scholarly journals Control of androgen biosynthesis in the human through the interaction of Arg347 and Arg358 of CYP17 with cytochrome b5

1998 ◽  
Vol 332 (2) ◽  
pp. 293-296 ◽  
Author(s):  
Peter LEE-ROBICHAUD ◽  
Monika E. AKHTAR ◽  
Muhammad AKHTAR
Keyword(s):  
Kinetic Analysis ◽  
Protein Interaction ◽  
Cytochrome B5 ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Mutant Proteins ◽  
Protein Protein Interaction ◽  
Genital Ambiguity ◽  
Lyase Activity ◽  
Male Patients

The lyase activity of human CYP17 (17α-hydroxylase-17,20-lyase also P-450c17 or P-45017α) is greatly dependent on the presence of cytochrome b5, and this effect has been ascribed an important regulatory role [Lee-Robichaud, Wright, Akhtar and Akhtar (1995) Biochem. J. 308, 901–908]. This facet was further investigated by site-directed mutagenesis of selected basic residues of human CYP17. The purified mutant proteins were subjected to detailed kinetic analysis. It was found that the mutation of Lys83, Arg347 and Arg358 produced proteins that were deficient in their responsiveness to cytochrome b5, and the effect was most pronounced for the two arginine mutants (Arg347 → His and Arg358 → Gln) which have been found in male patients suffering from genital ambiguity. These residues are invoked to mediate protein–protein interaction between cytochrome b5 and CYP17, which ‘awakens ’ the lyase activity of the enzyme required for androgen formation.

scholarly journals Lysine mutagenesis identifies cationic charges of human CYP17 that interact with cytochrome b5 to promote male sex-hormone biosynthesis

1999 ◽  
Vol 342 (2) ◽  
pp. 309-312 ◽  
Author(s):  
Peter LEE-ROBICHAUD ◽  
Monika E. AKHTAR ◽  
Muhammad AKHTAR
Keyword(s):  
Cytochrome B5 ◽  
Sex Hormone ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Hydroxylation Reaction ◽  
Lyase Activity ◽  
Hormone Biosynthesis ◽  
Male Patients ◽  
Male Sex ◽  
Amino Acid Mutations

Human CYP17 (17α-hydroxylase-17,20-lyase; also cytochrome P450c17 or cytochrome P45017α) catalyses a hydroxylation reaction and another reaction involving the cleavage of a C-C bond (the lyase activity) that is required only for androgen production. Single amino acid mutations in human CYP17, Arg347 → His and Arg358 → Gln, have been reported to result in the loss of the lyase activity and to cause sexual phenotypic changes in 46XY male patients. By using site-directed mutagenesis we show here that another mutation in human CYP17, Arg449 → Ala, for which human variants have yet not been described, also leads to selective lyase deficiency. Furthermore, all the three types of mutants display a loss of responsiveness to cytochrome b5, an interaction that is essential for lyase activity, and hence male sex-hormone biosynthesis. That the defect could be essentially reversed by lysine mutagenesis has led to the conclusion that the cationic charges on all three residues (at the positions of Arg347, Arg358, Arg449) are vital for the functional interaction of CYP17 with cytochrome b5 and that the loss of any one of these cationic charges is catastrophic.

scholarly journals Probing the Role of Cysteine Residues in Glucosamine-1-Phosphate Acetyltransferase Activity of the Bifunctional GlmU Protein fromEscherichia coli: Site-Directed Mutagenesis and Characterization of the Mutant Enzymes

1998 ◽  
Vol 180 (18) ◽  
pp. 4799-4803 ◽  
Author(s):  
Frédérique Pompeo ◽  
Jean van Heijenoort ◽  
Dominique Mengin-Lecreulx
Keyword(s):  
Active Site ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Acetyl Coa ◽  
Mutant Proteins ◽  
Acetyltransferase Activity ◽  
Phosphate Acetyltransferase ◽  
High Level

ABSTRACT The glucosamine-1-phosphate acetyltransferase activity but not the uridyltransferase activity of the bifunctional GlmU enzyme fromEscherichia coli was lost when GlmU was stored in the absence of β-mercaptoethanol or incubated with thiol-specific reagents. The enzyme was protected from inactivation in the presence of its substrate acetyl coenzyme A (acetyl-CoA), suggesting the presence of an essential cysteine residue in or near the active site of the acetyltransferase domain. To ascertain the role of cysteines in the structure and function of the enzyme, site-directed mutagenesis was performed to change each of the four cysteines to alanine, and plasmids were constructed for high-level overproduction and one-step purification of histidine-tagged proteins. Whereas the kinetic parameters of the bifunctional enzyme appeared unaffected by the C296A and C385A mutations, 1,350- and 8-fold decreases of acetyltransferase activity resulted from the C307A and C324A mutations, respectively. TheKm values for acetyl-CoA and GlcN-1-P of mutant proteins were not modified, suggesting that none of the cysteines was involved in substrate binding. The uridyltransferase activities of wild-type and mutant GlmU proteins were similar. From these studies, the two cysteines Cys307 and Cys324 appeared important for acetyltransferase activity and seemed to be located in or near the active site.

scholarly journals Experimental Characterization of the Interaction between the N-Terminal SH3 Domain of Crkl and C3G

2021 ◽  
Vol 22 (24) ◽  
pp. 13174
Author(s):  
Livia Pagano ◽  
Francesca Malagrinò ◽  
Caterina Nardella ◽  
Stefano Gianni ◽  
Angelo Toto
Keyword(s):  
Protein Interaction ◽  
Binding Pocket ◽  
Sh3 Domain ◽  
Sh2 Domain ◽  
Cellular Growth ◽  
Site Directed Mutagenesis ◽  
Experimental Conditions ◽  
Fast Kinetics ◽  
Sh3 Domains ◽  
Protein Protein Interaction

Crkl is a protein involved in the onset of several cancer pathologies that exerts its function only through its protein–protein interaction domains, a SH2 domain and two SH3 domains. SH3 domains are small protein interaction modules that mediate the binding and recognition of proline-rich sequences. One of the main physiological interactors of Crkl is C3G (also known as RAPGEF1), an interaction with key implications in regulating cellular growth and differentiation, cell morphogenesis and adhesion processes. Thus, understanding the interaction between Crkl and C3G is fundamental to gaining information about the molecular determinants of the several cancer pathologies in which these proteins are involved. In this paper, through a combination of fast kinetics at different experimental conditions and site-directed mutagenesis, we characterize the binding reaction between the N-SH3 domain of Crkl and a peptide mimicking a specific portion of C3G. Our results show a clear effect of pH on the stability of the complex, due to the protonation of negatively charged residues in the binding pocket of N-SH3. Our results are discussed under the light of previous work on SH3 domains.

Site directed mutagenesis of SOD1 (Gly72Ser) and analysis of mutant proteins oligomerization

2011 ◽  
Vol 44 (13) ◽  
pp. S263
Author(s):  
Asiye Sadat Javanian ◽  
Nikkhah Maryam ◽  
Hosseinkhani Saman
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Mutant Proteins
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