Abstract
The methyltetrahydrofolate (CH(3)-H(4)folate) corrinoid-iron-sulfur protein (CFeSP) methyltransferase (MeTr) catalyzes transfer of the methyl group of CH(3)-H(4)folate to cob(I)amide. This key step in anaerobic CO and CO(2) fixation is similar to the first half-reaction in the mechanisms of other cobalamin-dependent methyltransferases. Methyl transfer requires electrophilic activation of the methyl group of CH(3)-H(4)folate, which includes proton transfer to the N5 group of the pterin ring and poises the methyl group for reaction with the Co(I) nucleophile. The structure of the binary CH(3)-H(4)folate/MeTr complex (revealed here) lacks any obvious proton donor near the N5 group. Instead, an Asn residue and water molecules are found within H-bonding distance of N5. Structural and kinetic experiments described here are consistent with the involvement of an extended H-bonding network in proton transfer to N5 of the folate that includes an Asn (Asn-199 in MeTr), a conserved Asp (Asp-160), and a water molecule. This situation is reminiscent of purine nucleoside phosphorylase, which involves protonation of the purine N7 in the transition state and is accomplished by an extended H-bond network that includes water molecules, a Glu residue, and an Asn residue (Kicska, G. A., Tyler, P. C., Evans, G. B., Furneaux, R. H., Shi, W., Fedorov, A., Lewandowicz, A., Cahill, S. M., Almo, S. C., and Schramm, V. L. (2002) Biochemistry 41, 14489-14498). In MeTr, the Asn residue swings from a distant position to within H-bonding distance of the N5 atom upon CH(3)-H(4)folate binding. An N199A variant exhibits only approximately 20-fold weakened affinity for CH(3)-H(4)folate but a much more marked 20,000-40,000-fold effect on catalysis, suggesting that Asn-199 plays an important role in stabilizing a transition state or high energy intermediate for methyl transfer.
Highlights
The structure of the binary CH3-H4folate/MeTr complex lacks any obvious proton donor near the N5 group
The MeTr reaction is similar to a variety of cobalamin-dependent methylation reactions, including methyl transfer from CH3-H4folate to the bound cobalamin cofactor of methionine synthase, which subsequently transfers its methyl group from methylcobalamin to homocysteine [3, 4]
Cobalamin-dependent methyltransferases play an important role in redox homeostasis and amino acid metabolism in many organisms as well as in one-carbon metabolism and CO2 fixation in anaerobic microbes [3]
Summary
Its Co(I) center catalyzes an SN2 displacement of the N5 methyl group of CH3-H4folate to form the methylated CFeSP. The mechanism of proton transfer leading to electrophilic activation of the methyl group is unclear To solve this paradox, one might posit that the proton donor moves into place after CH3-H4folate binds; the recent crystal structure of the CH3-H4folate-MetH complex shows that there is no acidic group positioned near the N5 group of the pterin and, like the MeTr structure, Asn-199 (MeTr numbering) is within H-bonding distance to N5 [10] (Fig. 1). In addition to the structural homology, there are mechanistic similarities of the methyltransferases with PNP as well To facilitate their bond cleavage reactions, both sets of enzymes appear to require substrate protonation and subsequent stabilization of a protonated species in the transition state. Given the provocative location of Asn-199 in MeTr and its potential role in transition state stabilization in the transmethylation reaction, we performed site-directed mutagenesis, kinetic, and structural studies to evaluate the contribution of this residue to catalysis
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
