Glucuronyltransferase Activity of KfiC from Escherichia coli Strain K5 Requires Association of KfiA

Abstract

Heparan sulfate is a ubiquitous glycosaminoglycan in the extracellular matrix of most animals. It interacts with various molecules and exhibits important biological functions. K5 antigen produced by Escherichia coli strain K5 is a linear polysaccharide N-acetylheparosan consisting of GlcUA ␤1-4 and GlcNAc ␣1-4 repeating disaccharide, which forms the backbone of heparan sulfate. Region 2, located in the center of the K5-specific gene cluster, encodes four proteins, KfiA, KfiB, KfiC, and KfiD, for the biosynthesis of the K5 polysaccharide. Here, we expressed and purified the recombinant KfiA and KfiC proteins and then characterized these enzymes. Whereas the recombinant KfiC alone exhibited no GlcUA transferase activity, it did exhibit GlcUA transferase and polymerization activities in the presence of KfiA. In contrast, KfiA had GlcNAc transferase activity itself, which was unaffected by the presence of KfiC. The GlcNAc and GlcUA transferase activities were analyzed with various truncated and point mutants of KfiA and KfiC. The point mutants replacing aspartic acid of a DXD motif and lysine and glutamic acid of an ionic amino acid cluster, and the truncated mutants deleting the C-terminal and N-terminal sites, revealed the essential regions for GlcNAc and GlcUA transferase activity of KfiC and KfiA, respectively. The interaction of KfiC with KfiA is necessary for the GlcUA transferase activity of KfiC but not for the enzyme activity of KfiA. Together, these results indicate that the complex of KfiA and KfiC has polymerase activity to synthesize N-acetylheparosan, providing a useful tool toward bioengineering of defined heparan sulfate chains. Heparan sulfate (HS)3 is a linear polysaccharide of alternating hexuronic acid (D-glucuronic acid (GlcUA) or L-iduronic acid (IdoUA)) and D-glucosamine (GlcN) residues carrying sulfogroups at various sites of sugar residues. Usually, HS chains are covalently attached to a core protein in the form of proteoglycans and are present ubiquitously on the cell surface and in the extracellular matrices of animals. HS chains interact with cytokines, growth factors, coagulation factors, proteases and their inhibitors, and other molecules and contribute to several biological processes, including development, morphogenesis, cell proliferation and differentiation, and cancer cell invasion (1, 2).The biosynthesis of HS begins with the synthesis of the linkage tetrasaccharide, GlcUA-Gal-Gal-Xyl, on the serine residues of core proteins. The backbone of HS polysaccharide is then synthesized onto the linkage tetrasaccharide by alternating addition of monosaccharide units of GlcNAc and GlcUA with ␣1-4 and ␤1-4 bonds, respectively, using UDP-sugar donors with desired GlcNAc transferase (GlcNAc-T) and GlcUA transferase (GlcA-T). The elongation reaction is performed by HS co-polymerases, EXT1 and EXT2, in the Golgi apparatus (3, 4). The backbone is modified by several reactions, including N-deacetylation and N-sulfonation of GlcN by N-deacetylase/ N-sulfotransferase, C5-epimerization of GlcUA to f...

Metabolic engineering of Escherichia coli BL21 for biosynthesis of heparosan, a bioengineered heparin precursor

“…The single expression of KfiA or KfiC in E. coli BL21 could not produce heparosan at all, while the co-expression of KfiA and KfiC in recombinant sAC could produce 63 mg/L heparosan in shake flask. It was indicated that the KfiA and KfiC were the only exogenous genes required for heparosan synthesis in E. coli, and this result was consistent with what previously reported (Sugiura et al, 2010).…”

Interaction Network and Localization of Brucella abortus Membrane Proteins Involved in the Synthesis, Transport, and Succinylation of Cyclic β-1,2-Glucans

“…Recent studies of the glycosyltransferases involved in the synthesis of the K5 capsular polysaccharide have demonstrated that protein-protein interactions were essential for activity. In this large transenvelope complex, the glucuronyltransferase activity of KfiC is dependent on physical association with the N-acetylglucosaminyltransferase KfiA (29). Moreover, it has been demonstrated that this complex has a polar localization, consistent with the site where the biosynthesis and export of this polysaccharide occur (17).…”

“…The hypothesis that multienzymatic membrane complexes are implicated in the coordination of the biosynthesis (initiation, elongation, and termination) and transport of O polysaccharides and other bacterial polysaccharides is currently being strengthened experimentally (15,17,18,29). Some of these polysaccharides and the lipopolysaccharides (LPS) are important virulence factors in a wide spectrum of bacterial infections.…”

A Membrane-located Glycosyltransferase Complex Required for Biosynthesis of the d-Galactan I Lipopolysaccharide O Antigen in Klebsiella pneumoniae

“…In the related E. coli K5 system, recent studies of glycosyltransferases involved in the synthesis of the K5 capsular polysaccharide found that protein-protein interactions were essential for activity. The glucuronyltransferase activity of KfiC is dependent on physical association with the N-acetylglucosaminyltransferase, KfiA (43). These proteins may be part of a larger trans-envelope complex (44,45).…”