Bacterial and Viral Sialidases: Contribution of the Conserved Active Site Glutamate to Catalysis

Abstract

Mutagenesis of the conserved glutamic acid of influenza type A (E277) and Micromonospora viridifaciens (E260) sialidases was performed to probe the contribution of this strictly conserved residue to catalysis. Kinetic studies of the E260D and E260C M. viridifaciens mutant enzymes reveal that the overall mechanism of action has not changed. That is, the mutants are retaining sialidases in which glycosylation and deglycosylation are rate-limiting for k(cat)/K(m) and k(cat), respectively. The solvent kinetic isotope effect and proton inventory on k(cat) for the E260C mutant sialidase provide strong evidence that the newly installed cysteine residue provides little catalytic acceleration. The results are consistent with the conserved aspartic acid residue (D92) becoming the key general acid/base residue in the catalytic cycle. In addition, the E277D mutant influenza type A sialidase is catalytically active toward 4-nitrophenyl α-D-sialoside, although no measurable hydrolysis of natural substrates was observed. Thus, mutating the glutamate residue (E277) to an aspartate increases the activation free energy of hydrolysis for natural substrates by >22 kJ/mol.

Mutants of Micromonospora viridifaciens sialidase have highly variable activities on natural and non-natural substrates

“…Bacterial sialidases are classified in glycosyl hydrolase family 33 and their structures are of the six-bladed β-propeller type (Crennell et al, 1993). As retaining glycosidases, they employ a double-displacement mechanism and use a Tyr residue as catalytic nucleophile (Watts et al, 2003) while a conserved Asp-Glu pair is involved in general acid/base catalysis (Chan et al, 2012). Interestingly, this enzyme can retain function upon mutation of either of the catalytic residues (D92, E260 and Y370) (Watson et al, 2003;Chan et al, 2012).…”

Influenza neuraminidase operates via a nucleophilic mechanism and can be targeted by covalent inhibitors

“…Retaining NA mechanisms, on the other hand, appear to be quite distinct. Biochemical analysis of NA from various microorganisms has revealed that a conserved active-site tyrosine can function as a nucleophile and may be the most important catalytic residue [17][18][19] .…”

Neuraminidase Inhibitors from marine-derived actinomycete Streptomyces seoulensis

“…However, the viral and bacterial NA enzymes, all belonging to clan GH‐E, contain a sixfold β‐propeller as the prominent structural motif, and the release of progeny virus particles from infected host cells requires the presence of the exo‐sialidase composed commonly of three acidic amino acids, a tyrosine, and three arginines (Chan et al . ). In view of the highly conserved active site of the target enzymes of the viral and bacterial origin (Chan et al .…”

“…In view of the highly conserved active site of the target enzymes of the viral and bacterial origin (Chan et al . ), the three characterized NA inhibitors are of value in terms of drug discovery for the treatment of viral infections such as influenza.…”

“…Although short of the laboratory infrastructure allowing the experimentation with the virulent virus, the present work has characterized three NA inhibitors with structural scaffolds quite different from the existing ones, by using alternatively as target the commercially available bacterial NA. However, the viral and bacterial NA enzymes, all belonging to clan GH-E, contain a sixfold b-propeller as the prominent structural motif, and the release of progeny virus particles from infected host cells requires the presence of the exosialidase composed commonly of three acidic amino acids, a tyrosine, and three arginines (Chan et al 2012). In view of the highly conserved active site of the target enzymes of the viral and bacterial origin (Chan et al 2012), the three characterized NA inhibitors are of value in terms of drug discovery for the treatment of viral infections such as influenza.…”