Structural and functional analysis of KfiA, a UDP-GlcNAc transferase involved in Escherichia coli K5 heparosan biosynthesis.

Abstract

Heparosan is a linear glycosaminoglycan-related polysaccharide produced as the capsular polysaccharide of Escherichia coli K5. Its biosynthesis requires alternating transfer of N-acetylglucosamine and glucuronic acid by the glycosyltransferases KfiA and KfiC, respectively, but the structural basis of KfiA function has remained unclear. Here, we determined crystal structures of a C-terminally truncated KfiA in the apo state and in complex with UDP and a metal ion tentatively assigned as Mn2+, and combined structural analysis with functional characterization. KfiA adopts a conserved GT-A fold with a well-defined nucleotide- and metal-binding center. Structural and biochemical analyses further distinguish the core donor-associated catalytic region from peripheral structural elements that contribute to efficient glycosyl transfer. Comparison with related glycosyltransferases reveals a conserved structural framework for UDP-GlcNAc utilization. These results provide a structural basis for understanding KfiA function and heparosan chain extension in the two-enzyme KfiA-KfiC system.