Structural and functional analysis of the cAMP binding domain from the regulatory subunit of Mucor rouxii protein kinase A
The cAMP binding domain of the regulatory subunit (R) of Mucor rouxii protein kinase A was cloned. The deduced amino acid sequence was highly homologous in sequence and in size to the corresponding region in fungal and higher eukaryotic regulatory subunits (47-54%), but particularly homologous (62%)...
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todo:paper_00039861_v382_n2_p173_Sorol2023-10-03T13:57:02Z Structural and functional analysis of the cAMP binding domain from the regulatory subunit of Mucor rouxii protein kinase A Sorol, M.R. Pastori, R.L. Muro, A. Moreno, S. Rossi, S. cAMP binding Mucor rouxii Protein sequence amino acid arginine cyclic AMP cyclic AMP dependent protein kinase cyclic AMP derivative hybrid protein leucine lysine amino acid sequence article binding affinity chemical structure dissociation enzyme active site molecular model Mucor nonhuman nucleotide sequence polymerase chain reaction priority journal sequence homology Amylomyces rouxii Blastocladiella emersonii Blastocladiella emersonii Bovinae Eukaryota Fungi Mammalia Mucor Mucor rouxii The cAMP binding domain of the regulatory subunit (R) of Mucor rouxii protein kinase A was cloned. The deduced amino acid sequence was highly homologous in sequence and in size to the corresponding region in fungal and higher eukaryotic regulatory subunits (47-54%), but particularly homologous (62%) to Blastocladiella emersonii, a fungus classified in a different phylum. Amino acids reported to be important for interaction with cAMP, for cooperativity between the two cAMP binding domains, in the general folding of the domain, and for interaction with the catalytic subunit were conserved in all the fungal sequences. Based on either sequence or functional behavior, the M. rouxii R subunit cannot be classified as being more similar to RI or RII of mammalian systems. The M. rouxii protein sequence was modeled using as template the coordinates of the crystallized bovine regulatory subunit type I(α). The quality of the model is good. The two backbones could be perfectly overlapped, except for two loop regions of high divergence. The α helix C of domain A, proposed to have a strong interaction with the catalytic subunit, contains a leucine replacing a basic residue (arginine or lysine) commonly found in RI or RII. The domains A and B of the M. rouxii regulatory subunit were overexpressed as fusion proteins with GST. GST domain B protein was inactive. GST domain A was active; the kinetic parameters of affinity toward cAMP analogs, site selectivity, and dissociation kinetics of bound cAMP were analogous to the properties of the domain in the whole regulatory subunit. (C) 2000 Academic Press. Fil:Pastori, R.L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Muro, A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Moreno, S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Rossi, S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. JOUR info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar http://hdl.handle.net/20.500.12110/paper_00039861_v382_n2_p173_Sorol |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
cAMP binding Mucor rouxii Protein sequence amino acid arginine cyclic AMP cyclic AMP dependent protein kinase cyclic AMP derivative hybrid protein leucine lysine amino acid sequence article binding affinity chemical structure dissociation enzyme active site molecular model Mucor nonhuman nucleotide sequence polymerase chain reaction priority journal sequence homology Amylomyces rouxii Blastocladiella emersonii Blastocladiella emersonii Bovinae Eukaryota Fungi Mammalia Mucor Mucor rouxii |
| spellingShingle |
cAMP binding Mucor rouxii Protein sequence amino acid arginine cyclic AMP cyclic AMP dependent protein kinase cyclic AMP derivative hybrid protein leucine lysine amino acid sequence article binding affinity chemical structure dissociation enzyme active site molecular model Mucor nonhuman nucleotide sequence polymerase chain reaction priority journal sequence homology Amylomyces rouxii Blastocladiella emersonii Blastocladiella emersonii Bovinae Eukaryota Fungi Mammalia Mucor Mucor rouxii Sorol, M.R. Pastori, R.L. Muro, A. Moreno, S. Rossi, S. Structural and functional analysis of the cAMP binding domain from the regulatory subunit of Mucor rouxii protein kinase A |
