Distantly related plant and nematode core α1,3-fucosyltransferases display similar trends in structure–function relationships

Abstract

Here, we present a comparative structure-function study of a nematode and a plant core α1,3-fucosyltransferase based on deletion and point mutations of the coding regions of Caenorhabditis elegans FUT-1 and Arabidopsis thaliana FucTA (FUT11). In particular, our results reveal a novel "first cluster motif" shared by both core and Lewis-type α1,3-fucosyltransferases of the GT10 family. To evaluate the role of the conserved serine within this motif, this residue was replaced with alanine in FucTA (S218) and FUT-1 (S243). The S218A replacement completely abolished the enzyme activity of FucTA, while the S243A mutant of FUT-1 retained 20% of the "wild-type" activity. Based on the results of homology modeling of FucTA, other residues potentially involved in the donor substrate binding were examined, and mutations of N219 and R226 dramatically affected enzymatic activity. Finally, as both FucTA and FUT-1 were shown to be N-glycosylated, we examined the putative N-glycosylation sites. While alanine replacements at single potential N-glycosylation sites of FucTA resulted in a loss of up to 80% of the activity, a triple glycosylation site mutant still retained 5%, as compared to the control. In summary, our data indicate similar trends in structure-function relationships of distantly related enzymes which perform similar biochemical reactions and form the basis for future work aimed at understanding the structure of α1,3-fucosyltransferases in general.

Poplar carbohydrate‐active enzymes: whole‐genome annotation and functional analyses based on RNA expression data

“…Another coexpressed gene was Potri.019G082200 from GT10. In A. thaliana, Golgi localized GT10 a-1,3-or a-1,4-fucosyltransferases have been proposed to function in N-glycan biosynthesis (Both et al, 2011;Rips et al, 2017). Our data Fig.…”

“…Another co‐expressed gene was Potri.019G082200 from GT10. In A. thaliana , Golgi localized GT10 α‐1,3‐or α‐1,4‐fucosyltransferases have been proposed to function in N ‐glycan biosynthesis (Both et al ., ; Rips et al ., ). Our data suggest a role for the GT10 family in primary cell wall biosynthesis in the xylem.…”

Exploring the N-glycosylation Pathway in Chlamydomonas reinhardtii Unravels Novel Complex Structures

“…A putative fucosyltransferase, CrFUT1, exhibiting 20% and 21% identity with ␣1,3-fucosyltransferases from A. thaliana At-FUT11 and AtFUT12, respectively, was also predicted in the genome (Table IV). This protein sequence exhibited the expected type-II membrane protein topology and motifs required for ␣(1,3)-fucosyltransferase activity (73)(74)(75), as well as conserved Cys residues and a CXXC motif located at the C-terminal end that is involved in the formation of disulfide bonds in plant ␣(1,3)-fucosyltransferases (76). DISCUSSION Here, we developed an integrated genomic, glycomic, and glycoproteomic approach to unravel the N-glycosylation pathway of C. reinhardtii and shed light on N-glycan structures and N-glycosylated proteins.…”

Evolution of protein N-glycosylation process in Golgi apparatus which shapes diversity of protein N-glycan structures in plants, animals and fungi

“…In Arabidopsis, the genes encoding these enzymes were identified (AT3G19280 and AT1G49710 for α1,3-FucT, and AT1G71990 for α1,4-FucT)9697. The Arabidopsis genes encoding both the enzymes conferred domain PF00852.…”