Genome-wide analysis of the UDP-glucose dehydrogenase gene family in Arabidopsis, a key enzyme for matrix polysaccharides in cell walls

Arabidopsis cell walls contain large amounts of pectins and hemicelluloses, which are predominantly synthesized via the common precursor UDP-glucuronic acid. The major enzyme for the formation of this nucleotide-sugar is UDP-glucose dehydrogenase, catalysing the irreversible oxidation of UDP-glucose into UDP-glucuronic acid. Four functional gene family members and one pseudogene are present in the Arabidopsis genome, and they show distinct tissue-specific expression patterns during plant development. The analyses of reporter gene lines indicate gene expression of UDP-glucose dehydrogenases in growing tissues. The biochemical characterization of the different isoforms shows equal affinities for the cofactor NAD+ (~40 µM) but variable affinities for the substrate UDP-glucose (120–335 µM) and different catalytic constants, suggesting a regulatory role for the different isoforms in carbon partitioning between cell wall formation and sucrose synthesis as the second major UDP-glucose-consuming pathway. UDP-glucose dehydrogenase is feedback inhibited by UDP-xylose. The relatively (compared with a soybean UDP-glucose dehydrogenase) low affinity of the enzymes for the substrate UDP-glucose is paralleled by the weak inhibition of the enzymes by UDP-xylose. The four Arabidopsis UDP-glucose dehydrogenase isoforms oxidize only UDP-glucose as a substrate. Nucleotide-sugars, which are converted by similar enzymes in bacteria, are not accepted as substrates for the Arabidopsis enzymes

Understanding CHOKe: throughput and spatial characteristics

A recently proposed active queue management, CHOKe, is stateless, simple to implement, yet surprisingly effective in protecting TCP from UDP flows. We present an equilibrium model of TCP/CHOKe. We prove that, provided the number of TCP flows is large, the UDP bandwidth share peaks at (e+1)/sup -1/=0.269 when UDP input rate is slightly larger than link capacity, and drops to zero as UDP input rate tends to infinity. We clarify the spatial characteristics of the leaky buffer under CHOKe that produce this throughput behavior. Specifically, we prove that, as UDP input rate increases, even though the total number of UDP packets in the queue increases, their spatial distribution becomes more and more concentrated near the tail of the queue, and drops rapidly to zero toward the head of the queue. In stark contrast to a nonleaky FIFO buffer where UDP bandwidth shares would approach 1 as its input rate increases without bound, under CHOKe, UDP simultaneously maintains a large number of packets in the queue and receives a vanishingly small bandwidth share, the mechanism through which CHOKe protects TCP flows

alpha -Lactalbumin (LA) Stimulates Milk beta-1,4-Galactosyltransferase I (beta 4Gal-T1) to Transfer Glucose from UDP-glucose to N-Acetylglucosamine: CRYSTAL STRUCTURE OF beta 4Gal-T1·LA COMPLEX WITH UDP-Glc*

beta-1,4-Galactosyltransferase 1 (Gal-T1) transfers galactose (Gal) from UDP-Gal to N-acetylglucosamine (GlcNAc), which constitutes its normal galactosyltransferase (Gal-T) activity. In the presence of alpha -lactalbumin (LA), it transfers Gal to Glc, which is its lactose synthase (LS) activity. It also transfers glucose (Glc) from UDP-Glc to GlcNAc, constituting the glucosyltransferase (Glc-T) activity, albeit at an efficiency of only 0.3-0.4% of Gal-T activity. In the present study, we show that LA increases this activity almost 30-fold. It also enhances the Glc-T activity toward various N-acyl substituted glucosamine acceptors. Steady state kinetic studies of Glc-T reaction show that the Km for the donor and acceptor substrates are high in the absence of LA. In the presence of LA, the Km for the acceptor substrate is reduced 30-fold, whereas for UDP-Glc it is reduced only 5-fold. In order to understand this property, we have determined the crystal structures of the Gal-T1·LA complex with UDP-Glc·Mn2+ and with N-butanoyl-glucosamine (N-butanoyl-GlcN), a preferred sugar acceptor in the Glc-T activity. The crystal structures reveal that although the binding of UDP-Glc is quite similar to UDP-Gal, there are few significant differences observed in the hydrogen bonding interactions between UDP-Glc and Gal-T1. Based on the present kinetic and crystal structural studies, a possible explanation for the role of LA in the Glc-T activity has been proposed

Is Explicit Congestion Notification usable with UDP?

