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

Abstract

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 Characterization of Sialidases (Kdnases) from Ascomycete Fungal Pathogens

“… a Conditions were sodium acetate buffer (100 mM, pH 4.0) containing 0.01% bovine serum albumin, at 37 °C. Values represent the mean ± SD of three independent measurements. b Values taken from Yeung et al c This work. …”

“…Af S does display some activity on Neu5Ac substrates compared to At S and Tr S, which do not. The structure of an Af S R171L mutant in complex with Neu5Ac2en allows a direct comparison of the interactions with Af S and Kdn2en and may offer an explanation of how Af S tolerates the Neu5Ac substrates. Although the bulk of Arg171, which is conserved among the Kdnases, but not other sialidases, may contribute to the specificity for the smaller Kdn substrate, it is noteworthy that superimposition with the WT Af S structure in complex with Kdn2en shows that the carbonyl oxygen of the N-acetyl group in Neu5Ac2en is perfectly poised to hydrogen bond with Arg171.…”

“…This was confirmed experimentally by mutant analysis: replacement of arginine 171 with a leucine residue allowed for the tighter binding of the inhibitor Neu2en5Ac. Replacement of the tyrosine nucleophile with a histidine residue and the aspartic acid acid/base catalyst with an alanine residue significantly decreased catalytic activity in separate experiments A.…”

Kinetic and Structural Characterization of Sialidases (Kdnases) from Ascomycete Fungal Pathogens

“…Cloning and Purification of Fungal Sialidases. Recombinant AfKdnase was prepared according to Yeung et al 9 and putative Kdnases from A. terreus and T. rubrum were prepared as follows. Trichophyton rubrum (ATCC MYA-4607) spores (10 6 ) were grown in SD liquid media on a shaker at 30 C.…”

“…followed by re-N-acetylation with ( 2 H 6 )acetic anhydride) and(3,4,5,6,7,(8)(9)(10)(11)(12)(13) C 6 )Kdo (Scheme S1, Figures S19 and S20, Methods) was added to the homogenate aliquots for a final concentration of 10,000 ppb during analysis. The aliquots were then frozen in liquid N 2 , lyophilized overnight, and weighed to determine the dry mass of mycelia.…”