Along the path of bacterial nonulosonic acids. A study of the bio- and in vitro synthesis of sialic acid related compounds

Abstract

Nonulosonic acids (NulOs) are sugars expressed on the surface of bacterial cells as well as vertebrates and are involved in a multitude of physiological and pathogenic processes. Their biosynthesis is directed by gene clusters that vary in both gene content and organization, leading to a diverse family of compounds. The exploration of this diversity is relatively recent, helped by the amount of genetic data generated by new technologies. It is however limited by the availability of the compounds themselves and the means to produce them. The presented work describes, for the first part, the study of NulO biosynthesis gene clusters in the fish pathogens A. salmonicida, A. wodanis and M. viscosa in relation to the type of NulO they produce. The key enzyme of the biosynthesis, NeuB, is considered in terms of amino-acid sequence, active-site geometry, and substrate specificity. The second part deals with the bioprospecting of N-acetyl-2-glucosamine epimerases (AGEs) for the in vitro synthesis of N-acetylneuraminic acid, the most abundant NulO in nature. Two isolates of the cyanobacterium Nostoc sp. (KVJ10 and KV20) were targeted. Nostoc sp. KVJ20 was sequenced, and the AGEs from both strains (nAGE10 and nAGE20) were cloned, expressed, and purified. The crystal structure of nAGE10 was solved, revealing a new (and most likely biologically relevant) dimeric organization of AGEs. Together, these studies participate in the monumental task of understanding the determinants of NulO diversity as well as creating the means for the study of these compounds in general.