Chemical Insight into the Emergence of Influenza Virus Strains That Are Resistant to Relenza

A reagent panel containing ten 4-substituted 4-nitrophenyl α-d-sialosides and a second panel of the corresponding sialic acid glycals were synthesized and used to probe the inhibition mechanism for two neuraminidases, the N2 enzyme from influenza type A virus and the enzyme from <i>Micromonospora viridifaciens</i>. For the viral enzyme the logarithm of the inhibition constant (<i>K</i>_i) correlated with neither the logarithm of the catalytic efficiency (<i>k</i>_cat/<i>K</i>_m) nor catalytic proficiency (<i>k</i>_cat/<i>K</i>_m<i>k</i>_un). These linear free energy relationship data support the notion that these inhibitors, which include the therapeutic agent Relenza, are not transition state mimics for the enzyme-catalyzed hydrolysis reaction. Moreover, for the influenza enzyme, a correlation (slope, 0.80 ± 0.08) is observed between the logarithms of the inhibition (<i>K</i>_i) and Michaelis (<i>K</i>_m) constants. We conclude that the free energy for Relenza binding to the influenza enzyme mimics the enzyme–substrate interactions at the Michaelis complex. Thus, an influenza mutational response to a 4-substituted sialic acid glycal inhibitor can weaken the interactions between the inhibitor and the viral neuraminidase without a concomitant decrease in free energy of binding for the substrate at the enzyme-catalyzed hydrolysis transition state. The current findings make it clear that new structural motifs and/or substitution patterns need to be developed in the search for a bona fide influenza viral neuraminidase transition state analogue inhibitor

Neuraminidase Substrate Promiscuity Permits a Mutant <i>Micromonospora viridifaciens</i> Enzyme To Synthesize Artificial Carbohydrates

Mutation of the nucleophilic amino acid residue tyrosine to the small nonpolar residue glycine (Y370G) in the active site of <i>Micromonospora viridifaciens</i> neuraminidase (<i>Mv</i>NA) produces an efficient catalyst for the transfer of <i>N</i>-acetylneuraminic acid from an artificial substrate (i.e., phenyl <i>N</i>-acetyl-β-d-neuraminide) to a sugar acceptor (e.g., d-lactose, d-glucose, d-mannose, d-raffinose, d-allose, or d-fructose) to give <i>N</i>-acetyl-α-neuraminide coupled carbohydrate products. In addition, this mutant enzyme (<i>Mv</i>NA Y370G) catalyzes the transfer of a sugar residue from the artificial substrate 2-fluorophenyl <i>N</i>-acetyl-β-d-neuraminide to methyl glycopyranoside acceptors. Interestingly, when trans-glycosylation reactions are conducted in aqueous solutions containing 30% (v/v) acetonitrile, the α-anomeric acceptors of methyl glucopyranoside and galactopyranoside generate higher product yields than do their corresponding β-anomers. Specifically, a 64 h reaction with 2-fluorophenyl <i>N</i>-acetyl-β-d-neuraminide as the limiting reagent and the acceptors methyl α-d-galactopyranoside, methyl α-d-glucopyranoside, or methyl α-d-mannopyranoside gives trans-glycosylation product yields of 22%, 31%, or 34%, respectively. With methyl α-d-galactopyranoside as the acceptor, trans-glycosylations catalyzed by both <i>Mv</i>NA Y370G and a 2,6-sialyltransferase yield identical products, which we identified as methyl <i>N</i>-acetyl-α-d-neuraminyl-(2 → 6)-α-d-galactopyranoside. The <i>Mv</i>NA Y370G-catalyzed coupling of <i>N</i>-acetylneuraminic acid to these three methyl α-d-glycopyranoside acceptors is favored by factors of 18–27-fold over the competing hydrolysis reaction. These coupling efficiencies likely arise from nonselective interactions between the acceptor glycopyranoside and <i>Mv</i>NA Y370G, which preferentially places a carbohydrate hydroxyl group rather than water in close proximity to the active site where this functionality intercepts the nascent neuraminyl oxacarbenium ion that is formed during cleavage of the glycosidic bond in the aryl <i>N</i>-acetyl-β-d-neuraminide donor. The ability to transfer <i>N</i>-acetylneuraminic acid from a stable and readily accessible donor to acceptor carbohydrates that are not substrates for sialyltransferases is one step on the path for the production of pseudohuman glycoproteins from nonmammalian cell lines

Kinetic and structural evaluation of selected active site mutants of the Aspergillus fumigatus KDNase (sialidase)

