Selection of diphtheria toxin active-site mutants in yeast. Rediscovery of glutamic acid-148 as a key residue.
Saccharomyces cerevisiae was transformed with expression plasmids carrying the DTA gene under control of the GAL1 promoter; colonies that formed under inducing conditions were selected; and plasmids from these colonies were screened for mutations in DTA that failed to block expression of the protein. Substitutions at three sites were identified, all of which are in the active-site cleft; and each of the substitutions reduced ADP-ribosyltransferase activity by > 10(5). The substitutions include a charge reversal mutation of a catalytically important residue (Glu148Lys) and replacements of either of two glycines (Gly22 and Gly52) with bulky residues. The fact that multiple mutations were identified in these same residues implies that there are relatively few sites at which substitutions ablate ADP-ribosyltransferase activity without blocking expression of the full-length protein. Incorporation of a primary attenuating mutation into the DTA gene allowed S. cerevisiae also to be used to select complementary secondary mutations which altered activity less drastically. Besides elucidating structure-activity relationships, mutations identified by these approaches may be useful in designing new vaccines.
Fu
H
H
Blanke
S R
SR
Mattheakis
L C
LC
Collier
R J
RJ
D001665:Binding Sites; D004167:Diphtheria Toxin / Q000737:chemistry / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D005822:Genetic Vectors; D018698:Glutamic Acid; D015394:Molecular Structure; D009154:Mutation; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000302:isolation & purification
D004167:Diphtheria Toxin; D004791:Enzyme Inhibitors; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011993:Recombinant Fusion Proteins; D018698:Glutamic Acid