Identification and Molecular Cloning of a Heparosan Synthase fromPasteurella multocida Type D

DeAngelis, Paul L.; White, Carissa L.

doi:10.1074/jbc.m112130200

Cited by 80 publications

(38 citation statements)

References 21 publications

(32 reference statements)

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“…Reaction conditions were 50 mM Tris, pH 7.2, 5 mM MnCl 2 , 1 M ethylene glycol, 12.2 mM UDP-GlcUA, 12.2 mM UDP-GlcNAc, and 14 M PmHAS plus A-F-A acceptor at three different concentrations (8,80, and 800 M) in a total volume of 25 l. The reactions were incubated at 30°C overnight. The size of the products was analyzed using agarose gel electrophoresis (1.2%; 1ϫ TAE buffer (40 mM Tris acetate, 2 mM EDTA); 30 V) and Stains-All dye detection (0.005% w/v in ethanol) (13).…”

Section: Methodsmentioning

confidence: 99%

“…An acceptor molecule will bypass the slow PmHAS initiation step, resulting in synchronized reactions that yield relatively monodisperse polymer products. Polymerization reactions with A-F-A at three different concentrations (8,80, and 800 M) were performed and analyzed by gel electrophoresis (Fig. 4).…”

Section: Analysis Of Synthetic Analogs As Acceptors For Pmhas-mentioning

confidence: 99%

“…The Escherichia coli K4 chondroitin polymerase KfoC (7) is ϳ60% identical to PmHAS and PmCS; therefore, this system probably operates in a similar fashion. The recently described P. multocida heparosan synthases, one from Type D (PmHS1) (8) and another cryptic enzyme from Types A, D, and F (PmHS2) (9), promise to be interesting experimental models of dual action enzymes as well, but their overall amino acid sequence appears to differ from PmHAS and PmCS.…”

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Critical Elements of Oligosaccharide Acceptor Substrates for the Pasteurella multocida Hyaluronan Synthase

Williams

Halkes

Kamerling

et al. 2006

Journal of Biological Chemistry

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Three-dimensional structures are not available for polysaccharide synthases and only minimal information on the molecular basis for catalysis is known. The Pasteurella multocida hyaluronan synthase (PmHAS) catalyzes the polymerization of the alternating ␤1,3-N-acetylglucosamine-␤1,4-glucuronic acid sugar chain by the sequential addition of single monosaccharides to the non-reducing terminus. Therefore, PmHAS possesses both GlcNAc-transferase and glucuronic acid (GlcUA)-transferase activities. The recombinant Escherichia coli-derived PmHAS enzyme will elongate exogenously supplied hyaluronan chains in vitro with either a single monosaccharide or a long chain depending on the UDP-sugar availability. Competition studies using pairs of acceptors with distinct termini (where one oligosaccharide is a substrate that may be elongated, whereas the other cannot) were performed here; the lack of competition suggests that PmHAS contains at least two distinct acceptor sites. We hypothesize that the size of the acceptor binding pockets of the enzyme corresponds to the size of the smallest high efficiency substrates; thus we tested the relative activity of a series of authentic hyaluronan oligosaccharides and related structural analogs. The GlcUA-transferase site readily elongates (GlcNAcGlcUA) 2 , whereas the GlcNAc-transferase elongates GlcUA-GlcNAc-GlcUA. The minimally sized oligosaccharides, elongated with high efficiency, both contain a trisaccharide with two glucuronic acid residues that enabled the identification of a synthetic, artificial acceptor for the synthase. PmHAS behaves as a fusion of two complete glycosyltransferases, each containing a donor site and an acceptor site, in one polypeptide. Overall, this information advances the knowledge of glycosaminoglycan biosynthesis as well as assists the creation of various therapeutic sugars for medical applications in the future.Glycosaminoglycans, linear chains composed of alternating disaccharide repeats containing a hexosamine, are a class of heteropolysaccharides that includes hyaluronan (HA), 2 heparin, chondroitin, and keratan. These carbohydrates serve essential roles in vertebrates, including as intracellular adhesives, signaling molecules, anticoagulants, and structural elements. Certain pathogenic bacteria take advantage of these versatile molecules to produce extracellular glycosaminoglycan capsules, thus constructing molecular camouflage to avoid host defenses and increase virulence.The bacterial Pasteurella multocida synthases are dual action enzymes that add both sugars of the glycosaminoglycan repeat to the non-reducing terminus of the acceptor oligosaccharides (1). The P. multocida Type A hyaluronan synthase PmHAS and the P. multocida Type F chondroitin synthase PmCS have been amenable to study due to their two active center architectures (2, 3), their ability to polymerize long chains in vitro (4, 5), and their ability to elongate certain exogenously added acceptor oligosaccharides (6). The Escherichia coli K4 chondroitin polymerase KfoC (7) is ϳ60% ide...