| topic_facet |
cAMP binding Mucor rouxii Protein sequence amino acid arginine cyclic AMP cyclic AMP dependent protein kinase cyclic AMP derivative hybrid protein leucine lysine amino acid sequence article binding affinity chemical structure dissociation enzyme active site molecular model Mucor nonhuman nucleotide sequence polymerase chain reaction priority journal sequence homology Amylomyces rouxii Blastocladiella emersonii Blastocladiella emersonii Bovinae Eukaryota Fungi Mammalia Mucor Mucor rouxii |
| description |
The cAMP binding domain of the regulatory subunit (R) of Mucor rouxii protein kinase A was cloned. The deduced amino acid sequence was highly homologous in sequence and in size to the corresponding region in fungal and higher eukaryotic regulatory subunits (47-54%), but particularly homologous (62%) to Blastocladiella emersonii, a fungus classified in a different phylum. Amino acids reported to be important for interaction with cAMP, for cooperativity between the two cAMP binding domains, in the general folding of the domain, and for interaction with the catalytic subunit were conserved in all the fungal sequences. Based on either sequence or functional behavior, the M. rouxii R subunit cannot be classified as being more similar to RI or RII of mammalian systems. The M. rouxii protein sequence was modeled using as template the coordinates of the crystallized bovine regulatory subunit type I(α). The quality of the model is good. The two backbones could be perfectly overlapped, except for two loop regions of high divergence. The α helix C of domain A, proposed to have a strong interaction with the catalytic subunit, contains a leucine replacing a basic residue (arginine or lysine) commonly found in RI or RII. The domains A and B of the M. rouxii regulatory subunit were overexpressed as fusion proteins with GST. GST domain B protein was inactive. GST domain A was active; the kinetic parameters of affinity toward cAMP analogs, site selectivity, and dissociation kinetics of bound cAMP were analogous to the properties of the domain in the whole regulatory subunit. (C) 2000 Academic Press. |
| format |
JOUR |
| author |
Sorol, M.R. Pastori, R.L. Muro, A. Moreno, S. Rossi, S. |
| author_facet |
Sorol, M.R. Pastori, R.L. Muro, A. Moreno, S. Rossi, S. |
| author_sort |
Sorol, M.R. |
| title |
Structural and functional analysis of the cAMP binding domain from the regulatory subunit of Mucor rouxii protein kinase A |
| title_short |
Structural and functional analysis of the cAMP binding domain from the regulatory subunit of Mucor rouxii protein kinase A |
| title_full |
Structural and functional analysis of the cAMP binding domain from the regulatory subunit of Mucor rouxii protein kinase A |
| title_fullStr |
Structural and functional analysis of the cAMP binding domain from the regulatory subunit of Mucor rouxii protein kinase A |
| title_full_unstemmed |
Structural and functional analysis of the cAMP binding domain from the regulatory subunit of Mucor rouxii protein kinase A |
| title_sort |
structural and functional analysis of the camp binding domain from the regulatory subunit of mucor rouxii protein kinase a |
| url |
http://hdl.handle.net/20.500.12110/paper_00039861_v382_n2_p173_Sorol |
| work_keys_str_mv |
AT sorolmr structuralandfunctionalanalysisofthecampbindingdomainfromtheregulatorysubunitofmucorrouxiiproteinkinasea AT pastorirl structuralandfunctionalanalysisofthecampbindingdomainfromtheregulatorysubunitofmucorrouxiiproteinkinasea AT muroa structuralandfunctionalanalysisofthecampbindingdomainfromtheregulatorysubunitofmucorrouxiiproteinkinasea AT morenos structuralandfunctionalanalysisofthecampbindingdomainfromtheregulatorysubunitofmucorrouxiiproteinkinasea AT rossis structuralandfunctionalanalysisofthecampbindingdomainfromtheregulatorysubunitofmucorrouxiiproteinkinasea |
| _version_ |
1807321460200439808 |