Decarboxylation Of UDP-glucuronic Acid Research Articles

UDP-Glucuronic Acid Decarboxylases of Bacteroides fragilis and Their Prevalence in Bacteria

Xylose is rarely described as a component of bacterial glycans. UDP-xylose is the nucleotide-activated form necessary for incorporation of xylose into glycans and is synthesized by the decarboxylation of UDP-glucuronic acid (UDP-GlcA). Enzymes with UDP-GlcA decarboxylase activity include those that lead to the formation of UDP-xylose as the end product (Uxs type) and those synthesizing UDP-xylose as an intermediate (ArnA and RsU4kpxs types). In this report, we identify and confirm the activities of two Uxs-type UDP-GlcA decarboxylases of Bacteroides fragilis, designated BfUxs1 and BfUxs2. Bfuxs1 is located in a conserved region of the B. fragilis genome, whereas Bfuxs2 is in the heterogeneous capsular polysaccharide F (PSF) biosynthesis locus. Deletion of either gene separately does not result in the loss of a detectable phenotype, but deletion of both genes abrogates PSF synthesis, strongly suggesting that they are functional paralogs and that the B. fragilis NCTC 9343 PSF repeat unit contains xylose. UDP-GlcA decarboxylases are often annotated incorrectly as NAD-dependent epimerases/dehydratases; therefore, their prevalence in bacteria is underappreciated. Using available structural and mutational data, we devised a sequence pattern to detect bacterial genes encoding UDP-GlcA decarboxylase activity. We identified 826 predicted UDP-GlcA decarboxylase enzymes in diverse bacterial species, with the ArnA and RsU4kpxs types confined largely to proteobacterial species. These data suggest that xylose, or a monosaccharide requiring a UDP-xylose intermediate, is more prevalent in bacterial glycans than previously appreciated. Genes encoding BfUxs1-like enzymes are highly conserved in Bacteroides species, indicating that these abundant intestinal microbes may synthesize a conserved xylose-containing glycan.

Functional UDP-xylose Transport across the Endoplasmic Reticulum/Golgi Membrane in a Chinese Hamster Ovary Cell Mutant Defective in UDP-xylose Synthase

In mammals, xylose is found as the first sugar residue of the tetrasaccharide GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser, initiating the formation of the glycosaminoglycans heparin/heparan sulfate and chondroitin/dermatan sulfate. It is also found in the trisaccharide Xylalpha1-3Xylalpha1-3Glcbeta1-O-Ser on epidermal growth factor repeats of proteins, such as Notch. UDP-xylose synthase (UXS), which catalyzes the formation of the UDP-xylose substrate for the different xylosyltransferases through decarboxylation of UDP-glucuronic acid, resides in the endoplasmic reticulum and/or Golgi lumen. Since xylosylation takes place in these organelles, no obvious requirement exists for membrane transport of UDP-xylose. However, UDP-xylose transport across isolated Golgi membranes has been documented, and we recently succeeded with the cloning of a human UDP-xylose transporter (SLC25B4). Here we provide new evidence for a functional role of UDP-xylose transport by characterization of a new Chinese hamster ovary cell mutant, designated pgsI-208, that lacks UXS activity. The mutant fails to initiate glycosaminoglycan synthesis and is not capable of xylosylating Notch. Complementation was achieved by expression of a cytoplasmic variant of UXS, which proves the existence of a functional Golgi UDP-xylose transporter. A approximately 200 fold increase of UDP-glucuronic acid occurred in pgsI-208 cells, demonstrating a lack of UDP-xylose-mediated control of the cytoplasmically localized UDP-glucose dehydrogenase in the mutant. The data presented in this study suggest the bidirectional transport of UDP-xylose across endoplasmic reticulum/Golgi membranes and its role in controlling homeostasis of UDP-glucuronic acid and UDP-xylose production.

Origin of Lipid A Species Modified with 4-Amino-4-deoxy-l-arabinose in Polymyxin-resistant Mutants of Escherichia coli: AN AMINOTRANSFERASE (ArnB) THAT GENERATES UDP-4-AMINO-4-DEOXY-l-ARABINOSE

In Escherichia coli and Salmonella typhimurium, addition of the 4-amino-4-deoxy-l-arabinose (l-Ara4N) moiety to the phosphate group(s) of lipid A is required for resistance to polymyxin and cationic antimicrobial peptides. We have proposed previously (Breazeale, S. D., Ribeiro, A. A., and Raetz, C. R. H. (2002) J. Biol. Chem. 277, 2886-2896) a pathway for l-Ara4N biosynthesis that begins with the ArnA-catalyzed C-4" oxidation and C-6" decarboxylation of UDP-glucuronic acid, followed by the C-4" transamination of the product to generate the novel sugar nucleotide UDP-l-Ara4N. We now show that ArnB (PmrH) encodes the relevant aminotransferase. ArnB was overexpressed using a T7lac promoter-driven construct and shown to catalyze the reversible transfer of the amino group from glutamate to the acceptor, uridine 5'-(beta-l-threo-pentapyranosyl-4"-ulose diphosphate), the intermediate that is synthesized by ArnA from UDP-glucuronic acid. A 1.7-mg sample of the putative UDP-l-Ara4N product generated in vitro was purified by ion exchange chromatography, and its structure was confirmed by 1H and 13C NMR spectroscopy. ArnB, which is a cytoplasmic protein, was purified to homogeneity from an overproducing strain of E. coli and shown to contain a pyridoxal phosphate cofactor, as judged by ultraviolet/visible spectrophotometry. The pyridoxal phosphate was converted to the pyridoxamine form in the presence of excess glutamate. A simple quantitative radiochemical assay was developed for ArnB, which can be used to assay the enzyme either in the forward or the reverse direction. The enzyme is highly selective for glutamate as the amine donor, but the equilibrium constant in the direction of UDP-l-Ara4N formation is unfavorable (approximately 0.1). ArnB is a member of a very large family of aminotransferases, but closely related ArnB orthologs are present only in those bacteria capable of synthesizing lipid A species modified with the l-Ara4N moiety.

