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

Reid M. McCarty, Carsten Krebs, Vahe Bandarian

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34 Citations (Scopus)

Abstract

7-Carboxy-7-deazaguanine (CDG) synthase (QueE) catalyzes the complex heterocyclic radical-mediated conversion of 6-carboxy-5,6,7,8-tetrahydropterin (CPH4) 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 CPH4 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 Km of 20 ± 7 μM for CPH4 and a kcat of 5.4 ± 1.2 min-1 for the overall transformation. The kinetically determined Kapp for SAM is 45 ± 1 μM. QueE is also magnesium-dependent and exhibits a Kapp 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 CPH4 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 CPH4.

Original languageEnglish (US)
Pages (from-to)188-198
Number of pages11
JournalBiochemistry
Volume52
Issue number1
DOIs
StatePublished - Jan 8 2013

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Cyclization
Biosynthesis
Methionine
Kinetics
Enzymes
Atoms
Deuterium
Biosynthetic Pathways
Electron Spin Resonance Spectroscopy
Bacilli
Bacillus subtilis
Magnesium
Metal ions
Paramagnetic resonance
Hydrogen
Monomers
Metals
7-deazapurine
Ions
7-deazaguanine

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "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",
abstract = "7-Carboxy-7-deazaguanine (CDG) synthase (QueE) catalyzes the complex heterocyclic radical-mediated conversion of 6-carboxy-5,6,7,8-tetrahydropterin (CPH4) 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 CPH4 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 M{\"o}ssbauer spectroscopic features of the homodimeric QueE point to the presence of a single [4Fe-4S] cluster per monomer. Steady-state kinetic experiments indicate a Km of 20 ± 7 μM for CPH4 and a kcat of 5.4 ± 1.2 min-1 for the overall transformation. The kinetically determined Kapp for SAM is 45 ± 1 μM. QueE is also magnesium-dependent and exhibits a Kapp 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 CPH4 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 CPH4.",
author = "McCarty, {Reid M.} and Carsten Krebs and Vahe Bandarian",
year = "2013",
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doi = "10.1021/bi301156w",
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T1 - 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

AU - McCarty, Reid M.

AU - Krebs, Carsten

AU - Bandarian, Vahe

PY - 2013/1/8

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N2 - 7-Carboxy-7-deazaguanine (CDG) synthase (QueE) catalyzes the complex heterocyclic radical-mediated conversion of 6-carboxy-5,6,7,8-tetrahydropterin (CPH4) 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 CPH4 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 Km of 20 ± 7 μM for CPH4 and a kcat of 5.4 ± 1.2 min-1 for the overall transformation. The kinetically determined Kapp for SAM is 45 ± 1 μM. QueE is also magnesium-dependent and exhibits a Kapp 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 CPH4 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 CPH4.

AB - 7-Carboxy-7-deazaguanine (CDG) synthase (QueE) catalyzes the complex heterocyclic radical-mediated conversion of 6-carboxy-5,6,7,8-tetrahydropterin (CPH4) 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 CPH4 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 Km of 20 ± 7 μM for CPH4 and a kcat of 5.4 ± 1.2 min-1 for the overall transformation. The kinetically determined Kapp for SAM is 45 ± 1 μM. QueE is also magnesium-dependent and exhibits a Kapp 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 CPH4 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 CPH4.

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