tailieunhanh - Báo cáo khoa học: Identification and characterization of important residues in the catalytic mechanism of CMP-Neu5Ac synthetase from Neisseria meningitidis

Sialylated oligosaccharides, present on mammalian outer-cell surfaces, play vital roles in cellular interactions and some bacteria are able to mimic these structures to evade their host’s immune system. It would be of great benefit to the study of infectious and autoimmune diseases and cancers, to under-stand the pathway of sialylation in detail to enable the design and produc-tion of inhibitors and mimetics. | ỊFEBS Journal Identification and characterization of important residues in the catalytic mechanism of CMP-Neu5Ac synthetase from Neisseria meningitidis Louise E. Horsfall1 Adam Nelson1 2 and Alan Berry1 1 Astbury Centre for StructuralMolecular Biology University of Leeds UK 2 Schoolof Chemistry University of Leeds UK Keywords CMP-Neu5Ac enzyme kinetics N-acylneuraminate cytidylyltransferase sialic acid Correspondence A. Berry Astbury Centre for Structural Molecular Biology University of Leeds Leeds LS2 9JT UK Fax 44 113 343 7486 Tel 44 113 343 3158 E-mail Re-use of this article is permitted in accordance with the Terms and Conditions set out at http . com authorresources Received 4 March 2010 revised 22 April 2010 accepted 23 April 2010 doi Sialylated oligosaccharides present on mammalian outer-cell surfaces play vital roles in cellular interactions and some bacteria are able to mimic these structures to evade their host s immune system. It would be of great benefit to the study of infectious and autoimmune diseases and cancers to understand the pathway of sialylation in detail to enable the design and production of inhibitors and mimetics. Sialylation occurs in two stages the first to activate sialic acid and the second to transfer it to the target molecule. The activation step is catalysed by the enzyme CMP-Neu5Ac synthetase CNS . Here we used crystal structures of CNS and similar enzymes to predict residues of importance in the CNS from Neisseria meningitidis. Nine residues were mutated to alanine and the steady-state enzyme kinetic parameters were measured using a continuous assay to detect one of the products of the reaction pyrophosphate. Mutations that caused the greatest loss in activity included K142A D211A D209A and a series of mutations at residue Q104 highlighted from sequence-alignment studies of related enzymes demonstrating significant roles for these residues