Year: 2026
Type: preprint
Abstract: ABSTRACT Glycosyltransferases have broad diversity in their acceptor substrates, yet the molecular and evolutionary basis of this diversity remains poorly understood. NodZ is an α-1,6-fucosyltransferase that fucosylates Nod factors, lipochitooligosaccharide molecules involved in the establishment of rhizobium–legume symbioses. While NodZ is the only known bacterial member of the GT23 family of α-1,6-fucosyltransferases, the determinants of its acceptor selectivity and the evolutionary origins thereof are unclear. Here, a phylogenetic analysis of 332 NodZ-like proteins revealed a distinct rhizobial clade and significantly greater structural diversity in the N -terminus than in the C -terminus. Kinetic analysis of a Mesorhizobium japonicum NodZ demonstrated selectivity for longer chitooligosaccharide acceptors (comprised of four to five GlcNAc units), while alanine scanning revealed W139 as a primary determinant of this chain length preference. We propose that W139 forms an aromatic platform that engages distal GlcNAc units present only in longer acceptors, coupling productive binding to acceptor chain length. We then used the NodZ phylogeny to reconstruct ancestral NodZ homolog sequences, which indicated that W139 emerged at the base of the rhizobial clade, and thus may represent a key evolutionary step towards canonical NodZ function. Additionally, the most recent resurrected ancestor was capable of fucosylating chitooligosaccharides with lower efficiency than the extant NodZ, suggesting that efficiency towards chitooligosaccharides was improved during the evolution of this family. Results described here demonstrate that evolutionary diversification of the N -terminal region can remodel acceptor specificity while preserving the conserved catalytic architecture of GT23 fucosyltransferases, providing a framework for understanding and engineering fucosyltransferase specificity.
Institution: Queen's University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Awards: G1394658519, G2271590008
OpenAlex ID: W7220658094