Deprotonations in the Reaction of Flavin-Dependent Thymidylate Synthase

Abstract

Many microorganisms use flavin-dependent thymidylate synthase (FDTS) to synthesize the essential nucleotide 2′-deoxythymidine-5′-monophosphate (dTMP) from 2′-deoxyuridine-5′-monophosphate (dUMP), 5,10-methylenetetrahydrofolate (CH2THF), and NADPH. FDTSs have a structure that is unrelated to the thymidylate synthase used by humans, and a very different mechanism. Here we report NMR evidence that FDTS ionizes N3 of dUMP using an active-site arginine. The ionized form of dUMP is largely responsible for the changes in the flavin absorbance spectrum of FDTS upon dUMP binding. dUMP analogs also suggest that the phosphate of dUMP acts as the base that removes the proton from C5 of the dUMP-methylene intermediate in the FDTS-catalyzed reaction. These findings establish additional differences between the mechanisms of FDTS and human thymidylate synthase.

Unique Features and Anti-microbial Targeting of Folate- and Flavin-Dependent Methyltransferases Required for Accurate Maintenance of Genetic Information

“…Interestingly, structural data has indicated that both, CH 2 H 4 folate and dUMP are bound in close vicinity ( Koehn et al, 2012 ) at opposite sides of the FAD isoribityl ring system ( Figure 2A ), indicating that (N5) FAD shuttles a methylene group (and not electrons, as in up to 90% of flavoproteins) from one side of the FAD ring to the other during enzymatic formation of thymidylate ( Mishanina et al, 2016 ). A further key mechanistic difference between ThyA and ThyX is that the latter uses an electrostatic, and not covalent, activation of the methylene-receiving C5 of dUMP ( Stull et al, 2016 ). This observation convincingly explains why early efforts to identify nucleophilic amino acid residues of ThyX proteins involved in substrate activation were unsuccessful.…”

Probing the methylene and hydride transfers in flavin- dependent thymidylate synthase

“…This causes C4 and C2 carbonyl carbons to shift ~8 ppm downfield (with 13 C and 15 N labeled dUMP) with respect to free dUMP in solution at pH 8. 21 This observation further supports the role of R174 in stabilizing the negative charges on N3 and C4 carbonyl oxygen. The authors argue that deprotonation of N3 makes any negative charge at C5 more localized.…”

“…These observations ruled out the possibility of FDTS serving as a bifunctional enzyme in organisms lacking thyA/folA genes. 6,[11][12][13][14][15][16][17][18][19][20][21][22][23][24] The gene thyX produces an assembly with less than 30% sequence identity to TSase, DHFR, and all known proteins while producing a unique fold (Figure 1.3 B). [12][13] The center of the FDTS homotetramer is a large void intersecting four identical active sites each created at interfaces of three subunits and each binding dUMP, CH2H4folate, and FAD ( Figure 1.3B).…”

Flavin-dependent thymidylate synthase: N5 of flavin as a Methylene carrier

“…Additionally, recently published 13 C-NMR data on Tm FDTS-dUMP complex revealed N3 of dUMP is deprotonated. This causes C4 and C2 carbonyl carbons to shift ~8 ppm downfield (with 13 C and 15 N labeled dUMP) with respect to free dUMP in solution at pH 8 [21]. This observation further supports the role of R174 in stabilizing the negative charges on N3 and C4 carbonyl oxygen.…”

“…This mechanism is consistent with oxidation of flavin taking place at a later stage of the mechanism (step 4, Scheme 3d). Furthermore, studies by Frederick et al showed the phosphate moiety of dUMP might be the base that abstracts the proton from C5 of dUMP to yield the exocyclic methylene intermediate [21]. …”