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.

Probing the structure-activity relationship of E.coli LT-A by site directed mutagenesis / Pizza, M. G.; Domenighini, M.; Hol, W.; Giannelli, V.; Fontana, M. R.; Giuliani, M. M.; Magagnoli, C.; Peppoloni, Samuele; Manetti, R.; Rappuoli, R.. - In: MOLECULAR MICROBIOLOGY. - ISSN 0950-382X. - STAMPA. - 14:1(1994), pp. 51-60. [10.1111/j.1365-2958.1994.tb01266.x]

Probing the structure-activity relationship of E.coli LT-A by site directed mutagenesis

PEPPOLONI, Samuele;
1994

Abstract

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.
1994
14
1
51
60
Probing the structure-activity relationship of E.coli LT-A by site directed mutagenesis / Pizza, M. G.; Domenighini, M.; Hol, W.; Giannelli, V.; Fontana, M. R.; Giuliani, M. M.; Magagnoli, C.; Peppoloni, Samuele; Manetti, R.; Rappuoli, R.. - In: MOLECULAR MICROBIOLOGY. - ISSN 0950-382X. - STAMPA. - 14:1(1994), pp. 51-60. [10.1111/j.1365-2958.1994.tb01266.x]
Pizza, M. G.; Domenighini, M.; Hol, W.; Giannelli, V.; Fontana, M. R.; Giuliani, M. M.; Magagnoli, C.; Peppoloni, Samuele; Manetti, R.; Rappuoli, R.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11380/457062
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