Optimization of a direct spectrophotometric method to investigate the kinetics and inhibition of sialidases

Jasvinder Kaur Hayre,Garry L Taylor,Jean-Denis Docquier,Peter W Andrew,Marco R Oggioni,Guogang Xu,Luisa Borgianni

doi:10.1186/1471-2091-13-19

Abstract

BackgroundsStreptococcus pneumoniae expresses three distinct sialidases, NanA, NanB, and NanC, that are believed to be key virulence factors and thus, potential important drug targets. We previously reported that the three enzymes release different products from sialosides, but could share a common catalytic mechanism before the final step of product formation. However, the kinetic investigations of the three sialidases have not been systematically done thus far, due to the lack of an easy and steady measurement of sialidase reaction rate.ResultsIn this work, we present further kinetic characterization of pneumococcal sialidases by using a direct spectrophotometric method with the chromogenic substrate p-nitrophenyl-N-acetylneuraminic acid (p-NP-Neu5Ac). Using our assay, the measured kinetic parameters of the three purified pneumococcal sialidase, NanA, NanB and NanC, were obtained and were in perfect agreement with the previously published data. The major advantage of this alternative method resides in the direct measurement of the released product, allowing to readily determine of initial reaction rates and record complete hydrolysis time courses.ConclusionWe developed an accurate, fast and sensitive spectrophotometric method to investigate the kinetics of sialidase-catalyzed reactions. This fast, sensitive, inexpensive and accurate method could benefit the study of the kinetics and inhibition of sialidases in general.

Highlights

Sialidases catalyze the removal of terminal sialic acid residue from various glycoconjugates and have been implicated in pathogenesis of infectious diseases [1]
According to a recent NMR report, NanA is a classic hydrolytic sialidase, whereas NanB could be an IT trans-sialidase similar to the leech enzyme, and NanC can handle the dual functions of both producing 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en, DANA) and hydrating this general sialidase inhibitor when substrate is depleted [5,6,7]
Optimization of a direct spectrophotometric assay to investigate the kinetics of sialidase-catalyzed reactions UV-visible spectra of the substrate and reaction product of pneumococcal sialidases catalysis were compared in this work

Summary

Introduction

Sialidases catalyze the removal of terminal sialic acid residue from various glycoconjugates and have been implicated in pathogenesis of infectious diseases [1]. According to a recent NMR report, NanA is a classic hydrolytic sialidase, whereas NanB could be an IT trans-sialidase similar to the leech enzyme, and NanC can handle the dual functions of both producing 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en, DANA) and hydrating this general sialidase inhibitor when substrate is depleted [5,6,7]. It is proposed that the three could share a common catalytic mechanism before the final product formation step from a chemistry point of view. Based on these findings, a new sialidase triad is speculated, which might coordinate the sialidase action associated with pneumococcal virulence. The kinetic investigations of the three sialidases have not been systematically done far, due to the lack of an easy and steady measurement of sialidase reaction rate

Methods

Results

Conclusion