Formaldehyde, tetrahydrofolate

A subclass of lyases, involved in amino acid metabolism, utilizes pyridoxal 5-phosphate (PLP, 3-hydroxy-2-methyl-5-[(phosphonooxy)methyl]-4-pyridinecarbaldehyde) as a cofactor for imine/ enamine-type activation. These enzymes are not only an alternative to standard fermentation technology, but also offer a potential entry to nonnatural amino acids. Serine hydroxymethyl-tansferase (SHMT EC 2.1.2.1.) combines glycine as the donor with (tetrahydrofolate activated) formaldehyde to L-serine in an economic yield40, but will also accept a range of other aldehydes to provide /i-hydroxy-a-amino acids with a high degree of both absolute and relative stereochemical control in favor of the L-erythro isomers41. 

Recently, the enzymatic formation of folinic acid has been utilized to synthesize radioactively labeled products.34 The preparation of 5-formyl tetrahydrofolate, 9,3, 5 -3H and 5-formyl-14C-tetrahydrofolate starts with tritiated folic acid, which is reduced to dihydrofolate, incubated in the presence of formaldehyde, dihydrofolate reductase, and NADPH, and finally incubated with 5,10-methylenetetrahydrofolate dihydrogenase. The product,... 

Tetrahydrofolate derivatives are involved in one carbon unit transfer at the oxidation levels of formate, formaldehyde and methanol. At the formate level of oxidation, two derivatives, fV(5,10)-methenyltetrahydrofolate (23) andfV(10)-formyltetrahydrofolate (24), act as cofactors. Reactions involving one-carbon unit transfers at formaldehyde and methanol levels of oxidation utilize fV(5,10)-methylenetetrahydrofolate (25) and N(5)-methyltetrahydrofolate (26), respectively. 

This kind of reaction is exemplified by serine hydroxymethylase, an enzyme that requires both PLP and tetrahydrofolate as coenzymes. The aldimine produced upon the reaction of serine with (30), i.e. (31 R = CH2OH), loses formaldehyde via abstraction of the /3-hydroxyl proton (i.e. an aldol cleavage) to give (33). The formaldehyde that is produced is immediately trapped by the tetrahydrofolate cofactor to give iV(5,10)-methylenetetrahydrofolate... 

Side chain cleavage (Group c). In a third type of reaction the side chain of the Schiff base of Fig. 14-5 undergoes aldol cleavage. Conversely, a side chain can be added by (3 condensation. The best known enzyme of this group is serine hydroxymethyltransferase, which converts serine to glycine and formaldehyde.211-21313 The latter is not released in a free form but is transferred by the same enzyme specifically to tetrahydrofolic acid (Eq. 14-30), with which it forms a cyclic adduct. 

A number of pseudomonads and other bacteria convert C compounds to acetate via tetrahydrofolic acid-bound intermediates and C02 using the serine pathway179 192 193 shown in Fig. 17-15. This is a cyclic process for converting one molecule of formaldehyde (bound to tetrahydrofolate) plus one of C02 into acetate. The regenerating substrate is glyoxylate. Before condensation with the "active formaldehyde" of meth-... 

Tetrahydrofolates are cosubstrates for a variety of one-carbon transfer reactions. Tetrahydrofolates maintain formaldehyde and formate in chemically poised states, making them available for essential processes by specific enzymatic action. 

The pyridoxal-5 -phosphate dependent serine hydroxymethyltransferase (SHMT EC 2.1.2.1) in vivo catalyzes the interconversion of L-serine 158 and glycine 149 by transfer of the /1-carbon of L-serine to tetrahydrofolate (THF) by which the activated formaldehyde is physiologically made available as a C,-pool. The reaction is fully reversible and provides a means for the stereoselective synthesis of 158 in vitro from donor 149 and formaldehyde. Economical yields (88-94%) of L-serine have thus been obtained on a multimolar scale using raw cell extracts of recombinant Klebsiella aerogenes or E. coll in a controlled bioreactor at final product concentrations > 450 gl 1 [461,462], Several SHMTs have been purified and characterized from various organisms including animal tissues [463,464], eucaryotic [465] and procaryotic... 

