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.

9-Azido-9-deoxy-2,3-difluorosialic Acid as a Subnanomolar Inhibitor against Bacterial Sialidases.

A library of 2(a),3(a/e)-difluorosialic acids and their C-5 and/or C-9 derivatives were chemoenzymatically synthesized. Pasteurella multocida sialic acid aldolase (PmAldolase), but not its Escherichia coli homologue (EcAldolase), was found to catalyze the formation of C5-azido analogue of 3-fluoro(a)-sialic acid. In comparison, both PmAldolase and EcAldolase could catalyze the synthesis of 3-fluoro(a/e)-sialic acids and their C-9 analogues although PmAldolase was generally more efficient. The chemoenzymatically synthesized 3-fluoro(a/e)-sialic acid analogues were purified and chemically derivatized to form the desired difluorosialic acids and derivatives. Inhibition studies against several bacterial sialidases and a recombinant human cytosolic sialidase hNEU2 indicated that sialidase inhibition was affected by the C-3 fluorine stereochemistry and derivatization at C-5 and/or C-9 of the inhibitor. Opposite to that observed for influenza A virus sialidases and hNEU2, compounds with axial fluorine at C-3 were better inhibitors (up to 100-fold) against bacterial sialidases compared to their 3F-equatorial counterparts. While C-5-modified compounds were less-efficient antibacterial sialidase inhibitors, 9-N3-modified 2,3-difluoro-Neu5Ac showed increased inhibitory activity against bacterial sialidases. 9-Azido-9-deoxy-2-(e)-3-(a)-difluoro- N-acetylneuraminic acid [2(e)3(a)DFNeu5Ac9N3] was identified as an effective inhibitor with a long effective duration selectively against pathogenic bacterial sialidases from Clostridium perfringens (CpNanI) and Vibrio cholerae.

Facile chemoenzymatic synthesis of Lewis a (Lea) antigen in gram-scale and sialyl Lewis a (sLea) antigens containing diverse sialic acid forms.

An efficient streamlined chemoenzymatic approach has been developed for gram-scale synthesis of Lewis a angtigen (LeaßProN3) and a library of sialyl Lewis a antigens (sLeaßProN3) containing different sialic acid forms. Intially, commercially available inexpensive N-acetylglucosamine (GlcNAc) was converted to its N'-glycosyl p-toluenesulfonohydrazide in one step. Followed by chemical glycosylation, GlcNAcßProN3 was synthesized using this protecting group-free method in high yield (82%). Sequential one-pot multienzyme (OPME) ß1-3-galactosylation of GlcNAcßProN3 followed by OPME α1-4-fucosylation reactions produced target LeaßProN3 in gram-scale. Structurally diverse sialic acid forms was successfully introduced using a OPME sialylation reation containing a CMP-sialic acid synthetase and Pasteurella multocida α2-3-sialyltransferase 1 (PmST1) mutant PmST1 M144D with or without a sialic acid aldolase to form sLeaßProN3 containing naturally occurring or non-natural sialic acid forms in preparative scales.

Directed Evolution of Hyaluronic Acid Synthase from Pasteurella multocida towards High-Molecular-Weight Hyaluronic Acid.

Hyaluronic acid (HA), with diverse cosmetic and medical applications, is the natural glycosaminoglycan product of HA synthases. Although process and/or metabolic engineering are used for industrial HA production, the potential of protein engineering has barely been realised. Herein, knowledge-gaining directed evolution (KnowVolution) was employed to generate an HA synthase variant from Pasteurella multocida (pmHAS) with improved chain-length specificity and a twofold increase in mass-based turnover number. Seven improved pmHAS variants out of 1392 generated by error-prone PCR were identified; eight prospective positions were saturated and the most beneficial amino acid substitutions were recombined. After one round of KnowVolution, the longest HA polymer (<4.7 MDa), through an engineered pmHAS variant in a cell-free system, was synthesised. Computational studies showed that substitutions from the best variant (T40L, V59M and T104A) are distant from the glycosyltransferase sites and increase the flexibility of the N-terminal region of pmHAS. Taken together, these findings suggest that the N terminus may be involved in HA synthesis and demonstrate the potential of protein engineering towards improved HA synthase activity.

Covalent inhibitors of LgtC: A blueprint for the discovery of non-substrate-like inhibitors for bacterial glycosyltransferases.

Non-substrate-like inhibitors of glycosyltransferases are sought after as chemical tools and potential lead compounds for medicinal chemistry, chemical biology and drug discovery. Here, we describe the discovery of a novel small molecular inhibitor chemotype for LgtC, a retaining α-1,4-galactosyltransferase involved in bacterial lipooligosaccharide biosynthesis. The new inhibitors, which are structurally unrelated to both the donor and acceptor of LgtC, have low micromolar inhibitory activity, comparable to the best substrate-based inhibitors. We provide experimental evidence that these inhibitors react covalently with LgtC. Results from detailed enzymological experiments with wild-type and mutant LgtC suggest the non-catalytic active site residue Cys246 as a likely target residue for these inhibitors. Analysis of available sequence and structural data reveals that non-catalytic cysteines are a common motif in the active site of many bacterial glycosyltransferases. Our results can therefore serve as a blueprint for the rational design of non-substrate-like, covalent inhibitors against a broad range of other bacterial glycosyltransferases.

