Document Detail

Disulfide bond structure and domain organization of yeast beta(1,3)-glucanosyltransferases involved in cell wall biogenesis.
MedLine Citation:
PMID:  18468997     Owner:  NLM     Status:  MEDLINE    
The Gel/Gas/Phr family of fungal beta(1,3)-glucanosyltransferases plays an important role in cell wall biogenesis by processing the main component beta(1,3)-glucan. Two subfamilies are distinguished depending on the presence or absence of a C-terminal cysteine-rich domain, denoted "Cys-box." The N-terminal domain (NtD) contains the catalytic residues for transglycosidase activity and is separated from the Cys-box by a linker region. To obtain a better understanding of the structure and function of the Cys-box-containing subfamily, we identified the disulfide bonds in Gas2p from Saccharomyces cerevisiae by an improved mass spectrometric methodology. We mapped two separate intra-domain clusters of three and four disulfide bridges. One of the bonds in the first cluster connects a central Cys residue of the NtD with a single conserved Cys residue in the linker. Site-directed mutagenesis of the Cys residue in the linker resulted in an endoplasmic reticulum precursor that was not matured and underwent a gradual degradation. The relevant disulfide bond has a crucial role in folding as it may stabilize the NtD and facilitate its interaction with the C-terminal portion of a Gas protein. The four disulfide bonds in the Cys-box are arranged in a manner consistent with a partial structural resemblance with the plant X8 domain, an independent carbohydrate-binding module that possesses only three disulfide bonds. Deletion of the Cys-box in Gas2 or Gas1 proteins led to the formation of an NtD devoid of any enzymatic activity. The results suggest that the Cys-box is required for proper folding of the NtD and/or substrate binding.
Laura Popolo; Enrico Ragni; Cristina Carotti; Oscar Palomares; Ronald Aardema; Jaap Willem Back; Henk L Dekker; Leo J de Koning; Luitzen de Jong; Chris G de Koster
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2008-05-09
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  283     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  2008 Jul 
Date Detail:
Created Date:  2008-06-30     Completed Date:  2008-08-19     Revised Date:  2009-05-18    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  18553-65     Citation Subset:  IM    
Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, 20133 Milano, Italy.
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MeSH Terms
Amino Acid Substitution
Cell Wall / enzymology*,  genetics
Cysteine / chemistry,  genetics,  metabolism
Disulfides / chemistry*,  metabolism
Glucan Endo-1,3-beta-D-Glucosidase / chemistry*,  genetics,  metabolism
Mass Spectrometry
Membrane Glycoproteins / chemistry*,  genetics,  metabolism
Mutagenesis, Site-Directed
Protein Structure, Tertiary / physiology
Saccharomyces cerevisiae / enzymology*
Saccharomyces cerevisiae Proteins / chemistry*,  genetics,  metabolism
Reg. No./Substance:
0/Disulfides; 0/GAS1 protein, S cerevisiae; 0/Membrane Glycoproteins; 0/Saccharomyces cerevisiae Proteins; 52-90-4/Cysteine; EC 3.2.1.-/1,3-beta-glucanosyltransferase; EC Endo-1,3-beta-D-Glucosidase

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