Uridine diphosphate α-<scp>d</scp>-galactofuranose, an intermediate in the biosynthesis of galactofuranosyl residues

Trejo, Antonio García; Chittenden, G. J. F.; Buchanan, J. G.; Baddiley, J.

doi:10.1042/bj1170637

Cited by 40 publications

(16 citation statements)

References 17 publications

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“…Trejo et al (46) isolated a nucleotide sugar formed in cell-free enzyme preparations of P. charlesii, which was capable of acting as a donor of galactofuranosyl residues in the biosynthesis of a galactofuranosylcontaining polymer. Based on chemical analysis of the reaction products obtained by acid and alkaline hydrolosis as well as periodate oxidation, Trejo et al (46) predicted that the sugar nucleotide is UDP-Galf. More recently, Nassau et al (48) cloned the glf gene of E. coli and showed that it encodes UDP-galactopyranose mutase (EC 5.4.99.9).…”

Section: Discussionmentioning

confidence: 99%

“…In bacteria (this work, and Refs. 44, 45, and 48) and fungi (46), the biosynthetic precursor for Galf residues is believed to be UDPGalf. Galactofuranosyl-containing glycoconjugates have not been reported in humans so far, which makes UDP-Galf formation an interesting target for novel therapeutic compounds (38).…”

Section: Discussionmentioning

confidence: 99%

“…Early studies on the biosynthesis of the T1 antigen in S. enterica serovar Typhimurium (44,45) suggested that Galf precursors were derived from Galp at the level of UDP-linked sugars. The Galf precursor for a galactofuranosyl-containing polysaccharide in Penicillium charlesii was also proposed to be a UDP-linked derivative (46). More recently, the product of orf6, an enzyme encoded by the cryptic rfb locus in E. coli K-12 strains (47), was shown to have UDP-galactopyranose mutase (EC 5.4.99.9) activity, capable of the reversible formation of UDP-Galf from UDP-Galp (48).…”

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UDP-galactofuranose Precursor Required for Formation of the Lipopolysaccharide O Antigen of Klebsiella pneumoniae Serotype O1 Is Synthesized by the Product of the rfbDKPO1 Gene

Koplin

Brisson

Whitfield

1997

Journal of Biological Chemistry

123

100

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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UDP-galactofuranose Precursor Required for Formation of the Lipopolysaccharide O Antigen of Klebsiella pneumoniae Serotype O1 Is Synthesized by the Product of the rfbDKPO1 Gene

Koplin

Brisson

Whitfield

1997

Journal of Biological Chemistry

123

100

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“…Early experiments suggested that intact P. charlesii cells could not utilize exogenous galactose for galactocarolose biosynthesis (7). Cell extracts of this organism were subsequently found to incorporate either glucose or glucose-1-phosphate into galactocarolose (21). In the presence of Zn 2ϩ or F Ϫ ions, polysaccharide synthesis was inhibited and UDP-galactofuranose was found to accumulate, suggesting that this nucleotide sugar might be the activated galactofuranose precursor in galactocarolose biosynthesis.…”

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confidence: 99%

Galactofuranose biosynthesis in Escherichia coli K-12: identification and cloning of UDP-galactopyranose mutase

et al. 1996

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We have cloned two open reading frames (orf6 and orf8) from the Escherichia coli K-12 rfb region. The genes were expressed in E. coli under control of the T7lac promoter, producing large quantities of recombinant protein, most of which accumulated in insoluble inclusion bodies. Sufficient soluble protein was obtained, however, for use in a radiometric assay designed to detect UDP-galactopyranose mutase activity (the conversion of UDP-galactopyranose to UDP-galactofuranose). The assay is based upon high-pressure liquid chromatography separation of sugar phosphates released from both forms of UDP-galactose by phosphodiesterase treatment. The crude orf6 gene product converted UDP-[␣-D-U-14 C]-galactopyranose to a product which upon phosphodiesterase treatment gave a compound with a retention time identical to that of synthetic ␣-galactofuranose-1-phosphate. No mutase activity was detected in extracts from cells lacking the orf6 expression plasmid or from orf8-expressing cells. The orf6 gene product was purified by anion-exchange chromatography and hydrophobic interaction chromatography. Both the crude extract and the purified protein converted 6 to 9% of the UDP-galactopyranose to the furanose form. The enzyme was also shown to catalyze the reverse reaction; in this case an approximately 86% furanose-to-pyranose conversion was observed. These observations strongly suggest that orf6 encodes UDP-galactopyranose mutase (EC 5.4.99.9), and we propose that the gene be designated glf accordingly. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified UDPgalactopyranose mutase revealed one major band, and analysis by electrospray mass spectrometry indicated a single major species with a molecular weight of 42,960 ؎ 8, in accordance with that calculated for the Glf protein. N-terminal sequencing revealed that the first 15 amino acids of the recombinant protein corresponded to those expected from the published sequence. UV-visible spectra of purified recombinant enzyme indicated that the protein contains a flavin cofactor, which we have identified as flavin adenine dinucleotide.

