Spectroscopic and Computational Investigation of the Epoxyqueuosine Reductase QueG Reveals Intriguing Similarities with the Reductive Dehalogenase PceA

Elizabeth D. Greenhalgh, William Kincannon, Vahe Bandarian, Thomas C. Brunold

Research output: Contribution to journalArticlepeer-review

Abstract

Queuosine (Q) is a highly modified nucleoside of transfer RNA that is formed from guanosine triphosphate over the course of eight steps. The final step in this process, involving the conversion of epoxyqueuosine (oQ) to Q, is catalyzed by the enzyme QueG. A recent X-ray crystallographic study revealed that QueG possesses the same cofactors as reductive dehalogenases, including a base-off Co(II)cobalamin (Co(II)Cbl) species and two [4Fe-4S] clusters. While the initial step in the catalytic cycle of QueG likely involves the formation of a reduced Co(I)Cbl species, the mechanisms employed by this enzyme to accomplish the thermodynamically challenging reduction of base-off Co(II)Cbl to Co(I)Cbl and to convert oQ to Q remain unknown. In this study, we have used electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) spectroscopies in conjunction with whole-protein quantum mechanics/molecular mechanics (QM/MM) computations to further characterize wild-type QueG and select variants. Our data indicate that the Co(II)Cbl cofactor remains five-coordinate upon substrate binding to QueG. Notably, during a QM/MM optimization of a putative QueG reaction intermediate featuring an alkyl–Co(III) species, the distance between the Co ion and coordinating C atom of oQ increased to >3.3 Å and the C–O bond of the epoxide reformed to regenerate the oQ-bound Co(I)Cbl reactant state of QueG. Thus, our computations indicate that the QueG mechanism likely involves single-electron transfer from the transient Co(I)Cbl species to oQ rather than direct Co–C bond formation, similar to the mechanism that has recently been proposed for the tetrachloroethylene reductive dehalogenase PceA.

Original languageEnglish (US)
Pages (from-to)195-205
Number of pages11
JournalBiochemistry
Volume61
Issue number3
DOIs
StatePublished - Feb 1 2022

ASJC Scopus subject areas

  • Biochemistry

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