Role of Tryptophan Residues in Toxicity of Cry1Ab Toxin from Bacillus thuringiensis

ABSTRACT Bacillus thuringiensis produces insecticidal proteins (Cry protoxins) during the sporulation phase as parasporal crystals. During intoxication, the Cry protoxins must change from insoluble crystals into membrane-inserted toxins which form ionic pores. The structural changes of Cry toxins during oligomerization and insertion into the membrane are still unknown. The Cry1Ab toxin has nine tryptophan residues; seven are located in domain I, the pore-forming domain, and two are located in domain II, which is involved in receptor recognition. Eight Trp residues are highly conserved within the whole family of three-domain Cry proteins, suggesting an essential role for these residues in the structural folding and function of the toxin. In this work, we analyzed the role of Trp residues in the structure and function of Cry1Ab toxin. We replaced the Trp residues with phenylalanine or cysteine using site-directed mutagenesis. Our results show that W65 and W316 are important for insecticidal activity of the toxin since their replacement by Phe reduced the toxicity against Manduca sexta. The presence of hydrophobic residue is important at positions 117, 219, 226, and 455 since replacement by Cys affected either the crystal formation or the insecticidal activity of the toxin in contrast to replacement by Phe in these positions. Additionally, some mutants in positions 219, 316, and 455 were also affected in binding to brush border membrane vesicles (BBMV). This is the first report that studies the role of Trp residues in the activity of Cry toxins.

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Role of Tryptophan Residues in Gramicidin Channel Organization and Function

Biophysical Journal ◽

10.1529/biophysj.107.124206 ◽

2008 ◽

Vol 95 (1) ◽

pp. 166-175 ◽

Cited By ~ 29

Author(s):

Amitabha Chattopadhyay ◽

Satinder S. Rawat ◽

Denise V. Greathouse ◽

Devaki A. Kelkar ◽

Roger E. Koeppe

Keyword(s):

Gramicidin Channel ◽

Tryptophan Residues ◽

And Function

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Molecular Approaches to Improve the Insecticidal Activity of Bacillus thuringiensis Cry Toxins

Toxins ◽

10.3390/toxins6082393 ◽

2014 ◽

Vol 6 (8) ◽

pp. 2393-2423 ◽

Cited By ~ 17

Author(s):

Wagner Lucena ◽

Patrícia Pelegrini ◽

Diogo Martins-de-Sa ◽

Fernando Fonseca ◽

Jose Gomes ◽

...

Keyword(s):

Bacillus Thuringiensis ◽

Insecticidal Activity ◽

Cry Toxins ◽

Molecular Approaches

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The role of nitric oxide on matrix metalloproteinase 2 (MMP2) and MMP9 in placenta and fetus from diabetic rats

Reproduction ◽

10.1530/rep-06-0267 ◽

2007 ◽

Vol 134 (4) ◽

pp. 605-613 ◽

Cited By ~ 18

Author(s):

M C Pustovrh ◽

A Jawerbaum ◽

V White ◽

E Capobianco ◽

R Higa ◽

...

Keyword(s):

Nitric Oxide ◽

Structural Changes ◽

Diabetic Rats ◽

Matrix Metalloproteinase 2 ◽

No Donor ◽

Diaphorase Activity ◽

Nos Inhibitor ◽

And Function ◽

Fetal Organs

Matrix metalloproteinases (MMPs) play an important role in tissue remodeling that accompanies the rapid growth, differentiation, and structural changes of the placenta and several fetal organs. In the present study, we investigated whether the diabetic maternal environment may alter the regulatory homeostasis exerted by nitric oxide (NO) on MMPs activity in the feto-placental unit from rats at midgestation. We found that NADPH-diaphorase activity, which reflects the distribution and activity of NO synthases (NOS), was increased in both placenta and fetuses from diabetic rats when compared with controls. In addition, while a NO donor enhanced MMP2 and MMP9 activities, a NOS inhibitor reduced these activities in the maternal side of the placenta from control rats. This regulatory effect of NO was only observed on MMP9 in the diabetic group. On the other hand, the NO donor did not modify MMP2 and MMP9 activities, while the NOS inhibitor reduced MMP9 activity in the fetal side of both control and diabetic placentas. In the fetuses, MMP2 was enhanced by the NO donor and reduced by the NO inhibitor in both fetuses from control and diabetic rats. Overall, this study demonstrates that NO is able to modulate the activation of MMPs in the feto-placental unit, and provides supportive evidence that increased NOS activity leads to NO overproduction in the feto-placental unit from diabetic rats, an alteration closely related to the observed MMPs dysregulation that may have profound implications in the formation and function of the placenta and the fetal organs.

