Document Detail

Spectroscopic, steady-state kinetic, and mechanistic characterization of the radical SAM enzyme QueE, which catalyzes a complex cyclization reaction in the biosynthesis of 7-deazapurines.
MedLine Citation:
PMID:  23194065     Owner:  NLM     Status:  MEDLINE    
7-Carboxy-7-deazaguanine (CDG) synthase (QueE) catalyzes the complex heterocyclic radical-mediated conversion of 6-carboxy-5,6,7,8-tetrahydropterin (CPH(4)) to CDG in the third step of the biosynthetic pathway to all 7-deazapurines. Here we present a detailed characterization of QueE from Bacillus subtilis to delineate the mechanism of conversion of CPH(4) to CDG. QueE is a member of the radical S-adenosyl-l-methionine (SAM) superfamily, all of which use a bound [4Fe-4S](+) cluster to catalyze the reductive cleavage of the SAM cofactor to generate methionine and a 5'-deoxyadenosyl radical (5'-dAdo(•)), which initiates enzymatic transformations requiring hydrogen atom abstraction. The ultraviolet-visible, electron paramagnetic resonance, and Mössbauer spectroscopic features of the homodimeric QueE point to the presence of a single [4Fe-4S] cluster per monomer. Steady-state kinetic experiments indicate a K(m) of 20 ± 7 μM for CPH(4) and a k(cat) of 5.4 ± 1.2 min(-1) for the overall transformation. The kinetically determined K(app) for SAM is 45 ± 1 μM. QueE is also magnesium-dependent and exhibits a K(app) for the divalent metal ion of 0.21 ± 0.03 mM. The SAM cofactor supports multiple turnovers, indicating that it is regenerated at the end of each catalytic cycle. The mechanism of rearrangement of QueE was probed with CPH(4) isotopologs containing deuterium at C-6 or the two prochiral positions at C-7. These studies implicate 5'-dAdo(•) as the initiator of the ring contraction reaction catalyzed by QueE by abstraction of the H atom from C-6 of CPH(4).
Reid M McCarty; Carsten Krebs; Vahe Bandarian
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2012-12-24
Journal Detail:
Title:  Biochemistry     Volume:  52     ISSN:  1520-4995     ISO Abbreviation:  Biochemistry     Publication Date:  2013 Jan 
Date Detail:
Created Date:  2013-01-08     Completed Date:  2013-03-05     Revised Date:  2014-05-18    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  188-98     Citation Subset:  IM    
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MeSH Terms
Bacillus subtilis / chemistry,  enzymology*,  metabolism
Bacterial Proteins / chemistry,  metabolism*
Electron Spin Resonance Spectroscopy
Guanine / analogs & derivatives*,  chemistry,  metabolism
Iron-Sulfur Proteins / chemistry,  metabolism*
Protein Structure, Quaternary
Pterins / chemistry,  metabolism*
S-Adenosylmethionine / metabolism*
Spectroscopy, Mossbauer
Grant Support
Reg. No./Substance:
0/Bacterial Proteins; 0/Iron-Sulfur Proteins; 0/Pterins; 1008-35-1/tetrahydropterin; 5Z93L87A1R/Guanine; 7355-55-7/7-deazaguanine; 7LP2MPO46S/S-Adenosylmethionine

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