We present initial measurements to determine if ECN is usable with UDP traffic in the public Internet. This is interesting because ECN is part of current IETF proposals for congestion control of UDPbased interactive multimedia, and due to the increasing use of UDP as a substrate on which new transport protocols can be deployed. Using measurements from the author’s homes, their workplace, and cloud servers in each of the nine EC2 regions worldwide, we test reachability of 2500 servers from the public NTP server pool, using ECT(0) and not-ECT marked UDP packets. We show that an average of 98.97% of the NTP servers that are reachable using not-ECT marked packets are also reachable using ECT(0) marked UDP packets, and that ~98% of network hops pass ECT(0) marked packets without clearing the ECT bits. We compare reachability of the same hosts using ECN with TCP, finding that 82.0% of those reachable with TCP can successfully negotiate and use ECN. Our findings suggest that ECN is broadly usable with UDP traffic, and that support for use of ECN with TCP has increased

Substrate specificity provides insights into the sugar donor recognition mechanism of O-GlcNAc transferase (OGT).

O-Linked β-N-acetylglucosaminyl transferase (OGT) plays an important role in the glycosylation of proteins, which is involved in various cellular events. In human, three isoforms of OGT (short OGT [sOGT]; mitochondrial OGT [mOGT]; and nucleocytoplasmic OGT [ncOGT]) share the same catalytic domain, implying that they might adopt a similar catalytic mechanism, including sugar donor recognition. In this work, the sugar-nucleotide tolerance of sOGT was investigated. Among a series of uridine 5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc) analogs tested using the casein kinase II (CKII) peptide as the sugar acceptor, four compounds could be used by sOGT, including UDP-6-deoxy-GlcNAc, UDP-GlcNPr, UDP-6-deoxy-GalNAc and UDP-4-deoxy-GlcNAc. Determined values of Km showed that the substitution of the N-acyl group, deoxy modification of C6/C4-OH or epimerization of C4-OH of the GlcNAc in UDP-GlcNAc decreased its affinity to sOGT. A molecular docking study combined with site-directed mutagenesis indicated that the backbone carbonyl oxygen of Leu653 and the hydroxyl group of Thr560 in sOGT contributed to the recognition of the sugar moiety via hydrogen bonds. The close vicinity between Met501 and the N-acyl group of GlcNPr, as well as the hydrophobic environment near Met501, were responsible for the selective binding of UDP-GlcNPr. These findings illustrate the interaction of OGT and sugar nucleotide donor, providing insights into the OGT catalytic mechanism

Performance of TCP/UDP under Ad Hoc IEEE802.11

TCP is the De facto standard for connection oriented transport layer protocol, while UDP is the De facto standard for transport layer protocol, which is used with real time traffic for audio and video. Although there have been many attempts to measure and analyze the performance of the TCP protocol in wireless networks, very few research was done on the UDP or the interaction between TCP and UDP traffic over the wireless link. In this paper, we tudy the performance of TCP and UDP over IEEE802.11 ad hoc network. We used two topologies, a string and a mesh topology. Our work indicates that IEEE802.11 as a ad-hoc network is not very suitable for bulk transfer using TCP. It also indicates that it is much better for real-time audio. Although one has to be careful here since real-time audio does require much less bandwidth than the wireless link bandwidth. Careful and detailed studies are needed to further clarify that issue.Comment: 9 pages, 5 figures, ICT 2003 (10th International Conference on Telecommunication

Synthese des 18F-markierten Coenzyms Uridindiphosphatglucose als Basis für die 18F-Glykosylierung von Glykoproteinen

