Characterization of a Metal-independent CAZy Family 6 Glycosyltransferase from Bacteroides ovatus

Abstract

The myriad functions of complex carbohydrates include modulating interactions between bacteria and their eukaryotic hosts. In humans and other vertebrates, variations in the activity of glycosyltransferases of CAZy family 6 generate antigenic variation between individuals and species that facilitates resistance to pathogens. The well characterized vertebrate glycosyltransferases of this family are multidomain membrane proteins with C-terminal catalytic domains. Genes for proteins homologous with their catalytic domains are found in at least nine species of anaerobic commensal bacteria and a cyanophage. Although the bacterial proteins are strikingly similar in sequence to the catalytic domains of their eukaryotic relatives, a metal-binding Asp-X-Asp sequence, present in a wide array of metal ion-dependent glycosyltransferases, is replaced by Asn-X-Asn. We have cloned and expressed one of these proteins from Bacteroides ovatus, a bacterium that is linked to inflammatory bowel disease. Functional characterization shows it to be a metal-independent glycosyltransferase with a 200-fold preference for UDP-GalNAc as substrate relative to UDP-Gal. It efficiently catalyzes the synthesis of oligosaccharides similar to human blood group A and may participate in the synthesis of the bacterial O-antigen. The kinetics for GalNAc transfer to 2-fucosyl lactose are characteristic of a sequential mechanism, as observed previously for this family. Mutational studies indicate that despite the lack of a metal cofactor, there are pronounced similarities in structurefunction relationships between the bacterial and vertebrate family 6 glycosyltransferases. These two groups appear to provide an example of horizontal gene transfer involving vertebrates and prokaryotes.The structures of complex glycans are determined by the specificities of the glycosyltransferases (GTs) 2 that catalyze their biosynthesis. GTs fall into two groups that differ in mechanism, based on whether the anomeric configuration of the donor substrate (␣ for most UDP-sugars) is retained or inverted in the product (1-3). They are classified into 90 different families in the CAZy data base based on sequence similarities (4, 5), but the majority of those that have been structurally characterized fall into one of two fold types, designated GT-A and GT-B (2). The retaining GTs of CAZy family 6 (GT6) have a GT-A fold and catalyze the transfer of either galactose or GalNAc into an ␣-linkage with the 3-OH group of ␤-linked galactose or GalNAc. GT6 includes the histo-blood group A and B GTs (GTA and GTB), the ␣-galactosyltransferase (␣3GT) that catalyzes the synthesis of the xenoantigen or ␣-gal epitope, Forssman glycolipid synthase, isogloboside 3 synthase, and their homologues from other vertebrates (6). GT6 enzymes from vertebrates are type-2 membrane proteins with N-terminal cytosolic domains, a transmembrane helix, a spacer, and a C-terminal catalytic domain (6). Crystallographic studies of recombinant catalytic domains of GTA, GTB, and ␣3GT have provided detailed inform...

Family 6 Glycosyltransferases in Vertebrates and Bacteria: Inactivation and Horizontal Gene Transfer May Enhance Mutualism between Vertebrates and Bacteria

“…Although functional studies of more bacterial enzymes are needed, their apparent metal independence suggests that their catalytic mechanisms differ from those of the well studied metal-dependent enzymes from mammals or that the metal ion is functionally replaced by a substructure of the protein. The strong conservation of mammalian active-site residues in the bacterial enzymes suggests that the active-site structures in these groups are similar, consistent with the results of mutational studies with B. ovatus GT6 (39).…”

“…2, compare A and B). This region has a distinctive composition with high proportions of basic and nonpolar amino acids; truncation of this region in BoA shows that it is not required for activity (39). These features are reminiscent of previous observations with the unrelated bacterial GT8 enzymes.…”

“…However, the effects of mutating selected residues ( Fig. 2A, regions C and E-G) in the B. ovatus enzyme suggest that there are strong similarities in structure-function relationships with the mammalian GT6 group (39).…”

“…With new information from genome sequences, genes were identified that encode members of the GT6 family in two nonvertebrate eukaryotes, Branchiostoma floridae (an amphioxus) and Capsaspora owczarzaki (a unicellular opisthokont that is a symbiont of a snail) (38), plus 16 species of bacteria and the cyanophage P-SSM2 (supplemental Table 1); many other homologs are also in the human intestinal and marine metagenomes (39). The distribution pattern in eukaryotes is puzzling because the amphioxus is a chordate like the vertebrates, but C. owczarzaki represents an early branch from the line leading to metazoa and fungi.…”

“…Based on sequences in key substrate interaction regions, the members of each group appear to be similar in substrate specificity to each other, whereas the two groups may differ in both donor and acceptor substrate specificity; whether the second group is from phage and/or bacterial sources is unclear. Other homologs in the human intestinal metagenome have sequence characteristics of bacterial GT6 enzymes (39).…”