Dihydrofolate synthetase

In E. coli GTP cyclohydrolase catalyzes the conversion of GTP (33) into 7,8-dihydroneoptetin triphosphate (34) via a three-step sequence. Hydrolysis of the triphosphate group of (34) is achieved by a nonspecific pyrophosphatase to afford dihydroneopterin (35) (65). The free alcohol (36) is obtained by the removal of residual phosphate by an unknown phosphomonoesterase. The dihydroneoptetin undergoes a retro-aldol reaction with the elimination of a hydroxy acetaldehyde moiety. Addition of a pyrophosphate group affords hydroxymethyl-7,8-dihydroptetin pyrophosphate (37). Dihydropteroate synthase catalyzes the condensation of hydroxymethyl-7,8-dihydropteroate pyrophosphate with PABA to furnish 7,8-dihydropteroate (38). Finally, L-glutamic acid is condensed with 7,8-dihydropteroate in the presence of dihydrofolate synthetase. 

CARNOSINE SYNTHETASE CHAPERONES CHOLINE KINASE CHOLOYL-CoA SYNTHETASE COBALAMIN ADENOSYLTRANSFERASE 4-COUMAROYL-CoA SYNTHETASE CREATINE KINASE CTP SYNTHETASE CYTIDYLATE KINASE 2-DEHYDRO-3-DEOXYGLUCONOKINASE DEHYDROGLUCONOKINASE DEOXYADENOSINE KINASE DEOXYADENYLATE KINASE DEOXYCYTIDINE KINASE (DEOXYjNUCLEOSIDE MONOPHOSPHATE KINASE DEOXYTHYMIDINE KINASE DEPHOSPHO-CoA KINASE DETHIOBIOTIN SYNTHASE DIACYLGLYCEROL KINASE DIHYDROFOLATE SYNTHETASE DNA GYRASES DNA REVERSE GYRASE ETHANOLAMINE KINASE EXONUCLEASE V... 

DEOXYOCTULOSONATE 8-PHOS-PHATE SYNTHASE DETHIOBIOTIN SYNTHASE DIHYDROFOLATE SYNTHETASE... 

Both the sulfonamides and trimethoprim interfere with bacterial folate metabolism. For purine synthesis tetrahydrofolate is required. It is also a cofactor for the methylation of various amino acids. The formation of dihydrofolate from para-aminobenzoic acid (PABA) is catalyzed by dihydropteroate synthetase. Dihydrofolate is further reduced to tetrahydrofolate by dihydrofolate reductase. Micro organisms require extracellular PABA to form folic acid. Sulfonamides are analogues of PABA. They can enter into the synthesis of folic acid and take the place of PABA. They then competitively inhibit dihydrofolate synthetase resulting in an accumulation of PABA and deficient tetrahydrofolate formation. On the other hand trimethoprim inhibits dihydrofolate... 

FIGURE 33-2 Folic acid metabolism in bacterial cells. Certain antibacterial drugs [e.g., sulfonamides and trimethoprim] inhibit the dihydrofolate synthetase and reductase enzymes, thus interfering with DNA biosynthesis. 

Reaction of 1 with ATP in presence of Mg and kinase gives 2-amino-4-hy-droxy-6-hydroxymethyldihydropteridine pyrophosphate (2, DHP pyrophosphate). The latter is converted into 7,8-dihydropteroate (3, DHP) by its reaction with p-ami-nobenzoic acid (PABA) and DHP synthetase. Addition of L-glutamic acid to DHP in the presence of the enzyme dihydrofolate synthetase (DHF synthetase) yields 7,8-di-hydrofolate (4, DHF), which undergoes reduction by the enzyme dihydrofolate reductase to afford 5,6,7,8-tetrahydrofolate (5, THF). DHF may also lose a hydrogen molecule in the presence of DHF dehydrogenase to form folic acid (6). 

The key enzymes involved in the biosynthesis of DHFA and THFA are dihy-dropteroate synthetase, dihydrofolate synthetase and dihydrofolate reductase [1-3]. Drugs that block the synthesis of dihydropteroic acid are known as dihydropteroate synthetase inhibitors (PABA antagonists) and those which control the reduction of DHFA to THFA are called dihydrofolate reductase (DHFR) inhibitors. Collectively these drugs are known as antifolates. 

Salcedo, E., Cortese, J. F., Plowe, C. V., Sims, P. F., and Hyde, J. E. (2001). A bifunctional dihydrofolate synthetase-folylpolyglutamate synthetase in Plasmodium falciparum identified by functional complementation in yeast and bacteria. Mol. Biochem. Parasitol. 112, 239-252. 

Figure 2 Biosynthesis of foiates. a, GTP cyclohydrolase I b, dihydroneopterin epimerase c, nudix hydrolase d, phosphatase e, dihydroneopterin aldolase f, hydroxymethyidihydropterin pyrophosphokinase g, dihydropteroate synthase h, dihydrofolate synthetase I, dihydrofolate reductase j, folylpolyglutamate synthetase k, aminodeoxychorismate synthase I, aminodeoxychorismate lyase. 7, 7,8-dihydro-D-neopterin 3 -triphosphate 8, 7,8-dihydro-L-monapterin 3 -triphosphate 9, 7,8-dihydro-D-neopterin 3 -monophosphate 10, 7,8-dihydro-D-neopterin 11, glycolaldehyde ...

