Ancestral reconstruction of the GH5_18 beta-mannosidase from Bifidobacterium longum

Abstract

The family 5 of glycoside hydrolases (GH5) consists of more than 12000 members that recognize and hydrolyze a broad range of carbohydrates. For a better understanding of the great diversity of its enzymatic repertoire, Asperborg et al. (2012) classified the GH5 family into 51 subfamilies, from which 20 did not have available characterization data so far. In the current FAPESP doctoral project 2016/00740-2, one of the goals is to provide the first structural and functional data for the uncharacterized subfamily GH5_18. Using enzymatic assays, we discovered that one of the GH5_18 members, the enzyme we named BlMan5B, is a ²-mannosidase highly specific for the cleavage of disaccharide 2-Acetamido-2-deoxy-4-O-(bD-mannopyranosyl)-D-glucopyranose (Man-²1,4-GlcNAc), an intermediate product of N-glycans decomposition. The three-dimensional structure of BlMan5B was determined by X-ray crystallography and the generated model revealed unique structural features in the CAZyme universe: a flexible C-terminal region that is stabilized in the presence of N-acetylglucosamine (GlcNAc), acting as a lid on the active site; a Rossmann domain that appears to be important for the regulation of the flexible C-terminal; and a dimeric arrangement stabilized by an exchange of the Trp120 residue between the subunits of the dimer (tryptophan swapping), residue that also interacts with the substrate. BlMan5B is the first GH5 described acting on the decomposition of N-glycans and its unprecedented activity reflects its unique structural characteristics. The phylogeny of the subfamily GH5_18 suggests that this specialization occurred along the conquest of the intestinal microenvironment. To better understand the evolution of this enzyme and how it correlates with the adaptation to the human intestine, the technique of reconstructing ancestral sequences can be a powerful tool. Using this approach, we can resuscitate enzymes at different evolutionary stages, rescuing ancestral enzymatic characteristics in different geological periods. In addition to gradually showing the appearance of the recognition of GlcNAc by GH5_18 beta-mannosidases, the ancestral reconstruction technique usually results in more efficient, thermostable and generalist enzymes that can be used in various industrial and biotechnological processes. Therefore, during the period at the lab of Prof. Jimenez we intend to reconstruct the ancestral sequences of BlMan5B to understand how the specific recognition of GlcNAc emerged throughout its evolutionary history and to evaluate the biotechnological potential of their ancestors. (AU)