Formate cyclohydrolase

This interesting conversion of a five- into a six-membered heterocyclic ring was proven by the isolation of the enzyme GTP-cyclohydrolase from E. coli (71MI21600) and a similar one from Lactobacillus platarum (B-71MI21601) which catalyzes the reaction (300)(303). Dephosphorylation leads to 7,8-dihydro-D-neopterin (304), which is then cleaved in the side-chain to 6-hydroxymethyl-7,8-dihydropterin (305), the direct precursor of 7,8-dihy-dropteroic acid and 7,8-dihydrofolic acid (224). The alcohol (305) requires ATP and Mg " for the condensation with p-aminobenzoic and p-aminobenzoylglutamic acid, indicating pyrophosphate formation to (306) prior to the substitution step. 

The tetrahydrofolates do not function as tightly enzyme-bound coenzymes. Rather, they function as cosubstrates for a variety of enzymes associated with one-carbon metabolism. /Vl0-formyltetrahydrofolate is produced enzymatically from tetrahydrofolate and formate in an ATP-linked process in which formate is activated by phosphorylation to formyl phosphate the formyl group of formyl phosphate is then transferred to A10 of tetrahydrofolate. A10-Formyltetrahydrofolate is a formyl donor substrate for some enzymes and is interconvertible with A5,A10-methenyltetrahydrofolate by the action of cyclohydrolase. 

D-e/yf/iro-7,8-Dihydroneopterin triphosphate synthetase, or GTP cyclohydrolase I (EC 3.5.4.16), catalyzes the formation of D-eryr/iro-dihydroneopterin triphosphate (NH2TP) from GTP. This activity is required for the synthesis of tetrahydrobiopterin. The HPLC assay developed for this activity involves the direct measurement of neopterin phosphates after separation from GTP and its other hydrolytic products. 

The precursors for riboflavin biosynthesis in plants and microorganisms are guanosine triphosphate and ribulose 5-phosphate. As shown in Figure 7.3, the first step is hydrolytic opening of the imidazole ring of GTP, with release of carbon-8 as formate, and concomitant release of pyrophosphate. This is the same as the first reaction in the synthesis ofpterins (Section 10.2.4), but utilizes a different isoenzyme of GTP cyclohydrolase (Bacher et al., 2000, 2001). 

Even prior to the elucidation of the first committed step of the riboflavin pathway, it had been shown that the benzenoid ring of riboflavin is assembled from two identical 4-carbon precursors. More specifically, the final step in the biosynthesis of the vitamin involves a dismutation of 6,7-dimethyl-8-ribityllumazine (6), where one of the substrate molecules serves as donor and the other as acceptor of a 4-carbon segment.19,20 6,7-Dimethyl-8-ribityllumazine, in turn, is formed in the penultimate step of the biosynthetic pathway from 5-amino-6-ribitylamino-4(3f/)-pyrimidinedione (3), an intermediate that is obtained from the product of GTP cyclohydrolase II by a sequence of deamination, side chain reduction, and dephosphorylation (Figure 3). The nature of the 4-carbon precursor required for the formation of 6,7-dimethyl-8-ribityllumazine (6) from 5-amino-6-ribitylamino-4(3f/)-pyrimidinedione (3) remained controversial for quite a long period, with working hypotheses including, but not limited to, tetroses, pentoses, and acetoin. 

Grignani et al. (G14) studied several of the enzymes of folate metabolism in human epidermis—both normal and psoriatic. Increased levels of folate reductase were found in the psoriatic lesion, and further enzyme could be induced by treatment of the patients with amethopterin. By contrast, formate-activating enzyme, 5,10-methylenetetrahydrofolate dehydrogenase, serine hydroxylase, and cyclohydrolase were normal in the psoriatic lesion. Formiminotetrahydrofolate transferase could not be measured either in normal or psoriatic skin. The activities of the above enzymes as well as the absence of the transferase are similar to the findings for small bowel but not to other tissues studied. How these findings... 

In this reaction C-8 of GTP is lost as formate with the concomitant release of pyrophosphate. A mechanistic proposal is outlined in Fig. 7. This cyclohydrolase is different from the GTP cyclohydrolase I involved in folate biosynthesis (see section 4 of this review). 

Mechanistic studies on the formation of the molybdopterin cofactor are still at an early stage. Conversion of guanosine, presumably as the triphosphate, to precursor Z occurs with retention of C-8 [84]. A possible mechanism for this third type of cyclohydrolase, which is consistent with the labeling experiment, is outlined in Fig. 20. (The other two types of cyclohydrolase are cyclohydrolase 1 for folate biosynthesis and cyclohydrolase II for riboflavin biosynthesis. In both cases, C8 is removed as formate.)... 

Figure 3 Hypothetical mechanism for release of formate by GTP cyclohydrolase II.

In summary, GTP cyclohydrolase II appears to catalyze an ordered reaction that starts with the formation of a covalent guanyl adduct (15). This is followed by the hydrolytic opening of the imidazole ring (16, 17) and the hydrolysis of the resulting formamide-type intermediate (18, 19) the latter two reactions depend on the Zn ion acting as a Lewis acid, which sequentially activates two water molecules (17, 19) that attack first the imidazole carbon atom 8 of the covalent guanyl adduct 17 and then the formamide motif of the covalent intermediate 19. 

Plants and certain eubacteria specify bifunctional proteins where a cyclohydrolase II domain is fused with a domain catalyzing the formation of 3,4-dihydroxy-2-butanone 4-phosphate. Jointly, these two domains produce both committed starting materials of the convergent riboflavin pathway (see below). 

In plants and in many microorganisms (but not in E. coli), 3,4-dihydroxy-2-butanone-4-phosphate synthase and GTP cyclohydrolase II are expressed as a fusion protein with GTP cyclohydrolase II as the C-terminal domain. The ratio of product formation of the two initial reactions of the convergent biosynthetic pathway is thus rigidly coupled. 

Guanosine triphosphate and ribulose-5-phosphate are recruited in a 1 2 stoichiometric ratio by GTP cyclohydrolase II and DHBP synthase, respectively, for riboflavin biosynthesis. Since at substrate saturation the activity of B. subtilis DHBP is twice the activity of B. suhtilis cyclohydrolase II (DSM, unpublished observations) and since both enzymatic activities are associated with the same bifunctional protein encoded by rihA, the balanced formation of the pyrimidinedione and the dihydroxybutanone intermediates is ensured. However, the ifg.s constant of DHBP synthase ( 1 mmol is about 100-fold higher than the ifg.s constant of GTP cyclohydrolase II imposing the risk of excessive synthesis of the pyrimidinone and pyrimidinedione intermediates in case of reduced intracellular concentrations of pentose phosphate pathway intermediates. This can be expected, for instance, in glucose-limited fed-batch fermentations, which are frequentiy used in industrial applications. The pyrimidinone and pyrimidinedione intermediates are highly reactive, oxidative compounds, which can do serious damage on the bacteria. 

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