Evidence from 18O Exchange Studies for an Exocyclic Methylene Intermediate in the Reaction Catalyzed by T4 Deoxycytidylate Hydroxymethylase

Abstract

18O exchange experiments were designed to identify the final intermediate in the catalytic mechanism of bacteriophage T4 deoxycytidylate (dCMP) hydroxymethylase (CH). CH catalyzes the formation of 5-(hydroxymethyl)-dCMP (HmdCMP) from dCMP and methylenetetrahydrofolate (CH2-THF). CH resembles thymidylate synthase (TS), an enzyme of known three-dimensional structure, in both amino acid sequence and the reaction catalyzed. The final intermediate in the reaction catalyzed by TS or CH has been proposed to be the nucleotide with an exocyclic 5-methylene group covalently linked to the enzyme. This intermediate is then hydrated to HmdCMP (by CH) or reduced to deoxythymidylate (by TS). We report here that CH catalyzes the incorporation of 18O from solvent water into the product, HmdCMP, in the presence of tetrahydrofolate (THF). The cause of this exchange is a reverse reaction followed by a resynthesis. CH also catalyzes the exchange of 18O from solvent water into HmdCMP in the absence of exogenous THF and in the presence of THF analogues that lack N-5. N-5 is the nitrogen that is likely to be bound to the methylene as it is transferred to dCMP. A CH variant that lacks the nucleophilic Cys 148 is incapable of promoting these 18O exchange reactions. The THF analogues lacking N-5 do not promote a CH-catalyzed reverse reaction. Rather, we propose that the CH-catalyzed 18O exchange reaction promoted by these THF analogues occurs via 5-methylene-dCMP linked to the enzyme through Cys 148. We conclude here that enzyme-bound 5-methylene-dCMP is the final intermediate during catalysis by CH, as has also been proposed for TS and dUMP.

Crystal structure of deoxycytidylate hydroxymethylase from bacteriophage T4, a component of the deoxyribonucleoside triphosphate-synthesizing complex

“…During the initial steps, they both catalyze the transfer of a methylene group from CH 2 H 4 folate to C-5 of either dCMP or dUMP (Carreras and Santi, 1995; Figure 3). 18 O-exchange studies indicated the existence of a 5-exocyclic methylene intermediate in the reaction mechanism of T4 CH (Butler et al, 1994). The same intermediate was also proposed for TSs (Stroud and Finer-Moore, 1993).…”

Crystallization and preliminary X-ray crystallographic analysis of deoxycytidylate hydroxymethylase from bacteriophage T4

“…18 O-exchange experiments have identi®ed the ®nal exocyclic methylene intermediate formed during the proposed mechanism (Butler et al, 1994). Roles in cofactor binding and in catalysis of residues Glu60, Cys148, and Asp179 of T4 CH have been examined by site-directed mutagenesis (Graves et al, 1992;Hardy et al, 1995).…”

Structural basis of the substrate preference towards CMP for a thymidylate synthase MilA involved in mildiomycin biosynthesis

“…5hmC in mammalian DNA is produced post-replicatively by the Tet-catalyzed oxidation of 5-methyl cytosine (5mC)34. In T-even phage, the deoxycytidylate (dCMP) hydroxymethylase (CH) transfers the methylene group from methylene-tetrahydrofolate (CH 2 THF) to the C5 atom of dCMP, and then uses solvent water molecule to hydrate the methylene group to generate hydroxymethyl dCMP (hmdCMP)5, a precursor to be incorporated into DNA during replication6. Thereafter, its hydroxymethyl group serves as a substrate for glucosylation to form glucosylhydroxymethylated DNA to avoid cleavage by the host restriction systems78.…”