Document Detail


Characterizing the importance of the biotin carboxylase domain dimer for Staphylococcus aureus pyruvate carboxylase catalysis.
MedLine Citation:
PMID:  23286247     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Biotin carboxylase (BC) is a conserved component among biotin-dependent carboxylases and catalyzes the MgATP-dependent carboxylation of biotin, using bicarbonate as the CO₂ donor. Studies with Escherichia coli BC have suggested long-range communication between the two active sites of a dimer, although its mechanism is not well understood. In addition, mutations in the dimer interface can produce stable monomers that are still catalytically active. A homologous dimer for the BC domain is observed in the structure of the tetrameric pyruvate carboxylase (PC) holoenzyme. We have introduced site-specific mutations into the BC domain dimer interface of Staphylococcus aureus PC (SaPC), equivalent to those used for E. coli BC, and also made chimeras replacing the SaPC BC domain with the E. coli BC subunit (EcBC chimera) or the yeast ACC BC domain (ScBC chimera). We assessed the catalytic activities of these mutants and characterized their oligomerization states by gel filtration and analytical ultracentrifugation experiments. The K442E mutant and the ScBC chimera disrupted the BC dimer and were catalytically inactive, while the F403A mutant and the EcBC chimera were still tetrameric and retained catalytic activity. The R54E mutant was also tetrameric but was catalytically inactive. Crystal structures of the R54E, F403A, and K442E mutants showed that they were tetrameric in the crystal, with conformational changes near the mutation site as well as in the tetramer organization. We have also produced the isolated BC domain of SaPC. In contrast to E. coli BC, the SaPC BC domain is monomeric in solution and catalytically inactive.
Authors:
Linda P C Yu; Chi-Yuan Chou; Philip H Choi; Liang Tong
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2013-01-09
Journal Detail:
Title:  Biochemistry     Volume:  52     ISSN:  1520-4995     ISO Abbreviation:  Biochemistry     Publication Date:  2013 Jan 
Date Detail:
Created Date:  2013-01-22     Completed Date:  2013-03-14     Revised Date:  2014-01-24    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  488-96     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Substitution
Bacterial Proteins / chemistry*,  genetics,  metabolism*
Biocatalysis
Carbon-Nitrogen Ligases / chemistry*,  genetics,  metabolism*
Catalytic Domain
Chromatography, Gel
Crystallography, X-Ray
Holoenzymes / chemistry,  genetics,  metabolism
Kinetics
Models, Molecular
Mutagenesis, Site-Directed
Mutant Proteins / chemistry,  metabolism
Protein Conformation
Protein Multimerization
Protein Subunits / chemistry,  genetics,  metabolism
Pyruvate Carboxylase / chemistry*,  genetics,  metabolism*
Recombinant Fusion Proteins / chemistry,  metabolism
Recombinant Proteins / chemistry,  metabolism
Staphylococcus aureus / enzymology*
Ultracentrifugation
Grant Support
ID/Acronym/Agency:
DK067238/DK/NIDDK NIH HHS; GM007367/GM/NIGMS NIH HHS; GM008798/GM/NIGMS NIH HHS; P30 EB009998/EB/NIBIB NIH HHS; R01 DK067238/DK/NIDDK NIH HHS; T32 GM007367/GM/NIGMS NIH HHS; T32 GM008798/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Bacterial Proteins; 0/Holoenzymes; 0/Mutant Proteins; 0/Protein Subunits; 0/Recombinant Fusion Proteins; 0/Recombinant Proteins; EC 6.3.-/Carbon-Nitrogen Ligases; EC 6.3.4.14/biotin carboxylase; EC 6.4.1.1/Pyruvate Carboxylase
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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