The reaction pathway of C5 MTases in the presence and in the absence of mechanism-based inhibitors. (a) The reaction pathway for all C5 MTases involves the transfer of the labile methyl group from S-adenosyl-l-methionine (AdoMet) to the 5 position of the cytosine ring, proceeds through a covalent intermediate at position C6.14 The nucleophilic attack upon the C6 position of cytosine drives the subsequent acquisition of the labile methyl group from AdoMet. (Note, the protonation status of Glu119 in M.Hha I36). (b) The inhibition by FdC. Following covalent complex formation and methyl transfer, the analogue remains bound to the active-site Cys, since abstraction of F cannot be achieved. (c) The inhibition by AzaC. Following covalent complex formation at a C6 with enhanced reactivity, slow methyl transfer may take place, but there is no H at C5 to abstract and the covalent complex persists. (d) The inhibition by zebularine. Following covalent complex formation at a C6 with enhanced reactivity as with AzaC, facilitated deamination at C4 cannot proceed,33 since the amino moiety is absent from the analogue. Note that the water molecule nearest to the C4 atom is 3.6 Å away and the water molecule nearest to the C5 atom is 3.3 Å away.

Molecular orbital calculations. Ab initio geometries were calculated with the program SPARTAN (Wave-function, Irvine, CA) as described.37 1-Methylcytosine (instead of the sugar ring at position 1) was used to model the flipped base. Dark blue indicates a high value for the orbital, and the red indicates a low value, while green/yellow is in between. High values for the lowest unoccupied molecular orbital (LUMO) are seen over C6 and C4 for both 1-methylcytosine and N3 protonated 1-methylcytosine in all three cases, indicating that these are potential sites for nucleophilic attack. The 1-methyl-6-sulfhydryl-enol derivative of cytosine was used to model the intermediate formed by the enzyme after nucleophilic attack at C6. High values of the highest occupied molecular orbital (HOMO) are confined to C5 in cytosine and FdC, where the highly reactive orbital at C5 is then poised for attack on the methyl group of AdoMet, but between C4 and C5 in 2-H pyrimidinone. This is consistent with the observation that zebularine does not seem to be methylated at all.38

Structure of M.Hha I–AdoHcy–DNA containing zebularine. (a) The structural impasse between the proposed mechanism (outlined in Figure 1) and the barrier to substrate access in duplex DNA was overcome elegantly by the phenomenon of protein-induced base flipping.15 Once the target base (zebularine here) is released from the constraints of the Watson–Crick base-pair, conventional active-site chemistry is facilitated. (b) Zebularine difference electron density maps (F_o − F_c, α_c) superimposed on the refined coordinates with carbon atoms being yellow, oxygen atoms red, nitrogen atom blue, and sulfur atom green, respectively. The blue electron density map contoured at 5.0σ was computed with the zebularine moiety omitted from the atomic model. The green electron density maps contoured above 5.5σ were calculated with the C4, C5, and C6 atoms of zebularine omitted from the atomic model, respectively. The zebularine is constrained in the plane of the ring by a highly conserved network of hydrogen bonds (via E119 and R165) and van der Waals interactions between the main-chain C=O group of F79 and C4 and C5 atoms. (c) A view perpendicular to (b), looking edge-on at the flipped zebularine molecule. A covalent bond is observed between C6 of the zebularine ring and an invariant thiolate side-chain C81, approaching the C6 perpendicular to the ring. The red electron density map contoured above 10.0σ was calculated with the sulfur atom of C81 omitted from the atomic model.