Characterization of the <i>galU</i> Gene of <i>Streptococcus pneumoniae</i> Encoding a Uridine Diphosphoglucose Pyrophosphorylase: A Gene Essential for Capsular Polysaccharide Biosynthesis

Mollerach, Marta; López, Rubens; Garcı́a, Ernesto

doi:10.1084/jem.188.11.2047

Cited by 86 publications

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References 51 publications

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“…On the basis of sequence similarities and the observation that genes in the cps loci of S. pneumoniae seem to be arranged in the order that the assembly of the repeating unit is expected to occur, we anticipate that Cps9vG, Cps9vJ and Cps9vL exhibit α-1,3-galactosyltransferase, α-1,3-glucuronic acid transferase and α-1,4-glucosyltransferase activities, respectively. Some of the substrates used in these reactions are available in the pneumococcus via the activity of the GlcNAc-1-phosphate uridyltransferase (GlmU) (Sulzenbacher et al, 2001), UTP-glucose-1-phosphate uridylyltransferase (GalU) (Mollerach et al, 1998) and UDP-glucose-4-epimerase (GalE) (Tettelin et al, 2001) that are located outside the 9V locus. The substrates UPD-ManNAc and UDP-glucuronic acid, which do not seem to be part of 9V housekeeping, are synthesized with the help of the Cps9vH and Cps9vN proteins encoded by the 9V locus.…”

Section: Discussionmentioning

confidence: 99%

Organization and characterization of the capsule biosynthesis locus of Streptococcus pneumoniae serotype 9V The GenBank accession number for the sequence reported in this paper is AF402095.

Selm

Kolkman²,

Zeijst

et al. 2002

Microbiology

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The capsular polysaccharide (CPS) synthesis locus of Streptococcus pneumoniae serotype 9V was amplified by long-range PCR and sequenced. The locus was 17 368 bp in size and contained 15 ORFs. The genetic organization of the cluster shared many features with other S. pneumoniae capsule loci, including the presence of four putative regulatory genes at the 5' end. Comparative sequence analyses allowed putative functions to be assigned to each of the gene products. The ORFs appeared to encode, besides the four regulatory genes, five glycosyltransferases, two O-acetyltransferases, an Nacetylglucosamine 2-epimerase, a glucose 6-dehydrogenase, an oligosaccharide transporter protein and a polysaccharide repeating unit polymerase. These functions covered the steps proposed in the CPS biosynthesis of serotype 9V. TLC of carbohydrate intermediates formed after incubation of bacterial membrane preparations with14 C-labelled precursors demonstrated that the fifth ORF (cps9vE) encoded a UDP-glucosyl-1-phosphate transferase. This function was confirmed with the help of a cps9vE mutant that carried a deletion of a guanine residue located adjacent to a stretch of adenines. The identification and characterization of the serotype 9V locus is a major step in unravelling the 9V capsule biosynthesis pathway and broadens the insight into the genetic diversity of the S. pneumoniae capsule loci.

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Section: Discussionmentioning

confidence: 99%

Organization and characterization of the capsule biosynthesis locus of Streptococcus pneumoniae serotype 9V The GenBank accession number for the sequence reported in this paper is AF402095.

Selm

Kolkman²,

Zeijst

et al. 2002

Microbiology

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“…Interestingly, while the prokaryotic and eukaryotic forms of UGPase catalyze the same reaction, they are completely unrelated in amino acid sequence and three-dimensional structure (Flores-Diaz et al 1997;Mollerach et al 1998;Mollerach and Garcia 2000). Within recent years, the bacterial UGPases have become targets for drug design given their biological roles in various Gram-negative bacteria (Genevaux et al 1999).…”

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confidence: 99%

The molecular architecture of glucose‐1‐phosphate uridylyltransferase

Thoden

Holden

2007

Protein Science

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Glucose-1-phosphate uridylyltransferase, also referred to as UDP-glucose pyrophosphorylase or UGPase, catalyzes the formation of UDP-glucose from glucose-1-phosphate and UTP. Not surprisingly, given the central role of UDP-glucose in glycogen synthesis and in the production of glycolipids, glycoproteins, and proteoglycans, the enzyme is ubiquitous in nature. Interestingly, however, the prokaryotic and eukaryotic forms of the enzyme are unrelated in amino acid sequence and structure. Here we describe the cloning and structural analysis to 1.9 Å resolution of the UGPase from Escherichia coli. The protein is a tetramer with 222 point group symmetry. Each subunit of the tetramer is dominated by an eight-stranded mixed b-sheet. There are two additional layers of b-sheet (two and three strands) and 10 a-helices. The overall fold of the molecule is remarkably similar to that observed for glucose-1-phosphate thymidylyltransferase in complex with its product, dTDP-glucose. On the basis of this similarity, a UDP-glucose moiety has been positioned into the active site of UGPase. This protein/product model predicts that the side chains of Gln 109 and Asp 137, respectively, serve to anchor the uracil ring and the ribose of UDP-glucose to the protein. The b-phosphoryl group of the product is predicted to lie within hydrogen bonding distance to the e-nitrogen of Lys 202 whereas the carboxylate group of Glu 201 is predicted to bridge the 29-and 39-hydroxyl groups of the glucosyl moiety. Details concerning the overall structure of UGPase and a comparison with glucose-1-phosphate thymidylyltransferase are presented.Keywords: UDP-glucose; glucose-1-phosphate uridylyltransferase; UDP-glucose pyrophosphorylase; X-ray structure; carbohydrates The conversion of b-D-galactose to the more metabolically useful glucose-1-phosphate is accomplished in most organisms by the action of four enzymes that constitute the Leloir pathway (Holden et al. 2003). In the first step of this pathway, b-D-galactose is epimerized to a-D-galactose by galactose mutarotase. The next step involves the ATP-dependent phosphorylation of a-Dgalactose by galactokinase to yield galactose-1-phosphate. Subsequently, in the third step, the enzyme galactose-1-phosphate uridylyltransferase catalyzes the transfer of a UMP group from UDP-glucose to galactose-1-phosphate, thereby generating glucose-1-phosphate and UDP-galactose. To complete the pathway, UDP-galactose is converted to UDP-glucose by UDPgalactose 4-epimerase. In humans, mutations in the genes that encode the galactokinase, the galactose-1-phosphate uridylyltransferase, or the epimerase can result in the diseased state referred to as galactosemia with clinical manifestations including intellectual retardation, speech disorders, liver dysfunction, and cataract formation. The most common form of galactosemia arises from defects in galactose-1-phosphate uridylyltransferase and as such it has been the subject of intensive kinetic and structural investigations for many years (Holden et al. 2003). Studies have...