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“…Again, by analogy with other polysaccharide synthetic systems (33,34), G6P is converted to G1P and thence to UDP-glucose; an activity has been identified in mycobacteria which converts UDP-glucose to UDP-galactose for transfer into the AG complex (16). Inhibition of AG synthesis is lethal for the mycobacterial cell and is most likely the mechanism of action of ethambutol (29).…”

Section: Discussionmentioning

confidence: 99%

Interruption of the phosphoglucose isomerase gene results in glucose auxotrophy in Mycobacterium smegmatis

et al. 1997

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Two glycerol utilization mutants of Mycobacterium smegmatis that were unable to utilize most carbon sources except glucose were isolated. Supplementation of these media with small amounts of glucose restored growth in the mutants; these strains are therefore glucose auxotrophs. The mutant phenotype is complemented by the gene encoding phosphoglucose isomerase (pgi), and direct measurement of enzyme activities in the mutants suggests that this gene product is absent in the auxotrophic strains. Mapping of the mutant allele by Southern analysis demonstrates the presence of a 1-kb deletion extending into the coding sequence of pgi. The possible roles of phosphoglucose isomerase in mycobacterial cell wall synthesis and metabolic regulation are discussed.The resurgence of diseases caused by mycobacteria has spurred a new interest in these acid-fast organisms. Although current research is largely directed towards virulence mechanisms in pathogenic strains of mycobacteria, basic biochemical information about these organisms is essential for understanding their interactions with their hosts and for developing new therapeutics. Knowledge of intermediary metabolism of the mycobacteria has been fragmentary, in large part due to the low growth rates, tendency to clump in culture, and lack of genetics systems of members of this genus. Mycobacteria exhibit unusual and complex patterns of carbohydrate utilization. In Mycobacterium tuberculosis, Mycobacterium phlei, and Mycobacterium smegmatis, glycerol utilization is blocked until all other carbon sources, including glutamate and asparagine, have been consumed (3,23,38). Nevertheless, glycerol is considered the preferred carbon source under laboratory conditions. Growth of most species on this substrate results in greater mass per unit volume of growth medium (32).Glycerol and its metabolic derivatives are at the interface of catabolic (glycolytic and oxidative pathways) and anabolic (lipid biosynthesis and gluconeogenesis) processes and therefore play important growth regulatory roles in cells grown on a variety of carbon sources. In addition, glycerol is an important precursor of key cell wall constituents. The presence of glycolipids such as the acyl glycerols and acylglucoses varies widely depending on the carbon source in the medium (4). A number of the enzymes of glycolysis, the hexose monophosphate shunt, and the Entner-Doudoroff pathways have been studied (reviewed in reference 26). However, carbon metabolism in mycobacteria has not been characterized at the genetic level and few genes encoding glycolytic enzymes have been cloned.We have initiated a genetic approach to the study of carbohydrate metabolism in M. smegmatis by isolating and characterizing mutants unable to utilize certain substrates as sole carbon sources. In this study, we describe two strains obtained in a screen for glycerol utilization mutants. Unexpectedly, these mutants are unable to use any carbohydrate except glucose as a sole carbon source. Genetic complementation of the growth defect on glycerol ...

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Uridine diphosphate α-d-galactofuranose, an intermediate in the biosynthesis of galactofuranosyl residues

Cited by 40 publications

References 17 publications

UDP-galactofuranose Precursor Required for Formation of the Lipopolysaccharide O Antigen of Klebsiella pneumoniae Serotype O1 Is Synthesized by the Product of the rfbDKPO1 Gene

UDP-galactofuranose Precursor Required for Formation of the Lipopolysaccharide O Antigen of Klebsiella pneumoniae Serotype O1 Is Synthesized by the Product of the rfbDKPO1 Gene

Galactofuranose biosynthesis in Escherichia coli K-12: identification and cloning of UDP-galactopyranose mutase

Interruption of the phosphoglucose isomerase gene results in glucose auxotrophy in Mycobacterium smegmatis

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