Characterization of the Glycosyltransferase Enzyme from theEscherichia coli K5 Capsule Gene Cluster and Identification and Characterization of the Glucuronyl Active Site

Pasteurella multocida Type D, a causative agent of atrophic rhinitis in swine and pasteurellosis in other domestic animals, produces an extracellular polysaccharide capsule that is a putative virulence factor. It was reported previously that the capsule was removed by treating microbes with heparin lyase III. We molecularly cloned a 617-residue enzyme, pmHS, which is a heparosan (nonsulfated, unepimerized heparin) synthase. Recombinant Escherichia coli-derived pmHS catalyzes the polymerization of the monosaccharides from UDP-GlcNAc and UDP-GlcUA. Other structurally related sugar nucleotides did not substitute. Synthase activity was stimulated about 7-25-fold by the addition of an exogenous polymer acceptor. Molecules composed of ϳ500 -3,000 sugar residues were produced in vitro. The polysaccharide was sensitive to the action of heparin lyase III but resistant to hyaluronan lyase. The sequence of the pmHS enzyme is not very similar to the vertebrate heparin/heparan sulfate glycosyltransferases, EXT1 and 2, or to other Pasteurella glycosaminoglycan synthases that produce hyaluronan or chondroitin. The pmHS enzyme is the first microbial dual-action glycosyltransferase to be described that forms a polysaccharide composed of ␤4GlcUA-␣4GlcNAc disaccharide repeats. In contrast, heparosan biosynthesis in E. coli K5 requires at least two separate polypeptides, KfiA and KfiC, to catalyze the same polymerization reaction.

Section: Molecular Cloning Of the Type D P Multocida Heparosanmentioning

confidence: 99%

“…At first, KfiC was stated to be a dual-action glycosyltransferase responsible for the alternating addition of both GlcUA and GlcNAc to the heparosan chain (7). However, a later report by the same group reported that another protein, KfiA, was actually the ␣GlcNAc-transferase required for heparosan polymerization (8).…”

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confidence: 99%

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

DeAngelis

White

2002

“…The inactivation of the catalytic domain by substitution of both aspartic acids by asparagines has been described for P. multocida hyaluronan synthase PmHAS (9,10) and for PmHS1 (heparosan synthase 1) (11). Here we describe, based on amino acid sequence homology with PmHS1 and with Escherichia coli heparosan synthase KfiC and KfiA (12)(13)(14), the construction of the two PmHS2 single action transferases (PmHS2-GlcUA ϩ and PmHS2-GlcNAc ϩ ). Both the characterization of the single action transferases and a detailed study of the polymerization process of PmHS2 allowed us to investigate the initiation process of heparosan chains.…”

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confidence: 99%

Analysis of the Polymerization Initiation and Activity of Pasteurella multocida Heparosan Synthase PmHS2, an Enzyme with Glycosyltransferase and UDP-sugar Hydrolase Activity

Chavaroche

Broek

Springer

et al. 2011

Heparosan synthase catalyzes the polymerization of heparosan (-4GlcUA␤1-4GlcNAc␣1-) n by transferring alternatively the monosaccharide units from UDP-GlcUA and UDP-GlcNAc to an acceptor molecule. Details on the heparosan chain initiation by Pasteurella multocida heparosan synthase PmHS2 and its influence on the polymerization process have not been reported yet. By site-directed mutagenesis of PmHS2, the single action transferases PmHS2-GlcUA ؉ and PmHS2-GlcNAc ؉ were obtained.When incubated together in the standard polymerization conditions, the PmHS2-GlcUA ؉ /PmHS2-GlcNAc ؉ showed comparable polymerization properties as determined for PmHS2. We investigated the first step occurring in heparosan chain initiation by the use of the single action transferases and by studying the PmHS2 polymerization process in the presence of heparosan templates and various UDP-sugar concentrations. We observed that PmHS2 favored the initiation of the heparosan chains when incubated in the presence of an excess of UDP-GlcNAc. It resulted in a higher number of heparosan chains with a lower average molecular weight or in the synthesis of two distinct groups of heparosan chain length, in the absence or in the presence of heparosan templates, respectively. These data suggest that PmHS2 transfers GlcUA from UDP-GlcUA moiety to a UDP-GlcNAc acceptor molecule to initiate the heparosan polymerization; as a consequence, not only the UDP-sugar concentration but also the amount of each UDP-sugar is influencing the PmHS2 polymerization process. In addition, it was shown that PmHS2 hydrolyzes the UDP-sugars, UDP-GlcUA being more degraded than UDP-GlcNAc. However, PmHS2 incubated in the presence of both UDP-sugars favors the synthesis of heparosan polymers over the hydrolysis of UDP-sugars.

“…Both conservative and non-conservative mutations abolished N-acetylglucosaminyltransferase activity. Similarly, mutation of any one of several aspartic acid residues in KfiC resulted in total loss of the relevant GlcUA-transferase activity (10). These data, however, are not definitive because the mutagenized enzymes did not retain any activity, and it is difficult to draw conclusions from any inactive enzyme because of the potential for global misfolding.…”

mentioning

confidence: 71%

“…The K5 antigen region 2 encodes four proteins designated KfiA to KfiD involved in synthesizing the sugar chain (8). KfiA was discovered to act as an ␣4-N-acetylglucosaminyltransferase (9) whereas KfiC is the ␤4-glucuronyltransferase (although initially the latter was erroneously reported to be a dual action enzyme responsible for heparosan polymerization (10)). Therefore, in E. coli the formation of the heparosan polymer occurs by the concerted action of KfiA and KfiC proteins.…”

mentioning

confidence: 99%

Functional Characterization of PmHS1, a Pasteurella multocida Heparosan Synthase

Kane

White

DeAngelis

2006

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...