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“…Additional genes in this region may control the synthesis of nucleotide-linked sugars that are serotype-specific polysaccharide precursors, such as dTDP-Rha, while genes that reside outside CPS clusters control the synthesis of precursors with physiological roles that extend beyond CPS biosynthesis. Examples include UDP-Glc and UDP-Gal, which in addition to being CPS precursors (20,32) are essential for bacterial utilization of galactose by the Leloir pathway (16).…”

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confidence: 99%

Genetic Loci for Coaggregation Receptor Polysaccharide Biosynthesis inStreptococcus gordonii38

Thompson

Cisar

2003

J Bacteriol

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The cell wall polysaccharide of Streptococcus gordonii 38 functions as a coaggregation receptor for surface adhesins on other members of the oral biofilm community. The structure of this receptor polysaccharide (RPS) is defined by a heptasaccharide repeat that includes a GalNAc␤133Gal-containing recognition motif. The same RPS has now been identified from S. gordonii AT, a partially sequenced strain. PCR primers designed from sequences in the genomic database of strain AT were used to identify and partially characterize the S. gordonii 38 RPS gene cluster. This cluster includes genes for seven putative glycosyltransferases, a polysaccharide polymerase (Wzy), an oligosaccharide repeating unit transporter (Wzx), and a galactofuranose mutase, the enzyme that promotes synthesis of UDP-Galf, one of five predicted RPS precursors. Genes outside this region were identified for the other four nucleotide-linked sugar precursors of RPS biosynthesis, namely, those for formation of UDP-Glc, UDP-Gal, UDP-GalNAc, and dTDP-Rha. Two genes for putative galactose 4-epimerases were identified. The first, designated galE1, was identified as a pseudogene in the galactose operon, and the second, designated galE2, was transcribed with three of the four genes for dTDP-Rha biosynthesis (i.e., rmlA, rmlC, and rmlB). Insertional inactivation of galE2 abolished (i) RPS production, (ii) growth on galactose, and (iii) both UDP-Gal and UDP-GalNAc 4-epimerase activities in cell extracts. Repair of the galE1 pseudogene in this galE2 mutant restored growth on galactose but not RPS production. Cell extracts containing functional GalE1 but not GalE2 contained UDP-Gal 4-epimerase but not UDP-GalNAc 4-epimerase activity. Thus, provision of both UDP-Gal and UDP-GalNAc for RPS production by S. gordonii 38 depends on the dual specificity of the epimerase encoded by galE2.Specific interactions between different bacteria play an important role in development of the simple biofilm community that forms during primary colonization of the human tooth surface (36-38). The lactose-sensitive coaggregations between type 2 fimbriated strains of Actinomyces naeslundii and receptor-bearing strains of Streptococcus sanguinis, Streptococcus gordonii, Streptococcus oralis, and Streptococcus mitis are wellstudied examples of such interactions (10,19). Structural characterization of the cell wall polysaccharides isolated from over 20 receptor-bearing streptococcal strains has resulted in the identification of six different receptor polysaccharides (RPS) (1-4, 12, 30, 39), which are designated types 1Gn, 2Gn, 2G, 3G, 4Gn, and 5Gn RPS to reflect the structural relationships that exist between the different phosphodiester-linked, hexa-or heptasaccharide repeating units of these molecules (12). Each RPS repeating unit contains a host-like recognition motif consisting of Galf linked ␤136 to either Gal␤133GalNAc (G) or GalNAc␤133Gal (Gn). These features are recognized as receptors by Gal-and GalNAc-binding surface adhesins on other bacteria, such as A. naeslundii (11), while o...

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Characterization of the galU Gene of Streptococcus pneumoniae Encoding a Uridine Diphosphoglucose Pyrophosphorylase: A Gene Essential for Capsular Polysaccharide Biosynthesis

Cited by 86 publications

References 51 publications

Organization and characterization of the capsule biosynthesis locus of Streptococcus pneumoniae serotype 9V The GenBank accession number for the sequence reported in this paper is AF402095.

Organization and characterization of the capsule biosynthesis locus of Streptococcus pneumoniae serotype 9V The GenBank accession number for the sequence reported in this paper is AF402095.

The molecular architecture of glucose‐1‐phosphate uridylyltransferase

Genetic Loci for Coaggregation Receptor Polysaccharide Biosynthesis inStreptococcus gordonii38

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