Document Detail

Mutational analysis of the AtNUDT7 Nudix hydrolase from Arabidopsis thaliana reveals residues required for protein quaternary structure formation and activity.
MedLine Citation:
PMID:  19448856     Owner:  NLM     Status:  MEDLINE    
Arabidopsis thaliana AtNUDT7, a homodimeric Nudix hydrolase active on ADP-ribose and NADH, exerts negative control on the major signaling complex involved in plant defense activation and programmed cell death. The structural and functional consequences of altering several amino-acid residues of the AtNUDT7 protein have been examined by site-directed mutagenesis, far-UV circular dichroism (CD), attenuated total reflection-Fourier transform infrared (ATR-FTIR) and photon correlation (PCS) spectroscopy, biochemical analysis and protein-protein interaction studies. Alanine substitutions of F73 and V168 disallowed dimer formation. Both the F73A- and V168A-mutated proteins displayed no observable enzymatic activity. Alanine substitution of the V69 residue did not significantly alter the enzyme activity and had no influence on dimer arrangement. The non-conserved V26 residue, used as a negative control, did not contribute to the enzyme quaternary structure or activity. Detailed biophysical characterization of the wild-type and mutant proteins indicates that the mutations do not considerably alter the secondary structure of the enzyme but they affect dimer assembly. In addition, mutating residues V69, F73 and V168 disrupted the binding of AtNUDT7 to the regulatory 14.3.3 protein. These are the first studies of the structure-function relationship of AtNUDT7, a Nudix hydrolase of important regulatory function.
Kamil Olejnik; Danuta Płochocka; Marcin Grynberg; Grazyna Goch; Wiesław I Gruszecki; Teresa Basińska; Elzbieta Kraszewska
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Publication Detail:
Type:  Comparative Study; Journal Article; Research Support, Non-U.S. Gov't     Date:  2009-05-15
Journal Detail:
Title:  Acta biochimica Polonica     Volume:  56     ISSN:  1734-154X     ISO Abbreviation:  Acta Biochim. Pol.     Publication Date:  2009  
Date Detail:
Created Date:  2009-07-01     Completed Date:  2009-10-14     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  14520300R     Medline TA:  Acta Biochim Pol     Country:  Poland    
Other Details:
Languages:  eng     Pagination:  291-300     Citation Subset:  IM    
Department of Plant Biochemistry, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland.
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MeSH Terms
14-3-3 Proteins / metabolism
Amino Acid Sequence / genetics
Arabidopsis / enzymology*,  genetics
Arabidopsis Proteins / chemistry,  genetics,  metabolism
Catalytic Domain / genetics*
Cell Line
Chromatography, Liquid
Circular Dichroism
Escherichia coli
Models, Molecular
Molecular Sequence Data
Mutagenesis, Site-Directed*
Mutant Proteins / metabolism
Protein Multimerization / genetics*
Protein Structure, Quaternary / genetics*
Protein Structure, Secondary / genetics
Pyrophosphatases / chemistry,  genetics*,  isolation & purification,  metabolism*
Recombinant Proteins / metabolism
Saccharomyces cerevisiae
Sequence Alignment
Spectroscopy, Fourier Transform Infrared
Two-Hybrid System Techniques
Reg. No./Substance:
0/14-3-3 Proteins; 0/Arabidopsis Proteins; 0/Mutant Proteins; 0/Recombinant Proteins; EC 3.6.1.-/Pyrophosphatases; EC 3.6.1.-/nudix hydrolases

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