Converting Pasteurella multocidaα2-3-sialyltransferase 1 (PmST1) to a regioselective α2-6-sialyltransferase by saturation mutagenesis and regioselective screening.

A microtiter plate-based screening assay capable of determining the activity and regioselectivity of sialyltransferases was developed. This assay was used to screen two single-site saturation libraries of Pasteurella multocidaα2-3-sialyltransferase 1 (PmST1) for α2-6-sialyltransferase activity and total sialyltransferase activity. PmST1 double mutant P34H/M144L was found to be the most effective α2-6-sialyltransferase and displayed 50% reduced donor hydrolysis and 50-fold reduced sialidase activity compared to the wild-type PmST1. It retained the donor substrate promiscuity of the wild-type enzyme and was used in an efficient one-pot multienzyme (OPME) system to selectively catalyze the sialylation of the terminal galactose residue in a multigalactose-containing tetrasaccharide lacto-N-neotetraoside.

Auranofin Inhibits the Enzyme Activity of Pasteurella multocida Toxin PMT in Human Cells and Protects Cells from Intoxication.

The AB-type protein toxin from Pasteurella multocida (PMT) contains a functionally important disulfide bond within its catalytic domain, which must be cleaved in the host cell cytosol to render the catalytic domain of PMT into its active conformation. Here, we found that the reductive potential of the cytosol of target cells, and more specifically, the activity of the thioredoxin reductase (TrxR) is crucial for this process. This was demonstrated by the strong inhibitory effect of the pharmacological TrxR inhibitor auranofin, which inhibited the intoxication of target cells with PMT, as determined by analyzing the PMT-catalyzed deamidation of GTP-binding proteins (G-proteins) in the cytosol of cells. The amount of endogenous substrate levels modified by PMT in cells pretreated with auranofin was reduced compared to cells treated with PMT alone. Auranofin had no inhibitory effect on the activity of the catalytic domain of constitutively active PMT in vitro, demonstrating that auranofin did not directly inhibit PMT activity, but interferes with the mode of action of PMT in cells. In conclusion, the results show that TrxR is crucial for the mode of action of PMT in mammalian cells, and that the drug auranofin can serve as an efficient inhibitor, which might be a starting point for novel therapeutic options against toxin-associated diseases.

Metabolic engineering of Bacillus subtilis for biosynthesis of heparosan using heparosan synthase from Pasteurella multocida, PmHS1.

Heparosan, the capsular polysaccharide discovered in many pathogenic bacteria, is a promising material for heparin preparation. In this study, the Pasteurella multocida heparosan synthase 1 (PmHS1) module was used to synthesize heparosan with controlled molecular weight, while tuaD/gtaB module or gcaD module was responsible for UDP-precursors production in Bacillus subtilis 168. After metabolic pathway optimization, the yield of heparosan was as high as 237.6 mg/L in strain containing PmHS1 module and tuaD/gtaB module, which indicated that these two modules were key factors in heparosan production. The molecular weight of heparosan varied from 39 to 53 kDa, which indicated that heparosan molecular weight could be adjusted by the amount of PmHS1 and the ratio of two UDP precursors. The results showed that it would be possible to produce safe heparosan with appropriate molecular weight which is useful in heparin production.

Characterization of UDP-glucose dehydrogenase from Pasteurella multocida CVCC 408 and its application in hyaluronic acid biosynthesis.

Hyaluronic acid (HA), a vital acid mucopolysaccharide, has immense applied value in foodstuffs, medicaments, and cosmetics among others. UDP-glucose dehydrogenase (UGDH, EC 1.1.1.22) is an essential enzyme for HA synthesis. In this study, a UGDH (PmuHasB, 45.9 kDa) from Pasteurella multocida CVCC 408 was expressed in Escherichia coli BL21 (DE3). It was purified by two chromatographic columns with a specific activity of 6.58 IU/mg. The optimum pH and temperature were determined to be 10.0 and 37°C, respectively. The activity was stable across the pH range 6-10, and had a half-life of about 3 h at 45°C. The estimated apparent Km values for UDP-glucose and NAD(+) were 0.11 and 0.069 mM, respectively. The results indicated that PmuHasB was an alkaline and mesophilic UGDH. PmuHasB and PmuHasA (HA synthase, HAS) were co-expressed in E. coli BW25113 to obtain a HA high-producing strain pBPAB/BW25113. It produced about 2.39 g/L HA in shake flask by using the method of whole-cell catalysis. Investigation of the different UGDHs on HA synthesis revealed that intracellular UGDH activity and HA total yield of pBPAB/BW25113 (0.15 IU/mg and 5.4 g/L) were higher than from pBPASB/BW25113 (0.013 IU/mg and 2.8 g/L) and pBPAEB/BW25113 (0.010 IU/mg and 2.22 g/L). These results indicated that the activity and stability of UGDH plays a significant role in HA production, and should prove useful for further genetic engineering research with a view to construct other glucuronic acid polysaccharide synthesis pathways.