Aspergillus fumigatus is an airborne fungal pathogen. We previously cloned and characterized an exo-sialidase from A. fumigatus and showed that it preferred 2-keto-3-deoxynononic acid (KDN) as a substrate to N-acetylneuraminic acid (Neu5Ac). The purpose of this study was to investigate the structure-function relationships of critical catalytic site residues. Site-directed mutagenesis was used to create three mutant recombinant enzymes: the catalytic nucleophile (Y358H), the general acid/base catalyst (D84A), and an enlargement of the binding pocket to attempt to accommodate the N-acetyl group of Neu5Ac (R171L). Crystal structures for all enzymes were determined. The D84A mutation had an effect in decreasing the activity of AfKDNase that was stronger than that of the same mutation in the structurally similar sialidase from the bacterium Micromonospora viridifaciens. These data suggest that the catalytic acid is more important in the reaction of AfKDNase and that catalysis is less dependent on nucleophilic or electrostatic stabilization of the developing positive charge at the transition state for hydrolysis. Removal of the catalytic nucleophile (Y358H) significantly lowered the activity of the enzyme, but this mutant remained a retaining glycosidase as demonstrated by nuclear magnetic resonance spectroscopic analysis. This is a novel finding that has not been shown with other sialidases. Kinetic activity measured at pH 5.2 revealed that R171L had higher activity on a Neu5Ac-based substrate than wild-type KDNase; hence, leucine in place of arginine in the binding pocket improved catalysis toward Neu5Ac substrates. Hence, whether a sialidase is primarily a KDNase or a neuraminidase is due in part to the presence of an amino acid that creates a steric clash with the N-acetyl group

Kinetic and structural evaluation of selected active site mutants of the Aspergillus fumigatus KDNase (sialidase)

Aspergillus fumigatus is an airborne fungal pathogen. We previously cloned and characterized an exo-sialidase from A. fumigatus and showed that it preferred 2-keto-3-deoxynononic acid (KDN) as a substrate to N-acetylneuraminic acid (Neu5Ac). The purpose of this study was to investigate the structure-function relationships of critical catalytic site residues. Site-directed mutagenesis was used to create three mutant recombinant enzymes: the catalytic nucleophile (Y358H), the general acid/base catalyst (D84A), and an enlargement of the binding pocket to attempt to accommodate the N-acetyl group of Neu5Ac (R171L). Crystal structures for all enzymes were determined. The D84A mutation had an effect in decreasing the activity of AfKDNase that was stronger than that of the same mutation in the structurally similar sialidase from the bacterium Micromonospora viridifaciens. These data suggest that the catalytic acid is more important in the reaction of AfKDNase and that catalysis is less dependent on nucleophilic or electrostatic stabilization of the developing positive charge at the transition state for hydrolysis. Removal of the catalytic nucleophile (Y358H) significantly lowered the activity of the enzyme, but this mutant remained a retaining glycosidase as demonstrated by nuclear magnetic resonance spectroscopic analysis. This is a novel finding that has not been shown with other sialidases. Kinetic activity measured at pH 5.2 revealed that R171L had higher activity on a Neu5Ac-based substrate than wild-type KDNase; hence, leucine in place of arginine in the binding pocket improved catalysis toward Neu5Ac substrates. Hence, whether a sialidase is primarily a KDNase or a neuraminidase is due in part to the presence of an amino acid that creates a steric clash with the N-acetyl group

C2-Oxyanion Neighboring Group Participation: Transition State Structure for the Hydroxide-Promoted Hydrolysis of 4‑Nitrophenyl α‑d‑Mannopyranoside

The hydroxide-catalyzed hydrolysis of aryl 1,2-<i>trans</i>-glycosides proceeds through a mechanism involving neighboring group participation by a C2-oxyanion and rate-limiting formation of a 1,2-anhydro sugar (oxirane) intermediate. The transition state for the hydroxide-catalyzed hydrolysis of 4-nitrophenyl α-d-mannopyranoside in aqueous media has been studied by the use of multiple kinetic isotope effect (KIE) measurements in conjunction with <i>ab initio</i> theoretical methods. The experimental KIEs are C1-²H (1.112 ± 0.004), C2-²H (1.045 ± 0.005), anomeric 1-¹³C (1.026 ± 0.006), C2-¹³C (0.999 ± 0.005), leaving group oxygen 2-¹⁸O (1.040 ± 0.012), and C2-¹⁸O (1.044 ± 0.006). The transition state for the hydrolysis reaction was modeled computationally using the experimental KIE values as constraints. Taken together, the reported kinetic isotope effects and computational modeling are consistent with the reaction mechanism involving rate-limiting formation of a transient oxirane intermediate that opens in water to give α-d-mannopyranose. The transition state has significant nucleophilic participation by the C2-alkoxide, an essentially cleaved glycosidic bond, and a slight shortening of the endocyclic C1–O5 bond. The TS is late, consistent with the large, normal C2-¹⁸O isotope effect

