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., 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.
Schutzbach
J S
JS
Department of Microbiology, University of Alabama, Birmingham 35294-0019.
Zimmerman
J W
JW
Forsee
W T
WT
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D004286:Dolichols / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005800:Genes, Fungal; D008364:Mannosyltransferases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D004286:Dolichols; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D008364:Mannosyltransferases; C022755:dolichyl-phosphate beta-D-mannosyltransferase