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Importance of the two tryptophan residues in the Streptomyces R61 exocellular dd-peptidase

Biochemical Journal ◽

10.1042/bj2820361 ◽

1992 ◽

Vol 282 (2) ◽

pp. 361-367 ◽

Cited By ~ 12

Author(s):

C Bourguignon-Bellefroid ◽

J M Wilkin ◽

B Joris ◽

R T Aplin ◽

C Houssier ◽

...

Keyword(s):

Enzyme Activity ◽

Enzymic Activity ◽

Site Directed Mutagenesis ◽

Wild Type ◽

Beta Lactams ◽

Beta Lactamases ◽

Type Protein ◽

Wild Type Protein ◽

Tryptophan Residues

Modification of the Streptomyces R61 DD-peptidase by N-bromosuccinimide resulted in a rapid loss of enzyme activity. In consequence, the role of the enzyme's two tryptophan residues was investigated by site-directed mutagenesis. Trp271 was replaced by Leu. The modification yielded a stable enzyme whose structural and catalytic properties were similar to those of the wild-type protein. Thus the Trp271 residue, though almost invariant among the beta-lactamases of classes A and C and the low-Mr penicillin-binding proteins, did not appear to be essential for enzyme activity. Mutations of the Trp233 into Leu and Ser strongly decreased the enzymic activity, the affinity for beta-lactams and the protein stability. Surprisingly, the benzylpenicilloyl-(W233L)enzyme deacylated at least 300-fold more quickly than the corresponding acyl-enzyme formed with the wild-type protein and gave rise to benzylpenicilloate instead of phenylacetylglycine. This mutant DD-peptidase thus behaved as a weak beta-lactamase.

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Site Directed Mutagenesis of Human GLTP: Role of Tryptophan Residues

Biophysical Journal ◽

10.1016/j.bpj.2008.12.1639 ◽

2009 ◽

Vol 96 (3) ◽

pp. 326a

Author(s):

Ravi Kanth Kamlekar ◽

Yongguang Gao ◽

Helen Pike ◽

Roopa Kenoth ◽

Franklyn G. Prendergast ◽

...

Keyword(s):

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Tryptophan Residues

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Role of Spores in the Insecticidal Activity of Bacillus thuringiensis Delta-endotoxin

Japanese Journal of Applied Entomology and Zoology ◽

10.1303/jjaez.2008.183 ◽

2008 ◽

Vol 52 (4) ◽

pp. 183-192

Author(s):

Shoji Asano ◽

Kazuhisa Miyamoto

Keyword(s):

Bacillus Thuringiensis ◽

Insecticidal Activity ◽

Delta Endotoxin

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Analysis of the role of tryptophan residues in aspartate transcarbamylase by site-directed mutagenesis and fluorescence measurements

10.1117/12.58279 ◽

1992 ◽

Author(s):

Patrick Tauc ◽

Luc Fetler ◽

Guy Herve ◽

Moncef Ladjmi ◽

Jean-Claude Brochon

Keyword(s):

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Aspartate Transcarbamylase ◽

Fluorescence Measurements ◽

Tryptophan Residues

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Enzyme redesign and interactions of substrate analogues with sterol methyltransferase to understand phytosterol diversity, reaction mechanism and the nature of the active site

Biochemical Society Transactions ◽

10.1042/bst0331189 ◽

2005 ◽

Vol 33 (5) ◽

pp. 1189-1196 ◽

Cited By ~ 8

Author(s):

W.D. Nes

Keyword(s):

Active Site ◽

Functional Divergence ◽

Product Distribution ◽

Site Directed Mutagenesis ◽

Site Model ◽

Bifunctional Enzymes ◽

And Function ◽

The Relationship ◽

Sterol Methyltransferase

Several STM (sterol methyltransferase) genes have been cloned, sequenced and expressed in bacteria recently, making it possible to address questions of the relationship between sterol structure and function. The active site and mechanism of action of a set of phylogenetically diverse SMTs have been probed by site-directed mutagenesis as well as by using substrate and related analogues of the SMT-catalysed reaction. An active-site model has been developed that is in accord with the results presented, which is consistent with the hypothesis that SMTs are bifunctional enzymes kinetically responsible to bind Δ24-acceptor sterols of specific steric and electronic character and rigid orientation imposed by multiple hydrophobic active site contacts exacted from a common waxy core. Functional divergence influenced by the architectural role of sterols in membranes is considered to govern the evolution of product distribution and specificity of individual SMTs as discussed.

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