Functional Exploration of the Polysaccharide Lyase Family PL6.

Sophie Mathieu,William Helbert,Bernard Henrissat,Gudmund Skjåk-Bræk,Flavien Labre,Israel Silman

doi:10.1371/journal.pone.0159415

Abstract

Alginate, the main cell-wall polysaccharide of brown algae, is composed of two residues: mannuronic acid (M-residues) and, its C5-epimer, guluronic acid (G-residues). Alginate lyases define a class of enzymes that cleave the glycosidic bond of alginate by β-elimination. They are classified according to their ability to recognize the distribution of M- and G-residues and are named M-, G- or MG-lyases. In the CAZy database, alginate lyases have been grouped by sequence similarity into seven distinct polysaccharide lyase families. The polysaccharide lyase family PL6 is subdivided into three subfamilies. Subfamily PL6_1 includes three biochemically characterized enzymes (two alginate lyases and one dermatan sulfatase lyase). No characterized enzymes have been described in the two other subfamilies (PL6_2 and PL6_3). To improve the prediction of polysaccharide-lyase activity in the PL6 family, we re-examined the classification of the PL6 family and biochemically characterized a set of enzymes reflecting the diversity of the protein sequences. Our results show that subfamily PL6_1 includes two dermatan sulfates lyases and several alginate lyases that have various substrate specificities and modes of action. In contrast, subfamilies PL6_2 and PL6_3 were found to contain only endo-poly-MG-lyases.

Highlights

Alginate is the main structural cell-wall component of brown algae (Phaeophyceae) and is produced by a limited number of Gram-negative bacteria
All 15 genes were successfully amplified by PCR except the sven0074 gene whose sequence was optimized for expression in E. coli, and successfully cloned in pET28a
The cloned gene mase04180 was not expressed in pET28a and was cloned in pET32a in which there was satisfactory expression of the soluble protein coupled to thioredoxin

Summary

Introduction

Alginate is the main structural cell-wall component of brown algae (Phaeophyceae) and is produced by a limited number of Gram-negative bacteria. It is a linear copolymer made of two building units, β-D-mannuronic acid (M) and its C5 epimer α-L-guluronic acid (G). Alginate lyases define class of enzymes that cleave the alginate glycosidic bonds via a β-elimination mechanism leading to the formation of an unsaturated residue, 4-deoxy-erythro-hex-. Alginate lyases are classified according to their substrate specificities and refer to the block structures encountered in alginate. M-G blocks can be cleaved by M-G-lyase or G-M-lyase according to the recognition properties of the enzyme [2]

Methods

Results

Conclusion