Dataset Information

Analysis of the polymerization initiation and activity of Pasteurella multocida heparosan synthase PmHS2, an enzyme with glycosyltransferase and UDP-sugar hydrolase activity.

ABSTRACT: 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.

SUBMITTER: Chavaroche AA

PROVIDER: S-EPMC3023472 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Analysis of the polymerization initiation and activity of Pasteurella multocida heparosan synthase PmHS2, an enzyme with glycosyltransferase and UDP-sugar hydrolase activity.

Chavaroche Anais A E AA van den Broek Lambertus A M LA Springer Jan J Boeriu Carmen C Eggink Gerrit G

The Journal of biological chemistry 20101117 3

Heparosan synthase catalyzes the polymerization of heparosan (-4GlcUAβ1-4GlcNAcα1-)(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 ...[more]

PMID: 21084307

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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.

Dataset Information

Analysis of the polymerization initiation and activity of Pasteurella multocida heparosan synthase PmHS2, an enzyme with glycosyltransferase and UDP-sugar hydrolase activity.

Publications

Analysis of the polymerization initiation and activity of Pasteurella multocida heparosan synthase PmHS2, an enzyme with glycosyltransferase and UDP-sugar hydrolase activity.

Similar Datasets

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