A bifunctional Pasteurella multocida ß1-3-galactosyl/N-acetylgalactosaminyltransferase (PmNatB) for the highly efficient chemoenzymatic synthesis of disaccharides.

Glycosyltransferases are nature's key biocatalysts for the formation of glycosidic bonds. Discovery and characterization of new synthetically useful glycosyltransferases are critical for the development of efficient enzymatic and chemoenzymatic strategies for producing complex carbohydrates and glycoconjugates. Herein we report the identification of Pasteurella multocida PmNatB as a bifunctional single-catalytic-domain glycosyltransferase with both ß1-3-galactosyltransferase and ß1-3-N-acetylgalactosaminyltransferase activities. It is a novel glycosyltransferase for constructing structurally diverse GalNAcß3Galα/ßOR and Galß3GalNAcα/ßOR disaccharides in one-pot multienzyme systems with in situ generation of UDP-sugars.

A dimer between monomers and hexamers-Oligomeric variations in glucosamine-6-phosphate deaminase family.

In bacteria that live in hosts whose terminal sugar is a sialic acid, Glucosamine-6-phosphate deaminase (NagB) catalyzes the last step in converting sialic acid into Fructose-6-phosphate. These bacteria then use the Fructose-6-phosphate as an energy source. The enzyme NagB exists as a hexamer in Gram-negative bacteria and is allosterically regulated. In Gram-positive bacteria, it exists as a monomer and lacks allosteric regulation. Our identification of a dimeric Gram-negative bacterial NagB motivated us to characterize the structural basis of two closely related oligomeric forms. We report here the crystal structures of NagB from two Gram-negative pathogens, Haemophilus influenzae (Hi) and Pasturella multocida (Pm). The Hi-NagB is active as a hexamer, while Pm-NagB is active as a dimer. Both Hi-NagB and Pm-NagB contain the C-terminal helix implicated as essential for hexamer formation. The hexamer is described as a dimer of trimers. In the Pm-NagB dimer, the dimeric interface is conserved. The conservation of the dimer interface suggests that the three possible oligomeric forms of NagB are a monomer, a dimer, and a trimer of dimers. Computational modeling and MD simulations indicate that the residues at the trimeric interface have less stabilizing energy of oligomer formation than those in the dimer interface. We propose that Pm-NagB is the evolutionary link between the monomer and the hexamer forms.

A single point mutation in the hyaC gene affects Pasteurella multocida serovar A capsule production and virulence.

Pasteurella multocida (P. multocida) is a Gram-negative bacterium which causes diseases in poultry, livestock, and humans, resulting in huge economic losses. P. multocida serovar A CQ6 (PmCQ6) is a naturally occurring attenuated strain with a thin capsule. Thus, we aimed to explore why this strain is less virulent and produces less capsule compared with P. multocida serovar A strain CQ2 (PmCQ2). Analysis of capsular polysaccharide synthesis genes in PmCQ6 revealed that, compared with PmCQ2, there was only a single point mutation in the initiation codon sequence of the hyaC gene. To test whether this point mutation caused capsular deficiency and reduced virulence, we rescued this hyaC mutation and observed a restoration of capsule production and higher virulence. Transcriptome analysis showed that the hyaC point mutation led to a downregulation of capsule synthesis and/or iron utilization related-genes. Taken together, the results indicate that the start codon mutation of hyaC is an important factor affecting the capsule synthesis and virulence of PmCQ6.

Assays for hyaluronidases and heparanase using nonreducing end fluorophore-labeled hyaluronan and heparan sulfate proteoglycan.

Glycosaminoglycans (GAGs), such as hyaluronan (HA) and heparan sulfate (HS), are a large group of polysaccharides found in the extracellular matrix and on the cell surface. The turnover of these molecules is controlled by de novo synthesis and catabolism through specific endoglycosidases, which are the keys to our understanding of the homeostasis of GAGs and could hold opportunities for therapeutic intervention. Herein, we describe assays for endoglycosidases using nonreducing end fluorophore-labeled GAGs, in which GAGs were labeled via incorporation of GlcNAz by specific synthases and cycloaddition of alkyne fluorophores and then digested with corresponding endoglycosidases. Assays of various HA-specific hyaluronidases (HYALs), including PH-20 or SPAM1, and HS-specific heparanase (HPSE) are presented. We demonstrated the distinctive pH profiles, substrate specificities and specific activities of these enzymes and provided evidence that both HYAL3 and HYAL4 are authentic hyaluronidases. In addition, while all HYALs are active on high-molecular-weight HA, they are active on low-molecular-weight HA. Subsequently, we defined a new way of measuring the activities of HYALs. Our results indicate that the activities of HYALs must be under strict pH regulation. Our quantitative methods of measuring the activity GAG endoglycosidases could bring the opportunity of designing novel therapeutics by targeting these important enzymes.

Role of aspartate ammonia-lyase in Pasteurella multocida.

BACKGROUND: Pasteurella multocida is responsible for a highly infectious and contagious disease in birds, leading to heavy economic losses in the chicken industry. However, the pathogenesis of this disease is poorly understood. We recently identified an aspartate ammonia-lyase (aspA) in P. multocida that was significantly upregulated under iron-restricted conditions, the protein of which could effectively protect chicken flocks against P. multocida. However, the functions of this gene remain unclear. In the present study, we constructed aspA mutant strain △aspA::kan and complementary strain C△aspA::kan to investigate the function of aspA in detail. RESULT: Deletion of the aspA gene in P. multocida resulted in a significant reduction in bacterial growth in LB (Luria-Bertani) and MH (Mueller-Hinton) media, which was rescued by supplementation with 20 mM fumarate. The mutant strain △aspA::kan showed significantly growth defects in anaerobic conditions and acid medium, compared with the wild-type strain. Moreover, growth of △aspA::kan was more seriously impaired than that of the wild-type strain under iron-restricted conditions, and this growth recovered after supplementation with iron ions. AspA transcription was negatively regulated by iron conditions, as demonstrated by quantitative reverse transcription-polymerase chain reaction. Although competitive index assay showed the wild-type strain outcompetes the aspA mutant strain and △aspA::kan was significantly more efficient at producing biofilms than the wild-type strain, there was no significant difference in virulence between the mutant and the wild-type strains. CONCLUSION: These results demonstrate that aspA is required for bacterial growth in complex medium, and under anaerobic, acid, and iron-limited conditions.

