Structural and Kinetic Evidence for an Extended Hydrogen-bonding Network in Catalysis of Methyl Group Transfer

Abstract

The methyltetrahydrofolate (CH 3 -H 4 folate) corrinoid-ironsulfur 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.

Pulse-Chase Studies of the Synthesis of Acetyl-CoA by Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase

“…For example, one could argue that if acetylCoA synthesis follows an ordered mechanism with methyl binding first, the metal-carbonyl species could be mechanistically irrelevant. 5 Thus, the pulse-chase experiments described here, by demonstrating an active path with CO as the first substrate, provide unambiguous evidence supporting the NiFeC species as a central intermediate in acetyl-CoA synthesis.…”

“…An alternative is an Ni 2ϩ mechanism with the two activation electrons being provided by a reduced disulfide-Ni p -Ni d electron donor. 5 However, one should consider that the NiFeC species has firm experimental evidence, and the putative Ni(0) state on ACS has never been observed. Considering the lower branch of the random pathway (where methyl binds before CO), paramagnetic intermediates have not been identified when the methylated enzyme is reacted with CO and CoA.…”

Transient B12-Dependent Methyltransferase Complexes Revealed by Small-Angle X-ray Scattering

“…(A) The theoretical profile (black solid) of the homodimeric M. thermoacetica MeTr crystal structure 11 fits well to experimental data obtained from 470 μM MeTr (dark blue with error bars shown in cyan), while that of just one MeTr monomer (black dashed) gives a poor fit. (B) Theoretical profiles of the three CFeSP models (shown in C, with same coloring) are nearly superimposable with each other at 25 Å resolution (i.e., q < 0.25 Å –1 ).…”

“…Linear extrapolation to infinite dilution 19 yields an R g value of 27.6 ± 0.4 Å for MeTr, in good agreement with the theoretical value of 26.9 Å calculated from the previously reported crystal structure of the MeTr homodimer. 11 Furthermore, the theoretical scattering profile calculated from the crystal structure generates an excellent fit to the experimental scattering data (Figure 3A, black solid curve). Likewise, the extrapolated R g value of 31.1 ± 0.7 Å for CFeSP is consistent with the theoretical value calculated from the previously reported structure of a homologous CFeSP from Carboxydothermus hydrogenoformans (12) (30.2 Å) as well as those calculated from individual CFeSPs extracted from structures of the M. thermoacetica CFeSP/MeTr complex (30.1–30.4 Å).…”

Structural and Mechanistic Studies on Klebsiella pneumoniae 2-Oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline Decarboxylase

“…Based on this observation, we propose that Gln-88 instead acts to inductively destabilize the carbon-carbon bond between the carboxylate and C5 of the hydantoin ring in a manner analogous to purine nucleoside phosphorylase (33) and related enzymes (34). In the purine nucleoside phosphorylase-catalyzed reaction, an active site asparagine withdraws electrons from the purine ring through hydrogen bond formation causing a weakening and concomitant heterolysis of the C1Ј-N9 bond.…”