Sialidases, or neuraminidases (EC 3.2.1.18), belong to a class of glycosyl hydrolases that release terminal N-acylneuraminate residues from the glycans of glycoproteins, glycolipids, and polysaccharides. In bacteria, sialidases can be used to scavenge sialic acids as a nutrient from various sialylated substrates or to recognize sialic acids exposed on the surface of the host cell. Despite the fact that bacterial sialidases share many structural features, their biochemical properties, especially their linkage and substrate specificities, vary widely. Bacterial sialidases can catalyze the hydrolysis of terminal sialic acids linked by the α(2,3)-, α(2,6)-, or α(2,8)-linkage to a diverse range of substrates. In addition, some of these enzymes can catalyze the transfer of sialic acids from sialoglycans to asialoglycoconjugates via a transglycosylation reaction mechanism. Thus, some bacterial sialidases have been applied to synthesize complex sialyloligosaccharides through chemoenzymatic approaches and to analyze the glycan structure. In this review article, the biochemical features of bacterial sialidases and their potential applications in regioselective hydrolysis reactions as well as sialylation by transglycosylation for the synthesis of sialylated complex glycans are discussed.

The extremely thermoacidophilic archaeon Sulfolobus solfataricus utilizes D-glucose as a sole carbon and energy source through the non-phosphorylated Entner-Doudoroff pathway. It has been suggested that this micro-organism metabolizes D-gluconate, the oxidized form of D-glucose, to pyruvate and D-glyceraldehyde by using two unique enzymes, D-gluconate dehydratase and 2-keto-3-deoxy-D-gluconate aldolase. In the present study, we report the purification and characterization of D-gluconate dehydratase from S. solfataricus, which catalyses the conversion of D-gluconate into 2-keto-3-deoxy-D-gluconate. D-Gluconate dehydratase was purified 400-fold from extracts of S. solfataricus by ammonium sulphate fractionation and chromatography on DEAE-Sepharose, Q-Sepharose, phenyl-Sepharose and Mono Q. The native protein showed a molecular mass of 350 kDa by gel filtration, whereas SDS/PAGE analysis provided a molecular mass of 44 kDa, indicating that D-gluconate and Ni 2+ activated, whereas EDTA inhibited the enzyme. A metal analysis of the purified protein revealed the presence of one Co 2+ ion per enzyme monomer. Of the 22 aldonic acids tested, only D-gluconate served as a substrate, with K m = 0.45 mM and V max = 0.15 unit/mg of enzyme. From N-terminal sequences of the purified enzyme, it was found that the gene product of SSO3198 in the S. solfataricus genome database corresponded to D-gluconate dehydratase (gnaD). We also found that the D-gluconate dehydratase of S. solfataricus is a phosphoprotein and that its catalytic activity is regulated by a phosphorylation-dephosphorylation mechanism. This is the first report on biochemical and genetic characterization of D-gluconate dehydratase involved in the non-phosphorylated Entner-Doudoroff pathway.

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Dihydroxy-acid dehydratase (DHAD) is one of the key enzymes involved in the biosynthetic pathway of the branched chain amino acids. Although the enzyme has been purified and characterized in various mesophiles, including bacteria and eukarya, the biochemical properties of DHAD from hyperthermophilic archaea have not yet been reported. In this study we cloned, expressed in Escherichia coli, and purified a DHAD homologue from the thermoacidophilic archaeon Sulfolobus solfataricus, which grows optimally at 80 degrees C and pH 3. The recombinant S. solfataricus DHAD (rSso_DHAD) showed the highest activity on 2,3-dihydroxyisovalerate among 17 aldonic acids tested. Interestingly, this enzyme also displayed high activity toward d-gluconate and some other pentonic and hexonic sugar acids. The k(cat)/K(m) values were 140.3 mM(-1) s(-1) for 2,3-dihydroxyisovalerate and 20.0 mM(-1) s(-1) for d-gluconate, respectively. A possible evolutionary explanation for substrate promiscuity was provided through amino acid sequence alignments of DHADs and 6-phosphogluconate dehydratases from archaea, bacteria and eukarya.

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