Outstanding among drugs which inhibit the production of DNA from several stages back in the biosynthetic pathway are the sulfonamides and the 2,4-diaminopyrimidines used as antibacterials and anti-malarials. All of the chemotherapeutic sulfonamides, whether simple sulfanilamide 4.6a) or its more complex heterocyclic derivatives 4.6b) including sulfadiazine, competitively inhibit the enzyme dihydrofolate synthetase which produces dihydrofolic acid 2.14) (see p. 31). The basis of this inhibition, as outlined in Section 2.1 (p. 31), is the similarity in the steric and electronic properties of/ -aminobenzoic acid 2.12) (which the enzyme is ready to build into new molecules of dihydrofolic acid) and the sulfonamides 4.6) which, when taken up by the enzyme, merely block it. The basis of the selectivity of these antibacterial sulfonamides depends on two factors, which reinforce one another (i) mammals lack the enzymes necessary for the synthesis of dihydrofolic acid, and hence they tolerate these sulfonamides very well (ii) pathogenic bacteria lack the permease... 

A related kind of resistance, target withdrawal , falls somewhere in between Type 2 and Type 3. In an example, the clinical resistance Staphylococcus aureus to erythromycin, the 505 ribosome subunits were found to have been methylated by an enzyme peculiar to the resistant strain (Lai and Weisblum, 1971). Also, in some examples of clinical resistance of pneumococci to sulfonamides, the target enzyme, dihydrofolate synthetase, has been found chemically altered (Ortiz, 1970). 

The antibacterial sulfonamides have a very interesting relative, dapsone (9.77), the most used drug in the treatment of leprosy. Sulfadiazine, and the majority of antibacterial sulfonamides, have been shown to inhibit the relevant enzyme (dihydrofolate synthetase) best if in the form of the anion. However, dapsone is not capable of ionization, but forms an ion-dipole bond to the enzyme. The result is a milder agent with minimal side effects during the many long years that a cure of leprosy requires. 

There are antagonists of the biosynthesis of dihydrofolic acid, and antagonists of its utilization. The history of the discovery of the antibacterial sulfonamides, typical antagonists of biosynthesis, was given in Sections 2.1 and 6.3.1. In 1940, Woods showed that the anti-bacterial action of sulfanilamide depended on its competition with j -aminobenzoic acid (P.7), which is a natural metabolite (Woods, 1940). Later this competition was shown to take place at the site on the enzyme dihydrofolate synthetase, which uses j -aminobenzoic acid to build up the molecule of dihydrofolic acid (2.74) (G.M. Brown, 1962). 

Bacteria use/ -aminobenzoic acid only for conversion to 7.8-dihydrofolic acid (Griffin and Brown, 1964). Thus, E, coli condenses j -aminobenzoic acid (and, alternatively, -aminobenzoylglutamic acid) with 2-amino-4-oxo-6-hydroxy-methyl-7,8-dihydropteridine 9.21) (as the 6-pyrophosphate) to give dihydro-pteroic acid (and alternatively, dihydrofolic acid) (Jaenicke and Chan, 1960). The sulfonamides competitively inhibit the isolated enzyme dihydrofolate the-tase which catalyses these steps (G. Brown, 1962). From Lactobacillus plantamm two enzymes responsible for this synthesis have been isolated in a pure state (Shiota et al., 1969a). The first of these catalyses the esterification of the pteridine 9.21) to its pyrophosphoryl derivative. The second is Brown s dihydrofolate synthetase. This second enzyme has also been isolated from several strains of Pneumococcus, found to have a mol. wt. of 90000, and to need ATP and Mg " as coenzymes (Ortiz, 1970). 

Guanosine triphosphate cyclohydrolase I 2 pyrophosphorylase, phosphatase 3 diliydro-neopterin aldolase 4 hydroxymethyldihydropterin pyrophosphokinase 5 dihydropteroate synthase 6 dihydrofolate synthetase 7 dihydrofolate reductase 8 xanthine oxidase... 

Sulphanilamide and its derivatives. The concept that a drug receptor could be an enzyme was extended from pharmacodynamics to chemotherapy when Woods (1940) demonstrated the reversal of the antibacterial action of sulphanilamide 2.12) by p-aminobenzoic acid 2,13) and pointed out that this reversal depended on the structural similarity of these two substances. Later, the receptor for sulphonamides was found to be the enzyme dihydrofolate synthetase, which incorporates p-aminobenzoic acid into the molecule of dihydrofolic acid (2.74), an essential coenzyme for the biosynthesis of purines and thymine, and hence of DNA. This enzyme was isolated and purified by G. Brown (1962), and these functions confirmed. 

Lai and Weisblum, 1971). Also, in some examples of clinical resistance of pneumococci to sulphonamides, the target enzyme, dihydrofolate synthetase, has been found chemically altered (Ortiz, 1970). 

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