Probing the structure-activity relationship of Escherichia coli LT-A by site-directed mutagenesis.
Computer analysis of the crystallographic structure of the A subunit of Escherichia coli heat-labile toxin (LT) was used to predict residues involved in NAD binding, catalysis and toxicity. Following site-directed mutagenesis, the mutants obtained could be divided into three groups. The first group contained fully assembled, non-toxic new molecules containing mutations of single amino acids such as Val-53-->Glu or Asp, Ser-63-->Lys, Val-97-->Lys, Tyr-104-->Lys or Asp, and Ser-114-->Lys or Glu. This group also included mutations in amino acids such as Arg-7, Glu-110 and Glu-112 that were already known to be important for enzymatic activity. The second group was formed by mutations that caused the collapse or prevented the assembly of the A subunit: Leu-41-->Phe, Ala-45-->Tyr or Glu, Val-53-->Tyr, Val-60-->Gly, Ser-68-->Pro, His-70-->Pro, Val-97-->Tyr and Ser-114-->Tyr. The third group contained those molecules that maintained a wild-type level of toxicity in spite of the mutations introduced: Arg-54-->Lys or Ala, Tyr-59-->Met, Ser-68-->Lys, Ala-72-->Arg, His or Asp and Arg-192-->Asn. The results provide a further understanding of the structure-function of the active site and new, non-toxic mutants that may be useful for the development of vaccines against diarrhoeal diseases.
Pizza
M
M
Immunobiological Research Institute Siena (IRIS), Italy.
Domenighini
M
M
Hol
W
W
Giannelli
V
V
Fontana
M R
MR
Giuliani
M M
MM
Magagnoli
C
C
Peppoloni
S
S
Manetti
R
R
Rappuoli
R
R
D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D003196:Computer Graphics; D003198:Computer Simulation; D004768:Enterotoxins / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; C027538:heat-labile enterotoxin, E coli