The chemo-enzymatic radiosynthesis of no carrier added (n.c.a.) uridine diphospho-2-deoxy- 2-[$^{18}$F]fluoro-$\alpha$-D-glucose (UDP-[$^{18}$F]FGlc) was developed. In order to overcome the problem of poor regioselectivity when using the commonly strategy to label proteins via $^{18}$F-labelled prosthetic groups, the use of enzyme systems in addition to the corresponding $^{18}$F-labelled coenzymes was shown to be a reliable, regioselective and mild labelling method. With regard to the comparison and evaluation of the stereoselectivity of the phosphorylating agents used in the chemical synthesis of cold uridine diphospho-2-deoxy-2-fluoro-$\alpha$-Dglucose, $^{31}$P-decoupled and $^{1}$H-NMR-studies were successfully realized. Uridine diphospho- 2-deoxy-2-fluoro-$\alpha$-D-glucose was obtained in a 7 step synthesis. Tetrabenzylpyrophosphate was shown to be a highly stereoselective phosphorylating agent for FDG ($\alpha /\beta$=3:1). Moreover, a multienzymatic pathway for the synthesis of uridine diphospho-2-deoxy-2-fluoro-$\alpha$- D-glucose was adopted starting from FDG and four commercially available enzymes. This strategy was adjusted to a mg-scale synthesis providing 35% chemical yield. Within the scope of this procedure, a comparison of the natural substrate $\alpha$-D-glucose-1-phosphate with 2-fluoro-2-deoxy-$\alpha$-D-glucose-1-phosphate indicated that the enzyme activity of UDP-glucose pyrophosphorylase (UDP-Glc PPase) was decreased by a factor of 30. With regard to the adaptability of the multiple enzyme system for the radiosynthesis of n.c.a. uridine diphospho-2-deoxy-2-[$^{18}$F]fluoro-$\alpha$-D-glucose a rapid hexokinase-mediated phosphorylation of [$^{18}$F]FDG utilizing ATP or UTP as phosphate donor was performed. A further enzymatic isomerization of n.c.a [$^{18}$F]FDG-6-phosphate to n.c.a. [$^{18}$F]FDG-1-phosphate was limited due to the formation of [$^{18}$F]FDG-1.6-diphosphate as main product. Experiments using a multiple enzyme system to develop a fully enzymatic synthetic route to UDP-[$^{18}$F]FGlc turned out to be less efficient due to the necessity of carrier added conditions. Thus, a chemo-enzymatic synthesis of n.c.a. UDP-[$^{18}$8F]FGlc has been developed, starting from 1.3.4.6-tetra-O-acetyl-2-[$^{18}$F]fluoro-2-deoxy-D-glucose, which occurs as an intermediate in the [$^{18}$F]FDG synthesis. The chemical phosphorylation via MacDonald reaction and subsequent deprotection led to a radiochemical yield of 55% of [$^{18}$F]FDG-1-phosphate. UDP- [$^{18}$F]FGlc was synthesized enzymatically by condensation of [$^{18}$F]FDG-1-phosphate with UTP in presence of UDP-Glc PPase. In order to overcome the problem of decreased enzyme acitivty the reaction was performed in a minimized reaction volume and optimized UTP-concentration of 0.5 mmol/l leading to an overall radiochemical yield of 20% of UDP-[$^{18}$F]FGlc within 110 min. The $^{18}$F-labelled coenzyme UDP-[$^{18}$F]FGlc was used as a tool for $^{18}$F-glycosylation of N-acetylglucosamine mediated by $\beta$-1.4-galactosyltransferase. The $^{18}$F-glycosylated product was obtained in a radiochemical yield of 56% and was easily isolated by solid phase extraction. In addition to the general availability of [$^{18}$F]FDG worldwide, this new strategy for enzymatic transfer of "activated [$^{18}$F]FDG" has demonstrated its potential as a highly selective and mild $^{18}$F-labelling method of glycosylated biopolymers to study their pharmacokinetics using positron-emission-tomography

Phage display-derived inhibitor of the essential cell wall biosynthesis enzyme MurF

Background To develop antibacterial agents having novel modes of action against bacterial cell wall biosynthesis, we targeted the essential MurF enzyme of the antibiotic resistant pathogen Pseudomonas aeruginosa. MurF catalyzes the formation of a peptide bond between D-Alanyl-D-Alanine (D-Ala-D-Ala) and the cell wall precursor uridine 5'-diphosphoryl N-acetylmuramoyl-L-alanyl-D-glutamyl-meso-diaminopimelic acid (UDP-MurNAc-Ala-Glu-meso-A2pm) with the concomitant hydrolysis of ATP to ADP and inorganic phosphate, yielding UDP-N-acetylmuramyl-pentapeptide. As MurF acts on a dipeptide, we exploited a phage display approach to identify peptide ligands having high binding affinities for the enzyme. Results Screening of a phage display 12-mer library using purified P. aeruginosa MurF yielded to the identification of the MurFp1 peptide. The MurF substrate UDP-MurNAc-Ala-Glumeso-A2pm was synthesized and used to develop a sensitive spectrophotometric assay to quantify MurF kinetics and inhibition. MurFp1 acted as a weak, time-dependent inhibitor of MurF activity but was a potent inhibitor when MurF was pre-incubated with UDP-MurNAc-Ala-Glu-meso-A2pm or ATP. In contrast, adding the substrate D-Ala-D-Ala during the pre-incubation nullified the inhibition. The IC50 value of MurFp1 was evaluated at 250 μM, and the Ki was established at 420 μM with respect to the mixed type of inhibition against D-Ala-D-Ala. Conclusion MurFp1 exerts its inhibitory action by interfering with the utilization of D-Ala-D-Ala by the MurF amide ligase enzyme. We propose that MurFp1 exploits UDP-MurNAc-Ala-Glu-meso-A2pm-induced structural changes for better interaction with the enzyme. We present the first peptide inhibitor of MurF, an enzyme that should be exploited as a target for antimicrobial drug development