A semi-multifunctional sialyltransferase from Bibersteinia trehalosi and its comparison to the Pasteurella multocida ST1 mutants.

Sialic acids are well known for their crucial roles in many physiological and pathological processes. Improvement in the efficacy of protein drugs, an increase in the anti-inflammatory activity of intravenous immunoglobulin, preparation of infant milk and the diagnosis of diseases are examples of why there is a need for efficient in vitro sialylation. Sialyltransferases are crucial enzymes for the synthesis of sialo-oligosaccharides. Here, we introduce a new α2,3-sialyltransferase from bacteria Bibersteinia trehalosi (BtST1), which is homological to sialyltransferase from Pasteurella multocida (PmST1), Pasteurella dagmatis (PdST1) and Haemophilus ducreyi (Hd0053). BtST1 is active in a wide pH range and shows considerable acceptor flexibility. Very good specific activities have been detected with lactose and LacNAc as acceptors, and these activities were comparable to those of efficient multifunctional PmST1 and higher than PdST1, Hd0053 and also PmST1 M144D which was constructed to decrease the high sialidase activity of PmST1. Testing of PmST1 mutant forms revealed that mutations that included S143 caused only the restriction of sialyltransferase activity, whereas mutations including G142 resulted in the loss of activity with lactose. BtST1 possesses only low sialidase and trans-sialidase activities that are comparable to mutant PmST1 M144D, which are detected only in the presence of CMP. The combination of large acceptor flexibility, high activity for lactose and LacNAc and naturally low sialidase activity make BtST1 an attractive enzyme for biotechnological applications.

Modulating the regioselectivity of a Pasteurella multocida sialyltransferase for biocatalytic production of 3'- and 6'-sialyllactose.

Several bacterial sialyltransferases have been reported to be multifunctional also catalysing sialidase and trans-sialidase reactions. In this study, we examined the trans-sialylation efficacy and regioselectivity of mutants of the multifunctional Pasteurella multocida sialyltransferase (PmST) for catalysing the synthesis of 3'- and 6'-sialyllactose using casein glycomacropeptide as sialyl-donor and lactose as acceptor. The mutation P34H led to a 980-fold increase in α-2,6-sialyltransferase activity (with cytidine-5'-monophospho-N-acetylneuraminic acid as donor), while its α-2,3-sialyltransferase activity was abolished. Histidine in this position is conserved in α-2,6-sialyltransferases and has been suggested, and recently confirmed, to be the determinant for strict regiospecificity in the sialyltransferase reaction. Our data verified this theorem. In trans-sialidase reactions, the P34H mutant displayed a distinct preference for 6'-sialyllactose synthesis but low levels of 3'-sialyllactose were also produced. The sialyllactose yield was however lower than when using PmSTWT under optimal conditions for 6'-sialyllactose formation. The discrepancy in regiospecificity between the two reactions could indicate subtle differences in the substrate binding site in the two reactions. In contrast, the two mutations E271F and R313Y led to preferential synthesis of 3'-sialyllactose over 6'-sialyllactose and the double mutant (PmSTE271F/R313Y) exhibited the highest α-2,3-regioselectivity via reduced sialidase and α-2,6-trans-sialidase activity. The double mutant PmSTE271F/R313Y thus showed the highest α-2,3-regioselectivity and constitutes an interesting enzyme for regioselective synthesis of α-2,3-sialylated glycans. This study has expanded the understanding of the structure-function relationship of multifunctional, bacterial sialyltransferases and provided new enzymes for regioselective glycan sialylation.

Sialyltransferases with enhanced legionaminic acid transferase activity for the preparation of analogs of sialoglycoconjugates.

Legionaminic acids (Leg) are bacterial analogs of neuraminic acid, with the same stereochemistry but different substituents at C5, C7 and C9. Hence they may be incorporated into useful analogs of sialoglycoconjugates, and we previously reported two sialyltransferases that could utilize cytidine monophosphate (CMP)-Leg5Ac7Ac for preparation of Leg glycoconjugates, which were resistant to sialidases [Watson DC, Leclerc S, Wakarchuk WW, Young NM. 2011. Enzymatic synthesis and properties of glycoconjugates with legionaminic acid as a replacement for neuraminic acid. Glycobiology. 21:99-108.]. These were the porcine ST3Gal1 and Pasteurella multocida sialyltransferases. We now report two additional sialyltransferases with superior Leg-transferase properties to the previous two. These are (i) a truncated form of a Photobacterium α2,6-sialyltransferase with an Ala-Met mutation in its active site, and (ii) an α2,3-sialyltransferase from Neisseria meningitidis MC58 with a higher transferase activity than the P. multocida enzyme, with either CMP-Neu5Ac or CMP-Leg5Ac7Ac as the donor. These enzymes will enable the production of useful Leg5Ac7Ac glycoconjugate derivatives with either α2,6 or α2,3 linkages and unique biological properties.