Identification of a distinct, cryptic heparosan synthase from Pasteurella multocida types A, D, and F.
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SUBMITTER: Deangelis PL
PROVIDER: S-EPMC532432 | biostudies-literature | 2004 Dec
REPOSITORIES: biostudies-literature
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Project description:In vertebrates and bacteria, heparosan the precursor of heparin is synthesized by glycosyltransferases via the stepwise addition of UDP-N-acetylglucosamine and UDP-glucuronic acid. As heparin-like molecules represent a great interest in the pharmaceutical area, the cryptic Pasteurella multocida heparosan synthase PmHS2 found to catalyze heparosan synthesis using substrate analogs has been studied. In this paper, we report an efficient way to purify PmHS2 and to maintain its activity stable during 6 months storage at -80 degrees Celsius using His-tag purification and a desalting step. In the presence of 1 mM of each nucleotide sugar, purified PmHS2 synthesized polymers up to an average molecular weight of 130 kDa. With 5 mM of UDP-GlcUA and 5 mM of UDP-GlcNAc, an optimal specific activity, from 3 to 6 h of incubation, was found to be about 0.145 nmol/microg/min, and polymers up to an average of 102 kDa were synthesized in 24 h. In this study, we show that the chain length distribution of heparosan polymers can be controlled by change of the initial nucleotide sugar concentration. It was observed that low substrate concentration favors the formation of high molecular weight heparosan polymer with a low polydispersity while high substrate concentration did the opposite. Similarities in the polymerization mechanism between PmHS2, PmHS1, and PmHAS are discussed.
| S-EPMC2811250 | biostudies-literature
Project description:BackgroundHeparosan is the unsulfated precursor of heparin and heparan sulfate and its synthesis is typically the first step in the production of bioengineered heparin. In addition to its utility as the starting material for this important anticoagulant and anti-inflammatory drug, heparosan is a versatile compound that possesses suitable chemical and physical properties for making a variety of high-quality tissue engineering biomaterials, gels and scaffolds, as well as serving as a drug delivery vehicle. The selected production host was the Gram-positive bacterium Bacillus megaterium, which represents an increasingly used choice for high-yield production of intra- and extracellular biomolecules for scientific and industrial applications.ResultsWe have engineered the metabolism of B. megaterium to produce heparosan, using a T7 RNA polymerase (T7 RNAP) expression system. This system, which allows tightly regulated and efficient induction of genes of interest, has been co-opted for control of Pasteurella multocida heparosan synthase (PmHS2). Specifically, we show that B. megaterium MS941 cells co-transformed with pT7-RNAP and pPT7_PmHS2 plasmids are capable of producing heparosan upon induction with xylose, providing an alternate, safe source of heparosan. Productivities of ~ 250 mg/L of heparosan in shake flasks and ~ 2.74 g/L in fed-batch cultivation were reached. The polydisperse Pasteurella heparosan synthase products from B. megaterium primarily consisted of a relatively high molecular weight (MW) heparosan (~ 200-300 kD) that may be appropriate for producing certain biomaterials; while the less abundant lower MW heparosan fractions (~ 10-40 kD) can be a suitable starting material for heparin synthesis.ConclusionWe have successfully engineered an asporogenic and non-pathogenic B. megaterium host strain to produce heparosan for various applications, through a combination of genetic manipulation and growth optimization strategies. The heparosan products from B. megaterium display a different range of MW products than traditional E. coli K5 products, diversifying its potential applications and facilitating increased product utility.
| S-EPMC6691538 | biostudies-literature
Project description:Pasteurella multocida heparosan synthase PmHS2 is a dual action glycosyltransferase that catalyzes the polymerization of heparosan polymers in a non-processive manner. The two PmHS2 single-action transferases, obtained previously by site-directed mutagenesis, have been immobilized on Ni(II)-nitrilotriacetic acid agarose during the purification step. A detailed study of the polymerization process in the presence of non-equal amounts of PmHS2 single-action transferases revealed that the glucuronyl transferase (PmHS2-GlcUA(+)) is the limiting catalyst in the polymerization process. Using experimental design, it was determined that the N-acetylglucosaminyl transferase (PmHS2-GlcNAc(+)) plays an important role in the control of heparosan chain elongation depending on the number of heparosan chains and the UDP-sugar concentrations present in the reaction mixture. Furthermore, for the first time, the synthesis of heparosan oligosaccharides alternately using PmHS2-GlcUA(+) and PmHS2-GlcNAc(+) is reported. It was shown that the synthesis of heparosan oligosaccharides by PmHS2 single-action transferases do not require the presence of template molecules in the reaction mixture.
