The purification and characterization of recombinant yeast dolichyl-phosphate-mannose synthase. Site-directed mutagenesis of the putative dolichol recognition sequence

Yeast dolichyl-phosphate-mannose synthase was purified from cultures of Escherichia coli carrying the gene for this enzyme in a high expression vector. The synthase contains a highly conserved hydrophobic amino acid sequence proposed to be involved in the recognition of dolichols (Albright, C.F., Or...

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Bibliographic Details
Published in:The Journal of biological chemistry 1993-11, Vol.268 (32), p.24190-24196
Main Authors: SCHUTZBACH, J. S, ZIMMERMAN, J. W, FORSEE, W. T
Format: Article
Language:English
Subjects:
actividad enzimatica
activite enzymatique
Amino Acid Sequence
Analytical, structural and metabolic biochemistry
Base Sequence
Binding Sites
Biological and medical sciences
Catalysis
Dolichols - metabolism
Electrophoresis, Polyacrylamide Gel
Enzymes and enzyme inhibitors
enzymic activity
escherichia
Escherichia coli - genetics
Fundamental and applied biological sciences. Psychology
gene
genes
Genes, Fungal
genetic transformation
glicosiltransferasas
glycosyltransferase
glycosyltransferases
Mannosyltransferases - genetics
Mannosyltransferases - isolation & purification
Mannosyltransferases - metabolism
Molecular Sequence Data
mutacion
Mutagenesis, Site-Directed
mutation
Oligodeoxyribonucleotides
oligosacaridos
oligosaccharide
oligosaccharides
purificacion
purification
Recombinant Proteins - genetics
Recombinant Proteins - isolation & purification
Recombinant Proteins - metabolism
Saccharomyces cerevisiae
Saccharomyces cerevisiae - enzymology
Saccharomyces cerevisiae - genetics
Transferases
transformacion genetica
transformation genetique
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Description
Summary:Yeast dolichyl-phosphate-mannose synthase was purified from cultures of Escherichia coli carrying the gene for this enzyme in a high expression vector. The synthase contains a highly conserved hydrophobic amino acid sequence proposed to be involved in the recognition of dolichols (Albright, C.F., Orlean, P., and Robbins, P.W. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7366-7369) and amino acid residues in this sequence were altered by site-directed mutagenesis. Conservative substitutions had no effect on the affinity of the enzyme for dolichyl-P. The substitution of asparagine for isoleucine at position 253 resulted in higher values for the apparent Km for Dol-P when assayed in detergent solutions, but this substitution had no effect on Km when the enzyme was reconstituted with phosphatidylethanolamine. Enzyme containing a deletion of the entire putative dolichol recognition sequence retained catalytic activity. The apparent Km for Dol-P was increased when this enzyme was assayed in detergent solution but was the same as wild type enzyme when reconstituted in phosphatidylethanolamine. These results suggest that the amino acid composition and sequence of the conserved domain are not critically important for the recognition and binding of Dol-P when the synthase is present in a lipid matrix.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(20)80509-2