The role of tyrosine-114 in the enzymatic activity of the Shiga-like toxin I A-chain.
Shiga-like toxin I (SLT-I), the potent cytotoxin produced by certain pathogenic strains of Escherichia coli, is a member of a burgeoning family of ribosome-in-activating proteins (RIPs), which share common structural and mechanistic features. The prototype of the group is the plant toxin ricin. Recently we proposed a structural model for the Slt-IA active site, based in part on the known geometry of the enzymatic subunit of the ricin toxin. The model places three aromatic residues within the putative Slt-IA active site cleft: tyrosine 77, tyrosine 114, and tryptophan 203. Here we present biochemical and biophysical data regarding, the phenotypes of conservative point mutants of Slt-IA in which tyrosine 114 is altered. We used oligonucleotide-directed mutagenesis to replace tyrosine 114 with either phenylalanine (Y114F) or serine (Y114S). Periplasmic extracts of E. coli containing wild-type or mutant Slt-IA were tested for their ability to inhibit protein synthesis in vitro. Relative to wild-type, the activity of mutant Y114F was attenuated about 30-fold, while the mutant Y114S was attenuated about 500 to 1000-fold. In order to address the possibility that differential activation of the mutants rather than local effects at the active site might account for their diminished activity, we engineered the same mutations into a truncated slt-IA cassette that directs expression of a product corresponding to the activated A1 form of Slt-IA (wild-type-delta). The same general relationships held: relative to wild type-delta, Y114F-delta was attenuated about 7-fold, and Y114S-delta about 300-fold.(ABSTRACT TRUNCATED AT 250 WORDS)
Deresiewicz
R L
RL
Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115.
Austin
P R
PR
Hovde
C J
CJ
D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D022622:Shiga Toxin 1; D014443:Tyrosine / Q000378:metabolism*
D001427:Bacterial Toxins; D017931:DNA Primers; D022622:Shiga Toxin 1; D014443:Tyrosine