likely to be the optimal combination of reference genes for use in investigations of ginseng. The novel reference genes were validated by correlating the gene expression profiles of four pathogenesis­related protein genes generated from RT­qPCR, with their expression levels calculated from the RNA sequencing data. The expression levels were well correlated, which demonstrated their value in performing RT­qPCR analyses in ginseng.

Efficient chemoenzymatic synthesis of uridine 5'-diphosphate N-acetylglucosamine and uridine 5'-diphosphate N-trifluoacetyl glucosamine with three recombinant enzymes.

Uridine 5'-diphosphate N-acetylglucosamine (UDP-GlcNAc) is a natural UDP-monosaccharide donor for bacterial glycosyltransferases, while uridine 5'-diphosphate N-trifluoacetyl glucosamine (UDP-GlcNTFA) is its synthetic mimic. The chemoenzymatic synthesis of UDP-GlcNAc and UDP-GlcNTFA was attempted by three recombinant enzymes. Recombinant N-acetylhexosamine 1-kinase was used to produce GlcNAc/GlcNTFA-1-phosphate from GlcNAc/GlcNTFA. N-acetylglucosamine-1-phosphate uridyltransferase from Escherichia coli K12 MG1655 was used to produce UDP-GlcNAc/GlcNTFA from GlcNAc/GlcNTFA-1-phosphate. Inorganic pyrophosphatase from E. coli K12 MG1655 was used to hydrolyze pyrophosphate to accelerate the reaction. The above enzymes were expressed in E. coli BL21 (DE3) and purified, respectively, and finally mixed in one-pot bioreactor. The effects of reaction conditions on the production of UDP-GlcNAc and UDP-GlcNTFA were characterized. To avoid the substrate inhibition effect on the production of UDP-GlcNAc and UDP-GlcNTFA, the reaction was performed with fed batch of substrate. Under the optimized conditions, high production of UDP-GlcNAc (59.51 g/L) and UDP-GlcNTFA (46.54 g/L) were achieved in this three-enzyme one-pot system. The present work is promising to develop an efficient scalable process for the supply of UDP-monosaccharide donors for oligosaccharide synthesis.

Design, synthesis and bioactivity evaluation of Galf mimics as antitubercular agents.

A series of novel Galf mimics has been synthesized and characterized by IR, (1)H NMR, (13)C NMR, mass spectral and element analysis. All the newly prepared compounds were screened for their antitubercular activities. Bioactivity assays manifested that most of Galf mimics exhibited good antitubercular activities. Especially compound 4d and 4e displayed remarkable antitubercular efficacies, which were comparable to ethambutol.

Angelica Sinensis polysaccharides stimulated UDP-sugar synthase genes through promoting gene expression of IGF-1 and IGF1R in chondrocytes: promoting anti-osteoarthritic activity.

BACKGROUND: Osteoarthritis (OA) is a chronic joints disease characterized by progressive degeneration of articular cartilage due to the loss of cartilage matrix. Previously, we found, for the first time, that an acidic glycan from Angelica Sinensis Polysaccharides (APSs), namely the APS-3c, could protect rat cartilage from OA due to promoting glycosaminoglycan (GAG) synthesis in chondrocytes. In the present work, we tried to further the understanding of ASP-3c's anti-OA activity. METHODOLOGY/PRINCIPAL FINDINGS: Human primary chondrocytes were treated with APS-3c or/and recombinant human interleukin 1ß (IL-1ß). It turned out that APS-3c promoted synthesis of UDP-xylose and GAG, as well as the gene expression of UDP-sugar synthases (USSs), insulin like growth factor 1 (IGF1) and IGF1 receptor (IGF1R), and attenuated the degenerative phenotypes, suppressed biosynthesis of UDP-sugars and GAG, and inhibited the gene expression of USSs, IGF1 and IGF1R induced by IL-1ß. Then, we induced a rat OA model with papain, and found that APS-3c also stimulated GAG synthesis and gene expression of USSs, IGF1 and IGF1R in vivo. Additionally, recombinant human IGF1 and IGF1R inhibitor NP-AEW541 were applied to figure out the correlation between stimulated gene expression of USSs, IGF1 and IGF1R induced by APS-3c. It tuned out that the promoted GAG synthesis and USSs gene expression induced by APS-3c was mediated by the stimulated IGF1 and IGF1R gene expression, but not through directly activation of IGF1R signaling pathway. CONCLUSIONS/SIGNIFICANCES: We demonstrated for the first time that APS-3c presented anti-OA activity through stimulating IGF-1 and IGF1R gene expression, but not directly activating the IGF1R signaling pathway, which consequently promoted UDP-sugars and GAG synthesis due to up-regulating gene expression of USSs. Our findings presented a better understanding of APS-3c's anti-OA activity and suggested that APS-3c could potentially be a novel therapeutic agent for OA.