| S-EPMC3418500 | biostudies-literature
Project description:The biological activities of heparan sulfate (HS) and heparin (HP) are closely related to their molecular structures. Both Pasteurella multocida heparosan synthase 2 (PmHS2) and Escherichia coli K5 KfiA have been used for enzymatic and chemoenzymatic synthesis of HS and HP oligosaccharides and their derivatives. We show here that cloning using the pET15b vector and expressing PmHS2 as an N-His6-tagged fusion protein improve its expression level in E. coli. Investigation of the donor substrate specificity of the N-acetylglucosaminyltransferase activities of P. multocida heparosan synthase 2 (PmHS2) and E. coli K5 KfiA indicates the substrate promiscuities of PmHS2 and KfiA. Overall, both PmHS2 and KfiA can use uridine 5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc) and some of its C2'- and C6'-derivatives as donor substrates for their α1-4-GlcNAcT activities. Nevertheless, PmHS2 has a broader tolerance towards substrate modifications. Other than the UDP-sugars that can be used by KfiA, additional C6'-derivatives of UDP-GlcNAc, UDP-glucose, and UDP-N-acetylgalactosamine (UDP-GalNAc) are tolerable substrates for the α1-4-GlcNAcT activity of PmHS2. The substrate promiscuities of PmHS2 and KfiA will allow efficient chemoenzymatic synthesis of diverse HS and HP oligosaccharide derivatives which may have improved or altered activities compared to their natural counterparts.
| S-EPMC9416980 | biostudies-literature
Project description: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.
| S-EPMC3023472 | biostudies-literature
Project description:Pasteurella multocida heparosan synthase 2 (PmHS2) is a dual-function polysaccharide synthase having both α1-4-N-acetylglucosaminyltransferase (α1-4-GlcNAcT) and β1-4-glucuronyltransferase (β1-4-GlcAT) activities located in two separate catalytic domains. We found that removing PmHS2 N-terminal 80-amino acid residues improved enzyme stability and expression level while retaining its substrate promiscuity. We also identified the reverse glycosylation activities of PmHS2 which complicated its application in size-controlled synthesis of oligosaccharides longer than hexasaccharide. Engineered Δ80PmHS2 single-function-glycosyltransferase mutants Δ80PmHS2_D291N (α1-4-GlcNAcT lacking both forward and reverse β1-4-GlcAT activities) and Δ80PmHS2_D569N (β1-4-GlcAT lacking both forward and reverse α1-4-GlcNAcT activities) were designed and showed to minimize side product formation. They were successfully used in a sequential one-pot multienzyme (OPME) platform for size-controlled high-yield production of oligosaccharides up to decasaccharide. The study draws attention to the consideration of reverse glycosylation activities of glycosyltransferases, including polysaccharide synthases, when applying them in the synthesis of oligosaccharides and polysaccharides. The mutagenesis strategy has the potential to be extended to other multifunctional polysaccharide synthases with reverse glycosylation activities to generate catalysts with improved synthetic efficiency.
| S-EPMC7842274 | biostudies-literature
Project description:The Pasteurella multocida heparosan synthases, PmHS1 and PmHS2, are homologous (∼65% identical) bifunctional glycosyltransferase proteins found in Type D Pasteurella. These unique enzymes are able to generate the glycosaminoglycan heparosan by polymerizing sugars to form repeating disaccharide units from the donor molecules UDP-glucuronic acid (UDP-GlcUA) and UDP-N-acetylglucosamine (UDP-GlcNAc). Although these isozymes both generate heparosan, the catalytic phenotypes of these isozymes are quite different. Specifically, during in vitro synthesis, PmHS2 is better able to generate polysaccharide in the absence of exogenous acceptor (de novo synthesis) than PmHS1. Additionally, each of these enzymes is able to generate polysaccharide using unnatural sugar analogs in vitro, but they exhibit differences in the substitution patterns of the analogs they will employ. A series of chimeric enzymes has been generated consisting of various portions of both of the Pasteurella heparosan synthases in a single polypeptide chain. In vitro radiochemical sugar incorporation assays using these purified chimeric enzymes have shown that most of the constructs are enzymatically active, and some possess novel characteristics including the ability to produce nearly monodisperse polysaccharides with an expanded range of sugar analogs. Comparison of the kinetic properties and the sequences of the wild-type enzymes with the chimeric enzymes has enabled us to identify regions that may be responsible for some aspects of both donor binding specificity and acceptor usage. In combination with previous work, these approaches have enabled us to better understand the structure/function relationship of this unique family of glycosyltransferases.
