Document Detail

Aging-associated enzyme human clock-1: substrate-mediated reduction of the diiron center for 5-demethoxyubiquinone hydroxylation.
MedLine Citation:
PMID:  23445365     Owner:  NLM     Status:  MEDLINE    
The mitochondrial membrane-bound enzyme Clock-1 (CLK-1) extends the average longevity of mice and Caenorhabditis elegans, as demonstrated for Δclk-1 constructs for both organisms. Such an apparent impact on aging and the presence of a carboxylate-bridged diiron center in the enzyme inspired this work. We expressed a soluble human CLK-1 (hCLK-1) fusion protein with an N-terminal immunoglobulin binding domain of protein G (GB1). Inclusion of the solubility tag allowed for thorough characterization of the carboxylate-bridged diiron active site of the resulting GB1-hCLK-1 by spectroscopic and kinetic methods. Both UV-visible and Mössbauer experiments provide unambiguous evidence that GB1-hCLK-1 functions as a 5-demethoxyubiquinone-hydroxylase, utilizing its carboxylate-bridged diiron center. The binding of DMQn (n = 0 or 2) to GB1-hCLK-1 mediates reduction of the diiron center by nicotinamide adenine dinucleotide (NADH) and initiates O2 activation for subsequent DMQ hydroxylation. Deployment of DMQ to mediate reduction of the diiron center in GB1-hCLK-1 improves substrate specificity and diminishes consumption of NADH that is uncoupled from substrate oxidation. Both Vmax and kcat/KM for DMQ hydroxylation increase when DMQ0 is replaced by DMQ2 as the substrate, which demonstrates that an isoprenoid side chain enhances enzymatic hydroxylation and improves catalytic efficiency.
Tsai-Te Lu; Seung Jae Lee; Ulf-Peter Apfel; Stephen J Lippard
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2013-03-20
Journal Detail:
Title:  Biochemistry     Volume:  52     ISSN:  1520-4995     ISO Abbreviation:  Biochemistry     Publication Date:  2013 Apr 
Date Detail:
Created Date:  2013-04-02     Completed Date:  2013-05-21     Revised Date:  2014-04-04    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  2236-44     Citation Subset:  IM    
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MeSH Terms
Bacterial Proteins / chemistry,  genetics,  metabolism
CLOCK Proteins / chemistry*,  genetics,  metabolism*
Catalytic Domain
Cloning, Molecular
Electron Transport
Gene Expression
Iron Compounds / chemistry
Oxygenases / chemistry,  genetics,  metabolism
Protein Structure, Tertiary
Recombinant Fusion Proteins / chemistry,  genetics,  metabolism
Ubiquinone / analogs & derivatives*,  metabolism*
Grant Support
Reg. No./Substance:
0/Bacterial Proteins; 0/IgG Fc-binding protein, Streptococcus; 0/Iron Compounds; 0/Recombinant Fusion Proteins; 1339-63-5/Ubiquinone; EC 1.13.-/Oxygenases; EC Proteins

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