Antennal uridine diphosphate (UDP)-glycosyltransferases in a pest insect: diversity and putative function in odorant and xenobiotics clearance.

Uridine diphosphate UDP-glycosyltransferases (UGTs) are detoxification enzymes widely distributed within living organisms. They are involved in the biotransformation of various lipophilic endogenous compounds and xenobiotics, including odorants. Several UGTs have been reported in the olfactory organs of mammals and involved in olfactory processing and detoxification within the olfactory mucosa but, in insects, this enzyme family is still poorly studied. Despite recent transcriptomic analyses, the diversity of antennal UGTs in insects has not been investigated. To date, only three UGT cDNAs have been shown to be expressed in insect olfactory organs. In the present study, we report the identification of eleven putative UGTs expressed in the antennae of the model pest insect Spodoptera littoralis. Phylogenetic analysis revealed that these UGTs belong to five different families, highlighting their structural diversity. In addition, two genes, UGT40R3 and UGT46A6, were either specifically expressed or overexpressed in the antennae, suggesting specific roles in this sensory organ. Exposure of male moths to the sex pheromone and to a plant odorant differentially downregulated the transcription levels of these two genes, revealing for the first time the regulation of insect UGTs by odorant exposure. Moreover, the specific antennal gene UGT46A6 was upregulated by insecticide topical application on antennae, suggesting its role in the protection of the olfactory organ towards xenobiotics. This work highlights the structural and functional diversity of UGTs within this highly specialized tissue.

Structure of the human Parkin ligase domain in an autoinhibited state.

Mutations in the protein Parkin are associated with Parkinson's disease (PD), the second most common neurodegenerative disease in men. Parkin is an E3 ubiquitin (Ub) ligase of the structurally uncharacterized RING-in-between-RING(IBR)-RING (RBR) family, which, in an HECT-like fashion, forms a catalytic thioester intermediate with Ub. We here report the crystal structure of human Parkin spanning the Unique Parkin domain (UPD, also annotated as RING0) and RBR domains, revealing a tightly packed structure with unanticipated domain interfaces. The UPD adopts a novel elongated Zn-binding fold, while RING2 resembles an IBR domain. Two key interactions keep Parkin in an autoinhibited conformation. A linker that connects the IBR with the RING2 over a 50-Šdistance blocks the conserved E2∼Ub binding site of RING1. RING2 forms a hydrophobic interface with the UPD, burying the catalytic Cys431, which is part of a conserved catalytic triad. Opening of intra-domain interfaces activates Parkin, and enables Ub-based suicide probes to modify Cys431. The structure further reveals a putative phospho-peptide docking site in the UPD, and explains many PD-causing mutations.

Nuclear receptors and endobiotics glucuronidation: the good, the bad, and the UGT.

The recent progresses in molecular biology and pharmacology approaches allowed the characterization of a series of nuclear receptors (NRs) as efficient regulators of uridine diphosphate glucuronosyltransferase (UGT) genes activity. These regulatory processes ensure an optimized UGT expression in response to specific endo- and/or exogenous stimuli. Many of these NRs are activated by endobiotics that also are substrates for UGTs. Thus, by activating their receptors, these endogenous substances control their own conjugation, leading to the concept that glucuronidation is an important part of feed-forward/feedback mechanisms by which bioactive molecules control their own concentrations. On the other hand, numerous studies have established the pharmacological relevance of NR-UGT regulatory pathways in the response to therapeutic ligands. The present review article aims at providing a comprehensive view of the physiological and pharmacological importance of the NR regulation of the expression and activity of endobiotics-conjugating UGT enzymes. Selected examples will illustrate how the organism profits from the feed-forward/feedback mechanisms involving NR-UGT pathways, but also how such regulatory processes are involved in the initiation and/or progression of several pathological situations. Finally, we will discuss how the present pharmacopeia involves NR-dependent regulation of endobiotics glucuronidation, and whether the unexploited NR-UGT axes could serve as pharmacological targets for novel therapeutics to restore endobiotics homeostasis.

Tetrameric structure of the GlfT2 galactofuranosyltransferase reveals a scaffold for the assembly of mycobacterial Arabinogalactan.