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Section: Methodsmentioning

confidence: 99%

Section: Analysis Of Synthetic Analogs As Acceptors For Pmhas-mentioning

confidence: 99%

mentioning

confidence: 99%

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Critical Elements of Oligosaccharide Acceptor Substrates for the Pasteurella multocida Hyaluronan Synthase

Williams

Halkes

Kamerling

et al. 2006

Journal of Biological Chemistry

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“…P. multocida Type D strains encode the 617-residue PmHS1 enzyme in a typical Gram-negative bacterial Type 2 capsule biosynthesis locus (4). Another isozyme, the 651-residue PmHS2, is found in many Type A, D, and F strains (5); the genes are ϳ73% identical.…”

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Functional Characterization of PmHS1, a Pasteurella multocida Heparosan Synthase

Kane

White

DeAngelis

2006

Journal of Biological Chemistry

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Heparosan synthase 1 (PmHS1) from Pasteurella multocida Type D is a dual action glycosyltransferase enzyme that transfers monosaccharide units from uridine diphospho (UDP) sugar precursors to form the polysaccharide heparosan (N-acetylheparosan), which is composed of alternating (-␣4-GlcNAc-␤1,4-GlcUA-1-) repeats. We have used molecular genetic means to remove regions nonessential for catalytic activity from the amino-and the carboxyl-terminal regions as well as characterized the functional regions involved in GlcUA-transferase activity and in GlcNAc-transferase activity. Mutation of either one of the two regions containing aspartate-X-aspartate (DXD) residue-containing motifs resulted in complete or substantial loss of heparosan polymerizing activity. However, certain mutant proteins retained only GlcUA-transferase activity while some constructs possessed only GlcNAc-transferase activity. Therefore, it appears that the PmHS1 polypeptide is composed of two types of glycosyltransferases in a single polypeptide as was found for the Pasteurella multocida Type A PmHAS, the hyaluronan synthase that makes the alternating (-␤3-GlcNAc-␤1,4-GlcUA-1-) polymer. However, there is low amino acid similarity between the PmHAS and PmHS1 enzymes, and the relative placement of the GlcUA-transferase and GlcNAc-transferase domains within the two polypeptides is reversed. Even though the monosaccharide compositions of hyaluronan and heparosan are identical, such differences in the sequences of the catalysts are expected because the PmHAS employs only inverting sugar transfer mechanisms whereas PmHS1 requires both retaining and inverting mechanisms.The Gram-negative bacterial pathogen Pasteurella multocida Type D is known to cause atrophic rhinitis in swine and pasteurellosis in other domestic animals (1). This microbe produces an extracellular coating of polysaccharide, called a capsule, composed of heparosan (N-acetyl-heparosan or unsulfated, unepimerized heparin) (2) to enhance infection. It is thought that the capsule confers resistance to nonspecific host immunity and perhaps mediates adhesion to certain host cells. In general, most capsules are antigenic, but glycosaminoglycan polysaccharide capsules are a special case because they closely resemble the host molecules and thus may serve as molecular camouflage (3).Two heparosan synthase (PmHS) 2 enzymes have been identified from P. multocida that catalyze the formation of the repeating disaccharide (-4GlcNAc␣1-4GlcUA␤1-) units of the heparosan chain. P. multocida Type D strains encode the 617-residue PmHS1 enzyme in a typical Gram-negative bacterial Type 2 capsule biosynthesis locus (4). Another isozyme, the 651-residue PmHS2, is found in many Type A, D, and F strains (5); the genes are ϳ73% identical. The gene encoding PmHS2 is found in a different region of the chromosome not associated with typical carbohydrate biosynthesis genes and may be expressed during some stage of infection, but its role is not yet known.The dual action PmHS1 and PmHS2 are complex enzymes because the U...

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“…Heparosan is extracted from the capsular polysaccharide (CPS) of Escherichia coli K5, Pasteurella multocida, and E. coli strain Nissle 1917 (EcN) (8,10,11). The proteins encoded by the kps locus govern the biosynthesis and export of heparosan (Fig.…”

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Cyclic AMP (cAMP) Receptor Protein-cAMP Complex Regulates Heparosan Production in Escherichia coli Strain Nissle 1917

Yan

Bao

Zhao

et al. 2015

Appl Environ Microbiol

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cHeparosan serves as the starting carbon backbone for the chemoenzymatic synthesis of heparin, a widely used clinical anticoagulant drug. The availability of heparosan is a significant concern for the cost-effective synthesis of bioengineered heparin. The carbon source is known as the pivotal factor affecting heparosan production. However, the mechanism by which carbon sources control the biosynthesis of heparosan is unclear. In this study, we found that the biosynthesis of heparosan was influenced by different carbon sources. Glucose inhibits the biosynthesis of heparosan, while the addition of either fructose or mannose increases the yield of heparosan. Further study demonstrated that the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex binds to the upstream region of the region 3 promoter and stimulates the transcription of the gene cluster for heparosan biosynthesis. Site-directed mutagenesis of the CRP binding site abolished its capability of binding CRP and eliminated the stimulative effect on transcription.1 H nuclear magnetic resonance (NMR) analysis was further performed to determine the Escherichia coli strain Nissle 1917 (EcN) heparosan structure and quantify extracellular heparosan production. Our results add to the understanding of the regulation of heparosan biosynthesis and may contribute to the study of other exopolysaccharide-producing strains.

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Identification and Molecular Cloning of a Heparosan Synthase fromPasteurella multocida Type D

Cited by 80 publications

References 21 publications

Critical Elements of Oligosaccharide Acceptor Substrates for the Pasteurella multocida Hyaluronan Synthase

Critical Elements of Oligosaccharide Acceptor Substrates for the Pasteurella multocida Hyaluronan Synthase

Functional Characterization of PmHS1, a Pasteurella multocida Heparosan Synthase

Cyclic AMP (cAMP) Receptor Protein-cAMP Complex Regulates Heparosan Production in Escherichia coli Strain Nissle 1917

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