Chemoenzymatic synthesis of homogeneous chondroitin polymers and its derivatives.

Chondroitin sulfate is a linear glycosaminoglycan widely distributed as an important extracellular matrix component of mammalian cells. It participates in numerous pathological processes, however, illustration of its diverse biological roles is hampered by the unavailability of structurally defined chondroitin polymers and their derivatives. Herein, we report a novel homogeneous chondroitin polymers synthetic strategy which combines stepwise oligosaccharides synthesis with one-pot homogeneous chondroitin chain polymerization. Exogenous trisaccharide was proved to be the necessary acceptor for PmCS-catalyzed homogeneous chondroitin polymers synthetic reactions. The strategy exhibited a well-controlled relationship between the final sugar chain length and the molar ratios of reaction substrates that could synthesize homogenous chondroitin polymers with unprecedented narrow molecular weight distribution. More importantly, the strategy was further expanded to synthesis of unnatural zwitterionic and N-sulfonated chondroitin polymers by incorporation of sugar nucleotide derivatives into the synthetic approach.

Key Factors for A One-Pot Enzyme Cascade Synthesis of High Molecular Weight Hyaluronic Acid.

In the last decades, interest in medical or cosmetic applications of hyaluronic acid (HA) has increased. Size and dispersity are key characteristics of biological function. In contrast to extraction from animal tissue or bacterial fermentation, enzymatic in vitro synthesis is the choice to produce defined HA. Here we present a one-pot enzyme cascade with six enzymes for the synthesis of HA from the cheap monosaccharides glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc). The combination of two enzyme modules, providing the precursors UDP-GlcA and UDP-GlcNAc, respectively, with hyaluronan synthase from Pasteurella multocida (PmHAS), was optimized to meet the kinetic requirements of PmHAS for high HA productivity and molecular weight. The Mg2+ concentration and the pH value were found as key factors. The HA product can be tailored by different conditions: 25 mM Mg2+ and 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES)-NaOH pH 8 result into an HA product with high Mw HA (1.55 MDa) and low dispersity (1.05). Whereas with 15 mM Mg2+ and HEPES-NaOH pH 8.5, we reached the highest HA concentration (2.7 g/L) with a yield of 86.3%. Our comprehensive data set lays the basis for larger scale enzymatic HA synthesis.

Metabolic engineering of Bacillus megaterium for heparosan biosynthesis using Pasteurella multocida heparosan synthase, PmHS2.

BACKGROUND: Heparosan 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. RESULTS: We 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. CONCLUSION: We 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.

Surface display of sialyltransferase on the outer membrane of Escherichia coli and ClearColi.

α2,3-Sialyltransferase from Pasteurella multocida (PmST1) is an enzyme that transfers a sialyl group of donor substrates to an acceptor substrate called N-acetyl-d-lactosamine (LacNAc). In this study PmST1 was expressed on the outer membrane of wildtype Escherichia coli (BL21) with lipopolysaccharide (LPS) and ClearColi with no LPS, and then the enzyme activity and expression level of PmST1 were compared. As the first step, the expression levels of PmST1 on the outer membranes of wildtype E. coli (BL21) and ClearColi were compared according to the IPTG induction time, and the absolute amount of surface-displayed PmST1 was calculated using densitometry of SDS-PAGE. As the next step, the influence of LPS on the PmST1 activity was estimated by analyzing Michaelis-Menten plot. The enzyme activity of PmST1 was analyzed by measuring the concentration of CMP, which was a by-product after the transfer of the sialyl group of donor compounds to the acceptor compounds. From a Michaelis-Menten plot, the enzyme activity of the surface-displayed PmST1 and the maximum rate (Vmax) of ClearColi were higher than those of wildtype E. coli (BL21). However, the KM value, which represented the concentration of substrate to reach half the maximum rate (Vmax), was similar for both enzymes. These results represented such a difference in enzyme activity was occurred from the interference of LPS on the mass transport of the donor and acceptor to PmST1 for the sialyl group transfer.

Concise chemoenzymatic synthesis of heparan sulfate analogues as potent BACE-1 inhibitors.

Heparan sulfate (HS) and heparin, representative members of the glycosaminoglycans, possess distinct biological functions in terms of their specific interactions with hundreds of binding proteins. However, the structural properties of HS and heparin are complex due to their variable repeating motifs, different chain lengths and sulfation patterns. A concise chemoenzymatic approach has been developed to obtain well-defined low molecular weight (LMW) HS analogues. Pasteurella multocida heparosan synthase-2 (PmHS2) was utilized to fabricate the HS backbones with controllable chain lengths ranging from 14mer to 26mer. Moreover, regioselective and overall sulfation were conducted by chemical approach. The persulfated HS analogues exhibited more potent beta-site amyloid precursor protein (APP)-cleaving enzyme-1 (BACE-1) inhibitory activity than heparin and enoxaparin, and enhanced BACE-1 inhibitions were also found with the increasing molecular size of the HS analogues. This approach supplies the promising LMW HS analogues for the potential development of novel anti-Alzheimer's drugs.