The Biosynthesis of Glycosaminoglycans

The sites of nucleotide conjugation are few. Two pyrophosphorylase reactions are sufficient to account for the occurrence of the variety of sugar nucleotides previously cited. The pathways for each are different. Whereas UDP-glucose arises from a direct conjugation of glucose 1-phosphate with UTP by a pyrophosphorylase whose specificity has been studied by Albrecht et af. (1968), the hexosamine pathway is far more complex, involving the amination of fructose 6-phosphate by glutamine, the subsequent acetylation, mutation and, only then, conjugation by a specific pyrophosphorylase to form UDP-N-acetylglucosamine. Whereas considerable information is available on the UDPglucose pyrophosphorylase, information on the enzymology of the hexosamine pathway is scant. Fructose 6-phosphate-glutanline aminotransferase has been purified extensively from rat liver and its steady-state kinetic parameters have been established (Winterburn & Phelps, 1970, 1971). The acetylation by acetyl-CoA has been thoroughly investigated by P. J. Winterburn &A. Corfield (unpublished work), but scant information is available on the specific mutase interconverting N-acetylglucosamine 6-phosphate and its l-phosphate counterpart (Fernandez-Sorenson, 1968), and detailed information is similarly lacking on the synthesis of UDP-N-acetylglucosamine from this last intermediate and UTP (Strominger & Smith, 1959). Interconversion of nircleotide sitgars One of the most potentially productive areas of research in this field involves the study of the vast variety of interconversion reactions of which nucleotide sugars are capable. These involve precise inversions, dehydrogenations, reductions, decarboxylations and simultaneous performances of these reactions. Of immediate concern in the context of glycosaminoglycan biosynthesis is the formation of UDP-galactose from UDP-glucose by the 4’-epimerase. This enzyme has been studied in a variety of tissues. The n~ammalian enzyme is potently inhibited by NADH, though requiring catalytic amounts of NAD+ for its reaction (Maxwell, 1957). UDP-xylose arises from the decarboxylation of UDP-glucuronic acid and has been demonstrated in a wide variety of tissues, e.g. hen oviduct (Bdolah & Feingold, 1965), mouse cell mastocytoma (Silbert & Deluca, 1967) and cartilage (Castellani eta/., 1967). The enzyme from Cryptococciis lairventii and yeast has been intensively and elegantly studied by Schutzbach & Feingold (1970), who proposed an interesting mechanism of reaction. The enzyme is potently inhibited by NADH and UDP, though containing tighly bound NAD+. UDP-glucuronic acid arises by dehydrogenation of UDP-glucose (Strominger et a/., 1957). The reaction is noteworthy for being a four-electron oxidation. The enzyme from bovine liver contains six identical subunits, and some details of its protein chemistry have

Incorporation of D-xylose-C 14 into glycoprotein by particles from hen oviduct

D-Xylose, once thought to be restricted to polysaccharides of plants and microorganisms, has recently been found in a number of macromolecules of animal origin. This pentose occupies a unique position in chondroitin through a xylosyl-serine linkage ( Gregory et al ., 1964 ; Lindahl and Rodén, 1965; Rodén and Lindahl, 1965). Free xylosyl-serine has been isolated from human urine ( Tominaga et al ., 1965 ). Xylose ∗ ∗ The sugars are of the D-configuration. has been reported to be a constituent of mucopolysaccharides of mammalian brain ( Wardi et al ., 1965 ), and of glycoproteins from human lung ( Masamune et al ., 1957 ), cancerous ascitic fluid ( Sugimoto, 1956), placenta ( Tomoda and Murayama, 1965) and aorta ( Klemer and Burbaum, 1965), and from cockroach plasma (Lipke et al ., 1965). UDP-Xylose, the probable precursor of such glycosidically-bound xylose, has been isolated from the milk of sows and goats, and from cow colostrum ( Kobata and Zirô, 1965), while its biosynthesis by decarboxylation of UDP-glucuronic acid has been demonstrated in preparations from hen oviduct ( Bdolah and Feingold, 1965). The present communication describes a particulate system from the oviduct of laying hens, which catalyzes the transfer of xylose from UDP-xylose into glycoprotein.