\u3cem\u3eIn Vitro\u3c/em\u3e Biosynthesis and Chemical Identification of UDP-\u3cem\u3eN\u3c/em\u3e-acetyl-d-quinovosamine (UDP-d-QuiNAc)

N-acetyl-d-quinovosamine (2-acetamido-2,6-dideoxy-d-glucose, QuiNAc) occurs in the polysaccharide structures of many Gram-negative bacteria. In the biosynthesis of QuiNAc-containing polysaccharides, UDP-QuiNAc is the hypothetical donor of the QuiNAc residue. Biosynthesis of UDP-QuiNAc has been proposed to occur by 4,6-dehydration of UDP-N-acetyl-d-glucosamine (UDP-GlcNAc) to UDP-2-acetamido-2,6-dideoxy-d-xylo-4-hexulose followed by reduction of this 4-keto intermediate to UDP-QuiNAc. Several specific dehydratases are known to catalyze the first proposed step. A specific reductase for the last step has not been demonstrated in vitro, but previous mutant analysis suggested that Rhizobium etli gene wreQ might encode this reductase. Therefore, this gene was cloned and expressed in Escherichia coli, and the resulting His6-tagged WreQ protein was purified. It was tested for 4-reductase activity by adding it and NAD(P)H to reaction mixtures in which 4,6-dehydratase WbpM had acted on the precursor substrate UDP-GlcNAc. Thin layer chromatography of the nucleotide sugars in the mixture at various stages of the reaction showed that WbpM converted UDP-GlcNAc completely to what was shown to be its 4-keto-6-deoxy derivative by NMR and that addition of WreQ and NADH led to formation of a third compound. Combined gas chromatography-mass spectrometry analysis of acid hydrolysates of the final reaction mixture showed that a quinovosamine moiety had been synthesized after WreQ addition. The two-step reaction progress also was monitored in real time by NMR. The final UDP-sugar product after WreQ addition was purified and determined to be UDP-d-QuiNAc by one-dimensional and two-dimensional NMR experiments. These results confirmed that WreQ has UDP-2-acetamido-2,6-dideoxy-d-xylo-4-hexulose 4-reductase activity, completing a pathway for UDP-d-QuiNAc synthesis in vitro

Structural determination of archaeal UDP-N-acetylglucosamine 4-epimerase from Methanobrevibacter ruminantium M1 in complex with the bacterial cell wall intermediate UDP-N-acetylmuramic acid

The crystal structure of UDP-N-acetylglucosamine 4-epimerase (UDP-GlcNAc 4-epimerase; WbpP; EC 5.1.3.7), from the archaeal methanogen Methanobrevibacter ruminantium strain M1, was determined to a resolution of 1.65 Å. The structure, with a single monomer in the crystallographic asymmetric unit, contained a conserved N-terminal Rossmann fold for nucleotide binding and an active site positioned in the C-terminus. UDP-GlcNAc 4-epimerase is a member of the short-chain dehydrogenase/reductase superfamily, sharing sequence motifs and structural elements characteristic of this family of oxidoreductases and bacterial 4-epimerases. The protein was co-crystallized with coenzyme NADH and UDP-N-acetylmuramic acid, the latter an unintended inclusion and well known product of the bacterial enzyme MurB and a critical intermediate for bacterial cell wall synthesis. This is a non-native UDP sugar amongst archaea and was most likely incorporated from the Eschericha coli expression host during purification of the recombinant enzyme