Biosynthesis of the mycobacterial cell wall relies on the activities of many enzymes, including several glycosyltransferases (GTs). The polymerizing galactofuranosyltransferase GlfT2 (Rv3808c) synthesizes the bulk of the galactan portion of the mycolyl-arabinogalactan complex, which is the largest component of the mycobacterial cell wall. We used x-ray crystallography to determine the 2.45-Å resolution crystal structure of GlfT2, revealing an unprecedented multidomain structure in which an N-terminal ß-barrel domain and two primarily α-helical C-terminal domains flank a central GT-A domain. The kidney-shaped protomers assemble into a C(4)-symmetric homotetramer with an open central core and a surface containing exposed hydrophobic and positively charged residues likely involved with membrane binding. The structure of a 3.1-Å resolution complex of GlfT2 with UDP reveals a distinctive mode of nucleotide recognition. In addition, models for the binding of UDP-galactofuranose and acceptor substrates in combination with site-directed mutagenesis and kinetic studies suggest a mechanism that explains the unique ability of GlfT2 to generate alternating ß-(1→5) and ß-(1→6) glycosidic linkages using a single active site. The topology imposed by docking a tetrameric assembly onto a membrane bilayer also provides novel insights into aspects of processivity and chain length regulation in this and possibly other polymerizing GTs.

Biosynthesis of UDP-4-keto-6-deoxyglucose and UDP-rhamnose in pathogenic fungi Magnaporthe grisea and Botryotinia fuckeliana.

There is increasing evidence that in several fungi, rhamnose-containing glycans are involved in processes that affect host-pathogen interactions, including adhesion, recognition, virulence, and biofilm formation. Nevertheless, little is known about the pathways for the synthesis of these glycans. We show that rhamnose is present in glycans isolated from the rice pathogen Magnaporthe grisea and from the plant pathogen Botryotinia fuckeliana. We also provide evidence that these fungi produce UDP-rhamnose. This is in contrast to bacteria where dTDP-rhamnose is the activated form of this sugar. In bacteria, formation of dTDP-rhamnose requires three enzymes. Here, we demonstrate that in fungi only two genes are required for UDP-Rha synthesis. The first gene encodes a UDP-glucose-4,6-dehydratase that converts UDP-glucose to UDP-4-keto-6-deoxyglucose. The product was shown by time-resolved (1)H NMR spectroscopy to exist in solution predominantly as a hydrated form along with minor amounts of a keto form. The second gene encodes a bifunctional UDP-4-keto-6-deoxyglucose-3,5-epimerase/-4-reductase that converts UDP-4-keto-6-deoxyglucose to UDP-rhamnose. Sugar composition analysis and gene expression studies at different stages of growth indicate that the synthesis of rhamnose-containing glycans is under tissue-specific regulation. Together, our results provide new insight into the formation of rhamnose-containing glycans during the fungal life cycle. The role of these glycans in the interactions between fungal pathogens and their hosts is discussed. Knowledge of the metabolic pathways involved in the formation of rhamnose-containing glycans may facilitate the development of drugs to combat fungal diseases in humans, as to the best of our knowledge mammals do not make these types of glycans.

Leishmania UDP-sugar pyrophosphorylase: the missing link in galactose salvage?

The Leishmania parasite glycocalyx is rich in galactose-containing glycoconjugates that are synthesized by specific glycosyltransferases that use UDP-galactose as a glycosyl donor. UDP-galactose biosynthesis is thought to be predominantly a de novo process involving epimerization of the abundant nucleotide sugar UDP-glucose by the UDP-glucose 4-epimerase, although galactose salvage from the environment has been demonstrated for Leishmania major. Here, we present the characterization of an L. major UDP-sugar pyrophosphorylase able to reversibly activate galactose 1-phosphate into UDP-galactose thus proving the existence of the Isselbacher salvage pathway in this parasite. The ordered bisubstrate mechanism and high affinity of the enzyme for UTP seem to favor the synthesis of nucleotide sugar rather than their pyrophosphorolysis. Although L. major UDP-sugar pyrophosphorylase preferentially activates galactose 1-phosphate and glucose 1-phosphate, the enzyme is able to act on a variety of hexose 1-phosphates as well as pentose 1-phosphates but not hexosamine 1-phosphates and hence presents a broad in vitro specificity. The newly identified enzyme exhibits a low but significant homology with UDP-glucose pyrophosphorylases and conserved in particular is the pyrophosphorylase consensus sequence and residues involved in nucleotide and phosphate binding. Saturation transfer difference NMR spectroscopy experiments confirm the importance of these moieties for substrate binding. The described leishmanial enzyme is closely related to plant UDP-sugar pyrophosphorylases and presents a similar substrate specificity suggesting their common origin.

A novel xylosylphosphotransferase activity discovered in Cryptococcus neoformans.

Cryptococcus neoformans is a fungal pathogen that causes serious disease in immunocompromised individuals. The organism produces a distinctive polysaccharide capsule that is necessary for its virulence, a predominantly polysaccharide cell wall, and a variety of protein- and lipid-linked glycans. The glycan synthetic pathways of this pathogen are of great interest. Here we report the detection of a novel glycosylphosphotransferase activity in C. neoformans, identification of the corresponding gene, and characterization of the encoded protein. The observed activity is specific for UDP-xylose as a donor and for mannose acceptors and forms a xylose-alpha-1-phosphate-6-mannose linkage. This is the first report of a xylosylphosphotransferase activity in any system.