Figure 2. Aerobic catabolism of methylated sulfides (adapted from Kelly, 1988). 1) DMSO reductase (Hyphomicrobium sp.) 2) DMDS reductase (Thiobacillus sp. 3) trimethylsulfonium-tetrahydrofolate methyltransferase (Pseudomonas sp.) 4) DMS monooxygenase 5) methanethiol oxidase 6) sulfide oxidizing enzymes 7) catalase 8) formaldehyde dehydrogenase 9) formate dehydrogenase 10) Calvin cycle for CO2 assimilation (Thiobacillus sp.) 11) serine pathway for carbon assimilation (Hyphomicrobium sp.).

Answer See the mechanism below. The formaldehyde produced in the second step reacts rapidly with tetrahydrofolate at the enzyme active site to produce 7V5,W10-methylene tetrahy-drofolate (see Fig. 18-17). 

Tetrahydrofolate functions as a carrier of one-carbon units. There are numerous metabolic reactions that require either the addition or removal of a one-carbon unit of some specific oxidation state. THF binds one-carbon units of three oxidation levels the methanol, formaldehyde, and formate states. These are shown in Table 6.4 along with their origins and uses. The various one-carbon units are interconvertible, as shown in Figure 6.5. Nicotinamide coenzymes are involved. In addition, the one-carbon unit may be released as C02. The methanol-level THF-bound one-carbon unit 5-methyl-THF is the storage and transport form. Once formed, its main pathway of metabolism is to form methionine from homocysteine, a reaction that requires vitamin B12 in the form of methylcobalamin (see Figure 6.2 and Chapter 20) ... 

Dihydrofolate reductase activates folate to tetrahydrofolate with dihydrofolate as an intermediate. Methotrexate, an antitumor agent, inhibits this enzyme. The 5-methyl group is first oxidized to the formaldehyde level, then to the formate level, then to C02. Three steps require three molecules of NAD or an equivalent, for a total of 3 x 3 = 9 ATP molecules. 

In order to understand the bewildering variety of reactions involving tetrahydrofolate, it is essential to realize that in biological systems, one-carbon compounds may exist in five different oxidation states. The most reduced form is methane, CH4, and the most oxidized form is C02. In between these two extremes are methanol (CH3OH), formaldehyde (CH20), and formate (HCOO ). 

Extracts of cells - of Escherichia coli infected with a T-even bacteriophage contain an enzyme deoxycytidylate hydroxymethylase which is not detected in uninfected cells it catalyzes the formation of 6 -(hydroxymethyl)-2-deoxyc3didylic acid from formaldehyde and 2-deoxycytidyUc acid in the presence of tetrahydrofolic acid and magnesium ions. In a large-scale experiment, 10.6 mg. of the barium salt was prepared. 

There is at present no unifying concept which would explain the presence of optically active TIQs, TIQ-1-carboxylic acids, and noralkaloids in mammalian systems. The conclusion reached by several investigators that methylenetetrahydrofolate is responsible for the N-methylation of phenethylamines and indolylethylamines (221,222) makes it likely that the one-carbon unit present at C-1 in the noralkaloids is derived from formaldehyde, formed via nonenzymatic disassociation of methylene-tetrahydrofolates (223,224). 

Dimethylglycine and sarcosine dehydrogenases in the catabolism of choline (Section 14.2.1). In these reactions, a methyl group in the substrate is oxidized by FAD, then the intermediate adduct undergoes hydrolysis to release formaldehyde, which reacts with tetrahydrofolate to form 5,10-methylene tetrahydrofolate. 

Figure 4-2. Structures of the various 1-carbon carriers of tetrahydrofolate (THF). THF can carry one-carbon units in the oxidation states of methanol (N -methyl THF), formaldehyde (N, N °-methylene THF) or formic acid (remaining structures).

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