Abstract
Folate enzyme cofactors and their derivatives have the unique ability to provide a single carbon unit at different oxidation levels for the de novo synthesis of amino-acids, purines, or thymidylate, an essential DNA nucleotide. How these cofactors mediate methylene transfer is not fully settled yet, particularly with regard to how the methylene is transferred to the methylene acceptor. Here, we uncovered that the bacterial thymidylate synthase ThyX, which relies on both folate and flavin for activity, can also use a formaldehyde-shunt to directly synthesize thymidylate. Combining biochemical, spectroscopic and anaerobic crystallographic analyses, we showed that formaldehyde reacts with the reduced flavin coenzyme to form a carbinolamine intermediate used by ThyX for dUMP methylation. The crystallographic structure of this intermediate reveals how ThyX activates formaldehyde and uses it, with the assistance of active site residues, to methylate dUMP. Our results reveal that carbinolamine species promote methylene transfer and suggest that the use of a CH2O-shunt may be relevant in several other important folate-dependent reactions.
Highlights
Folate enzyme cofactors and their derivatives have the unique ability to provide a single carbon unit at different oxidation levels for the de novo synthesis of amino-acids, purines, or thymidylate, an essential DNA nucleotide
flavin adenine dinucleotide (FAD) in its two-electron reduced FADH− form could activate CH2O leading to a flavincarbinolamine species, which should naturally be in equilibrium with its iminium counterpart[28] (Supplementary Fig. 2)
The analysis was repeated with 13C-labeled CH2O and showed that the methylene incorporated in dTMP did not come from sources other than CH2O since only [13C7]-dTMP was formed, as unambiguously detected by nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) analyses (Fig. 2 and Supplementary Figs. 3 and 4)
Summary
Folate enzyme cofactors and their derivatives have the unique ability to provide a single carbon unit at different oxidation levels for the de novo synthesis of amino-acids, purines, or thymidylate, an essential DNA nucleotide. How these cofactors mediate methylene transfer is not fully settled yet, with regard to how the methylene is transferred to the methylene acceptor. The postulated iminium species have never been directly observed in any folate-dependent enzymes, while alternative mechanisms involving carbinolamine species or formaldehyde (CH2O) as a reaction intermediate have been considered[9,12,14], but remained difficult to demonstrate (Fig. 1). ThyX relies on the flavin adenine dinucleotide (FAD) as well as N5,N10-
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