A kinetic model for chain length modulation of Streptococcus pneumoniae cellubiuronan capsular polysaccharide by nucleotide sugar donor concentrations.

The chain length of Streptococcus pneumoniae type 3 capsular polysaccharide (cellubiuronic acid) is tightly regulated by the cellubiuronic acid synthase through an assembly process involving a catalytic motif that is potentially conserved over a wide range of related processive beta-glucan synthases. Cellubiuronic acid is initiated on a lipid and is composed of alternating beta-1,3-Glc and beta-1,4-glucuronic acid (GlcUA) linkages. The entire assembly process is carried out by a polypeptide synthase thought to contain a single active site, suggesting that the donor specificity is controlled by the terminal nonreducing sugar in the acceptor subsite. Shortly after initiation, the synthase undergoes an allosteric transition accompanied by the tight binding of the nascent chain via its nonreducing oligosaccharide terminal segment to the carbohydrate acceptor recognition site. The chain length of polysaccharide assembled by recombinant synthase in Escherichia coli membranes was determined by an ejection mechanism that appeared to be a reversal of the allosteric transition of the synthase from the transitory to the fully processive state. The rates of both ejection and transition were shown to be highly sensitive to the concentration of UDP-GlcUA. As the concentration of UDP-GlcUA was increased, both the rate of synthesis and the processive turnover time increased. The product of the processive turnover time and the rate of synthesis predicted a marked increase in polysaccharide chain size (from 50 to 1150 kDa) over a relatively narrow concentration range of 1-11.5 microm UDP-GlcUA. The kinetic model chain length predictions were in close agreement with chemically determined sizes of polysaccharides synthesized at the same UDP-sugar concentrations. The model indicates that translocation occurs following the addition of GlcUA to the chain terminus, whereas UDP-Glc drives chain termination when inadequate levels of UDP-GlcUA are present. In sum, type 3 synthase appears to modulate polysaccharide chain length by functioning as a concentration-dependent kinetic timing device.

Structures of human deoxycytidine kinase product complexes.

Human deoxycytidine kinase (dCK) is involved in the nucleotide-biosynthesis salvage pathway and has also been shown to phosphorylate several antitumor and antiviral prodrugs. The structures of dCK alone and the dead-end complex of dCK with substrate nucleoside and product ADP or UDP have previously been reported; however, there is currently no structure available for a substrate or product complex. Here, the structures of dCK complexes with the products dCMP, UDP and Mg2+ ion, and with dAMP, UDP and Mg2+ ion are reported. Structural comparisons show that the product complexes with UDP and a dead-end complex with substrate and UDP have similar active-site conformations.

Fluorescent reagents for in vitro studies of lipid-linked steps of bacterial peptidoglycan biosynthesis: derivatives of UDPMurNAc-pentapeptide containing d-cysteine at position 4 or 5.

UDPMurNAc-L-Ala-gamma-D-Glu-X-D-Ala-DAla (X = L-Lys or m-DAP) is the cytoplasmic precursor for the lipid-linked cycle of bacterial peptidoglycan biosynthesis, consisting of at least four enzymatic reactions, which are targets for antibacterial agents. Fluorescent derivatives of the UDPMurNAc-pentapeptide labelled at the 3rd, 4th, and 5th position of the peptide chain were prepared chemoenzymatically, in order to study the reactions catalysed by enzymes in this cycle. Derivatives labelled on the epsilon-amino group of the 3rd amino acid (N-dansyl, N-fluorescamine and N-phthalaldehyde) were prepared by chemical modification. Two methods were developed for preparation of analogues of UDPMurNAc-pentapeptide containing D-cysteine at position 4 or 5: either by MurF-catalysed ligation of the UDPMurNAc-tripeptide to synthetic D-Ala-D-Cys or D-Cys-D-Ala dipeptides; or by enzymatic synthesis of D-Ala-D-Cys by ligase VanD. D-Cys-containing UDPMurNAc-pentapeptides were labelled with pyrene maleimide, to give 4-pyrene and 5-pyrene labelled derivatives. The fluorescent UDPMurNAc-pentapeptides were processed as substrates by Escherichia coli MraY or E. coli membranes, giving 1.5-150-fold changes in fluorescence upon transformation to lipid intermediate I. Subsequent processing to lipid intermediate II gave rise only to small changes in fluorescence. Pyrene-labelled lipid intermediates I and II can be generated using Micrococcus flavus membranes, enabling the study of the later lipid-linked steps.