Abstract
In the yeast Saccharomyces cerevisiae, members of the Kre2/Mnt1 protein family have been shown to be α-1,2-mannosyltransferases or α-1,2-mannosylphosphate transferases, utilising an Mn2+-coordinated GDP-mannose as the sugar donor and a variety of mannose derivatives as acceptors. Enzymes in this family are localised to the Golgi apparatus, and have been shown to be involved in both N- and O-linked glycosylation of newly-synthesised proteins, including cell wall glycoproteins. Our knowledge of the nine proteins in this family is however very incomplete at present. Only one family member, Kre2p/Mnt1p, has been studied by structural methods, and three (Ktr4p, Ktr5p, Ktr7p) are completely uncharacterised and remain classified only as putative glycosyltransferases. Here we use in vitro enzyme activity assays to provide experimental confirmation of the predicted glycosyltransferase activity of Ktr4p. Using GDP-mannose as the donor, we observe activity towards the acceptor methyl-α-mannoside, but little or no activity towards mannose or α-1,2-mannobiose. We also present the structure of the lumenal catalytic domain of S. cerevisiae Ktr4p, determined by X-ray crystallography to a resolution of 2.2 Å, and the complex of the enzyme with GDP to 1.9 Å resolution.
Highlights
IntroductionGlycosyltransferase enzymes catalyse glycosidic bond formation by transferring a carbohydrate moiety from an activated ‘donor’ sugar substrate, most frequently a nucleoside diphosphate sugar, to an acceptor molecule which may be another sugar, a protein or nucleic acid, or a small molecule such as an antibiotic
Ktr4p is a member of the Kre2/Mnt1 family of glycosyltransferases
Despite extensive efforts to produce a structure of Ktr4p in complex with GDP-mannose, both by co-crystallisation and by soaking native crystals with GDP-mannose, we have not been able to observe the non-hydrolysed state of the ligand, but only the GDP moiety
Summary
Glycosyltransferase enzymes catalyse glycosidic bond formation by transferring a carbohydrate moiety from an activated ‘donor’ sugar substrate, most frequently a nucleoside diphosphate sugar, to an acceptor molecule which may be another sugar, a protein or nucleic acid, or a small molecule such as an antibiotic. They constitute a large and diverse class of enzymes, with more than 190,000 identified in sequenced genomes according to the CAZy database, (www.cazy.org)[1], and these are classified as belonging to more than 90 sequence-based families. There are no homologues of the proteins in higher eukaryotes.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
