An Engineered Hyaluronan Synthase

Abstract

The Class I hyaluronan synthase (HAS) is a unique glycosyltransferase synthesizing hyaluronan (HA), a polysaccharide composed of GlcUA and GlcNAc, by using one catalytic domain that elongates two different monosaccharides. As for the synthetic mechanism, there are two alternative manners for the sugar elongation process. Some bacterial HASs add new sugars to the non-reducing end of the acceptor to grow polymers. On the other hand, some vertebrate enzymes seem to transfer sugars to the reducing end. Expression of vertebrate HASs as active and soluble proteins will accelerate further precise insight into mechanisms of sugar elongation reactions by natural HASs. Since large scale production of HA polymers and oligomers would become powerful tools both for basic studies and new biotechnology to create functional carbohydrates in medicinal purposes, advent of an efficient method for the expression of HASs in Escherichia coli is strongly expected. Here we communicate the first success of the production of recombinant human HAS2 proteins composed of only the catalytic region in E. coli as the active form. It was demonstrated that an engineered HAS2 expressed in E. coli exhibited significant activity to synthesize a mixture of HAS oligomers from 8-mer (HA8) to 16-mer (HA16). Engineered HAS2 prepared herein elongated sugars from exogenous tetrasaccharide to form polymers with a direction to the non-reducing end. According to the present results, large scale production of engineered recombinant HASs is to be performed using E. coli that will provide practical and economic advantages in manufacturing enzymes for use in the synthesis of various oligomeric HA molecules and their industrial applications.

Biosynthesis of Hyaluronan

“…Our conclusion is in agreement with recent studies of a truncated form of recombinant mammalian HAS2. This enzyme, contrary to the intact protein, was found to use exogenously supplied HA oligosaccharides as sugar acceptors in chain elongation reactions (16). Acceptor capacity thus was demonstrated for a tetrasaccharide with an intact non-reducing terminal GlcUA residue, generated by the digestion of HA with testicular hyaluronidase.…”

“…Both of these native enzyme systems failed to further extend preformed, exogenously added HA oligosaccharides. However, in more recent studies, recombinant HAS from P. multocida (15) as well as a recombinant truncated form of the vertebrate HAS2 isoform (16) were found to catalyze the addition of sugar units at the non-reducing ends of added oligosaccharide primers.…”

Characterization of the Lipid Linkage Region and Chain Length of the Cellubiuronic Acid Capsule of Streptococcus pneumoniae

“…In view of the conflicting history for the direction of growth of hyaluronan (27)(28)(29)(30)(31)(32), we sought to more firmly establish this feature of cellubiuronan assembly. The pulse-chase feature of the direction of growth experiment, in conjunction with the dual isotope labeling pattern, demonstrated both kinetically and structurally that cellubiuronan polysaccharide chains are first initiated as oligosaccharide-lipids and that the transition into the fully processive state occurs by successive nonreducing terminal additions of Glc and GlcUA, in agreement with the previous cellubiuronan biosynthetic model (12).…”

Genetic basis for hyper production of hyaluronic acid in natural and engineered microorganisms

“…HASs are divided into two categories, designated Class I and Class II, based on their amino acid sequence homology and structural topology [ 30 , 40 ]. HA synthases belonging to Class I are present in some species of Streptococcus , viruses and vertebrates.…”