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.