An enzymatic activation of formaldehyde for nucleotide methylation.

Folate enzyme cofactors and their derivatives have the unique ability to provide a single carbon unit at different oxidation levels for the de novo synthesis of amino-acids, purines, or thymidylate, an essential DNA nucleotide. How these cofactors mediate methylene transfer is not fully settled yet, particularly with regard to how the methylene is transferred to the methylene acceptor. Here, we uncovered that the bacterial thymidylate synthase ThyX, which relies on both folate and flavin for activity, can also use a formaldehyde-shunt to directly synthesize thymidylate. Combining biochemical, spectroscopic and anaerobic crystallographic analyses, we showed that formaldehyde reacts with the reduced flavin coenzyme to form a carbinolamine intermediate used by ThyX for dUMP methylation. The crystallographic structure of this intermediate reveals how ThyX activates formaldehyde and uses it, with the assistance of active site residues, to methylate dUMP. Our results reveal that carbinolamine species promote methylene transfer and suggest that the use of a CH2O-shunt may be relevant in several other important folate-dependent reactions.

Deprotonations in the Reaction of Flavin-Dependent Thymidylate Synthase.

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 nuclear magnetic resonance 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 analogues 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.

Comparative ease of separation of mixtures of selected nuisance anions (nitrate, nitrite, sulfate, phosphate) using Octolig.

Mixtures of sodium salts of nitrate, nitrite, sulfate, and phosphate were prepared in relative amounts present in atomic waste containers with a view to effect removal by chromatography over Octolig, commercially available material with polyethylenediamine moieties covalently attached to high-surface area silica gel. Separation was attempted using aqueous solutions and column chromatography with Octolig. It is presumed that this material is capable of removing the anions by means of encapsulation. Matrix effects were tested by varying the relative concentrations. Rates of elution were varied 5-fold without adverse effect. The order of selectivity was found to be phosphate > sulfate > nitrite > nitrate through experiments altering the volume and relative concentrations. Quantitative removal of all anions (375 ppm of each) could be achieved given reasonable volumes of Octolig. An effort at regeneration by altering the pH of the eluant indicated the stability of the encapsulated anions.

Comparison of effectiveness of removal of nuisance anions by metalloligs, metal derivatives of Octolig.

The present study describes the effectiveness of removal of selected aqueous anions by several metalloligs, i.e., metal derivatives of a commercially available immobilized ligand, IMLIG, Octolig. This material consists of polyethylenediamine moieties covalently bound to a high-surface area silica gel (CAS Registry number = 404899-06-5). The metals involved are copper, cobalt, iron, nickel, manganese, and thorium. The nuisance anions, studied as aqueous solutions, were arsenate, orthophosphate, selenite, sulfate, nitrate, and nitrite. All six metalloligs tested were able to remove arsenate (280 ppb, > 99% removal) effectively. The effectiveness for other anions varied, but all anions were removed by one metallolig or another. For example, for Colbaltilig, in deionized water samples, removal was 96% for 20.9 ppm NO(3)-N, 98% for 29.6 ppm sulfate (as sodium sulfate). For Cuprilig, removal was 98% for 9.82 ppm boron as boric acid, > 99% for Cr as 212 ppm dichromate, 97% for P as 10 ppm HPO(4)-P, and 90% for sulfate as 29 ppm sulfate. Removal of nitrate by Cobaltilig appeared to be adversely affected by the presence of sulfate. Manganilig and Nickelig were able to remove > 97% of 10 ppm P as Na(2)HPO(4). Mechanisms of interaction are proposed that suggest six types of behavior and several classes of materials are represented by these metalloligs.