Kinetic and Structural Evaluation of Selected Active Site Mutants of the <i>Aspergillus fumigatus</i> KDNase (Sialidase)

<i>Aspergillus fumigatus</i> is an airborne fungal pathogen. We previously cloned and characterized an <i>exo</i>-sialidase from <i>A. fumigatus</i> and showed that it preferred 2-keto-3-deoxynononic acid (KDN) as a substrate to <i>N</i>-acetylneuraminic acid (Neu5Ac). The purpose of this study was to investigate the structure–function relationships of critical catalytic site residues. Site-directed mutagenesis was used to create three mutant recombinant enzymes: the catalytic nucleophile (Y358H), the general acid/base catalyst (D84A), and an enlargement of the binding pocket to attempt to accommodate the <i>N</i>-acetyl group of Neu5Ac (R171L). Crystal structures for all enzymes were determined. The D84A mutation had an effect in decreasing the activity of AfKDNase that was stronger than that of the same mutation in the structurally similar sialidase from the bacterium <i>Micromonospora viridifaciens</i>. These data suggest that the catalytic acid is more important in the reaction of AfKDNase and that catalysis is less dependent on nucleophilic or electrostatic stabilization of the developing positive charge at the transition state for hydrolysis. Removal of the catalytic nucleophile (Y358H) significantly lowered the activity of the enzyme, but this mutant remained a retaining glycosidase as demonstrated by nuclear magnetic resonance spectroscopic analysis. This is a novel finding that has not been shown with other sialidases. Kinetic activity measured at pH 5.2 revealed that R171L had higher activity on a Neu5Ac-based substrate than wild-type KDNase; hence, leucine in place of arginine in the binding pocket improved catalysis toward Neu5Ac substrates. Hence, whether a sialidase is primarily a KDNase or a neuraminidase is due in part to the presence of an amino acid that creates a steric clash with the <i>N</i>-acetyl group

Both Chemical and Non-Chemical Steps Limit the Catalytic Efficiency of Family 4 Glycoside Hydrolases

The glycoside hydrolase family 4 (GH4) α-galactosidase from <i>Citrobacter freundii</i> (MelA) catalyzes the hydrolysis of fluoro-substituted phenyl α-d-galactopyranosides by utilizing two cofactors, NAD⁺ and a metal cation, under reducing conditions. In order to refine the mechanistic understanding of this GH4 enzyme, leaving group effects were measured with various metal cations. The derived β_lg value on <i>V</i>/<i>K</i> for strontium activation is indistinguishable from zero (0.05 ± 0.12). Deuterium kinetic isotope effects (KIEs) were measured for the activated substrates 2-fluorophenyl and 4-fluorophenyl α-d-galactopyranosides in the presence of Sr²⁺, Y³⁺, and Mn²⁺, where the isotopic substitution was on the carbohydrate at C-2 and/or C-3. To determine the contributing factors to the virtual transition state (TS) on which the KIEs report, kinetic isotope effects on isotope effects were measured on these KIEs using doubly deuterated substrates. The measured ^D<i>V</i>/<i>K</i> KIEs for MelA-catalyzed hydrolysis of 2-fluorophenyl α-d-galactopyranoside are closer to unity than the measured effects on 4-fluorophenyl α-d-galactopyranoside, irrespective of the site of isotopic substitution and of the metal cation activator. These observations are consistent with hydride transfer at C-3 to the on-board NAD⁺, deprotonation at C-2, and a non-chemical step contributing to the virtual TS for <i>V</i>/<i>K</i>

Novel small molecules potentiate premature termination codon readthrough by aminoglycosides

International audienceNonsense mutations introduce premature termination codons and underlie 11% of genetic disease cases. High concentrations of aminoglycosides can restore gene function by eliciting premature termination codon readthrough but with low efficiency. Using a high-throughput screen, we identified compounds that potentiate readthrough by aminoglyco-sides at multiple nonsense alleles in yeast. Chemical optimization generated phthalimide derivative CDX5-1 with activity in human cells. Alone, CDX5-1 did not induce readthrough or increase TP53 mRNA levels in HDQ-P1 cancer cells with a homozygous TP53 nonsense mutation. However, in combination with aminoglycoside G418, it enhanced readthrough up to 180-fold over G418 alone. The combination also increased readthrough at all three nonsense codons in cancer cells with other TP53 nonsense mutations, as well as in cells from rare genetic disease patients with nonsense mutations in the CLN2, SMARCAL1 and DMD genes. These findings open up the possibility of treating patients across a spectrum of genetic diseases caused by nonsense mutations