Structure of the Glycosyltransferase Ktr4p from Saccharomyces cerevisiae

Dominik D D Possner; Magnus Claesson; Jodie E Guy

doi:10.1371/journal.pone.0136239

Structure of the Glycosyltransferase Ktr4p from Saccharomyces cerevisiae

PLoS One. 2015 Aug 21;10(8):e0136239. doi: 10.1371/journal.pone.0136239. eCollection 2015.

Authors

Dominik D D Possner¹, Magnus Claesson², Jodie E Guy¹

Affiliations

¹ Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Tomtebodavägen 6, S 171-77, Stockholm, Sweden.
² Department of Biochemistry and Biophysics, Stockholm University, Svante Arrhenius väg 16C, S 106-91, Stockholm, Sweden.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Motifs
Catalysis
Catalytic Domain
Cell Wall / chemistry*
Cell Wall / enzymology
Cloning, Molecular
Crystallography, X-Ray
Escherichia coli / genetics
Escherichia coli / metabolism
Gene Expression
Golgi Apparatus / chemistry*
Golgi Apparatus / enzymology
Guanosine Diphosphate Mannose / chemistry*
Kinetics
Mannans / chemistry
Methylmannosides / chemistry
Models, Molecular
Molecular Sequence Data
Protein Structure, Secondary
Recombinant Proteins / chemistry
Recombinant Proteins / genetics
Saccharomyces cerevisiae / chemistry*
Saccharomyces cerevisiae / enzymology
Saccharomyces cerevisiae Proteins / chemistry*
Saccharomyces cerevisiae Proteins / genetics
Substrate Specificity
Transcription Factors / chemistry*
Transcription Factors / genetics

Substances

Mannans
Methylmannosides
Recombinant Proteins
SMI1 protein, S cerevisiae
Saccharomyces cerevisiae Proteins
Transcription Factors
mannobiose
Guanosine Diphosphate Mannose
methylmannoside

Grants and funding

This work was supported by funding awarded to JEG from the Åke Wibergs Stiftelse (ake-wiberg.se). Access to BESSY was supported by the European Community’s Seventh Framework Programme (FP7/2007-2013) under BioStruct-X (grant agreement N°283570). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.