| S-EPMC3293577 | biostudies-literature
Project description:UnlabelledP. multocida is a Gram-negative pathogen responsible for causing diseases in animals of economic significance to livestock industries throughout the world. Current vaccines include bacterins, which provide only limited protection against homologous serotypes. Therefore there is a need for more effective vaccines to control diseases caused by P. multocida. As a step towards developing vaccines against fowl cholera, a genomics based approach was applied for the identification of novel immunogens.ResultsBioinformatics analysis of the P. multocida genome predicted 129 proteins as secreted, located in the outer membrane, or lipoproteins. 105 of the genes encoding these proteins were cloned and recombinant protein expressed in Escherichia coli. Polyclonal serum from P. multocida-infected chickens reacted with a subset of these proteins.ConclusionThese data show the range of bacterial immunogens recognized by the chicken immune system, including 6 novel immunoreactive proteins.
| S-EPMC1781955 | biostudies-literature
Project description:Pasteurella multocida, an encapsulated gram-negative bacterium, is a significant veterinary pathogen. The P. multocida is classified into 5 serogroups (A, B, D, E, and F) based on the bacterial capsular polysaccharide (CPS), which is important for virulence. Serogroups B and E are the primary causative agents of bovine hemorrhagic septicemia that is associated with significant yearly losses of livestock worldwide, primarily in low- and middle-income countries. The P. multocida disease is currently managed by whole-cell vaccination, albeit with limited efficacy. CPS is an attractive antigen target for an improved vaccine: CPS-based vaccines have proven highly effective against human bacterial diseases and could provide longer-term protection against P. multocida. The recently elucidated CPS repeat units of serogroups B and E both comprise a N-acetyl-β-D-mannosaminuronic acid/N-acetyl-β-D-glucosamine disaccharide backbone with β-D-fructofuranose (Fruf) side chain, but differ in their glycosidic linkages, and a glycine (Gly) side chain in serogroup B. Interestingly, the Haemophilus influenzae types e and d CPS have the same backbone residues. Here, comparative modeling of P. multocida serogroups B and E and H. influenzae types e and d CPS identifies a significant impact of small structural differences on both the chain conformation and the exposed potential antibody-binding epitopes (Ep). Further, Fruf and/or Gly side chains shield the immunogenic amino-sugar CPS backbone-a possible common strategy for immune evasion in both P. multocida and H. influenzae. As the lack of common epitopes suggests limited potential for cross-reactivity, a bivalent CPS-based vaccine may be necessary to provide adequate protection against P. multocida types B and E.
| S-EPMC10627249 | biostudies-literature
Project description:Pasteurella multocida is a highly versatile pathogen capable of causing infections in a wide range of domestic and wild animals as well as in humans and nonhuman primates. Despite over 135 years of research, the molecular basis for the myriad manifestations of P. multocida pathogenesis and the determinants of P. multocida phylogeny remain poorly defined. The current availability of multiple P. multocida genome sequences now makes it possible to delve into the underlying genetic mechanisms of P. multocida fitness and virulence. Using whole-genome sequences, the genotypes, including the capsular genotypes, lipopolysaccharide (LPS) genotypes, and multilocus sequence types, as well as virulence factor-encoding genes of P. multocida isolates from different clinical presentations can be characterized rapidly and accurately. Putative genetic factors that contribute to virulence, fitness, host specificity, and disease predilection can also be identified through comparative genome analysis of different P. multocida isolates. However, although some knowledge about genotypes, fitness, and pathogenesis has been gained from the recent whole-genome sequencing and comparative analysis studies of P. multocida, there is still a long way to go before we fully understand the pathogenic mechanisms of this important zoonotic pathogen. The quality of several available genome sequences is low, as they are assemblies with relatively low coverage, and genomes of P. multocida isolates from some uncommon host species are still limited or lacking. Here, we review recent advances, as well as continuing knowledge gaps, in our understanding of determinants contributing to virulence, fitness, host specificity, disease predilection, and phylogeny of P. multocida.
| S-EPMC6759666 | biostudies-literature