A comparison of the effectiveness with which p-aminobenzoic acid and p-aminobenzoylglutamic acid are used as substrate by dihydropteroate synthetase from Escherichia coli

Richey, David P.; Brown, Gene M.

doi:10.1016/0304-4165(70)90376-4

Cited by 12 publications

(13 citation statements)

References 12 publications

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“…Certainly the molecular weights found were larger than those reported for the E. coli kinase (15,000) (15), E. coli synthetase (50,000) (15), or the Diplococcus pneumoniae synthetase (75,000-90,000) ( 13). The P. berghei enzymes were separated easily on a DEAE-Sephadex column, as had been found for E. coli (15) and L. plantarum enzymes (18). I t was not determined if both enzymes of P. berghei have the same large molecular weight or if they are found in a complex in crude extracts which can be dissociated on a DEAE column.…”

Section: Discussionsupporting

confidence: 53%

“…Several differences are apparent between the kinase and synthetase from Plasmodium and these enzymes from other sources. The specific activity of the kinase (= 2 pmole/min/mg protein) in crude extracts of P. berghei was much lower than that from E. coli (74) (15) and L. plantarum (= 1500) (18). The P. berghei synthetase specific activity (c 1) was 200-1000 times lower than that of bacteria (13, 15, 17), 20-30 times lower than that of Plasmodium chabaudi (20), but 3 times higher than that reported for this enzyme from pea seedlings (11).…”

Section: Discussionmentioning

confidence: 97%

“…The radioactive assay measures the formation of ["C] H, pteroate from [7-14C]pAB, after separation of product and substrate by paper chromatography (based on ref. 15). The reaction systems were the same as above, except 0.01 mM [7-W]pAB (5-10 mC/mmole, New England Nuclear) was used instead of has an Rf of 0.8.…”

Section: Assaymentioning

confidence: 99%

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The Enzymic Synthesis of Dihydropteroate and Dihydrofolate by Plasmodium berghei

Ferone

1973

The Journal of Protozoology

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SYNOPSIS. Cell‐free extracts of the rodent malaria parasite Plasmodium berghei synthesized dihydropteroate (H2pteroate) and dihydrofolate (H2folate) from 2‐amino‐4‐hydroxy‐6‐hydroxymethyl‐7,8‐dihydropteridine (hydroxymethyldihydropteridine) and p‐aminobenzoate (pAB) or p‐aminobenzoylglutamate (pABG). The reaction was demonstrated also in extracts of Plasmodium gallinaceum, Plasmodium lophurae and Plasmodium knowlesi, by the use of a microbiologic assay method and pABG as cosubstrate. Some of the properties of the enzymes involved were investigated in P. berghei preparations, utilizing a radioactive assay which measures the conversion of [7‐14C]pAB to [14C]H2pteroate. Apparent Km values of 0.28 μM for [7‐14C]pAB, 0.037 mM for pABG and 0.8 μM for hydroxymethyldihydropteridine were obtained. The reaction had absolute requirements for ATP and Mg++, and was stimulated by dithiothreitol. The enzymes required for the reaction were eluted together from Sephadex G‐200 columns in a molecular weight range of 200,000–250,000. In bacteria hydroxymethyldihydropteridine is converted 1st by a pyrophosphokinase to pyrophosphorylmethyldihydropteridine, and this compound is then condensed with pAB to form H2pteroate by H2pteroate synthetase. Both enzymic activities were demonstrated in P. berghei preparations and separated by DEAE‐Sephadex chromatography. The enzymic synthesis of H2pteroate by P. berghei is inhibited by several sulfonamides and diaminodiphenylsulfone (DDS). The latter compound is shown to be competitive with pAB, with a Ki value of 0.38 μM; pABG is also a competitive inhibitor. These data establish an enzymic basis of support for the evidence obtained in vivo which indicate that malaria synthesize their folate cofactors de novo. It is suggested that the antimalarial action of sulfonamides and DDS is due to their inhibition of plasmodial H2pteroate synthetase.

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Section: Discussionsupporting

confidence: 53%

Section: Discussionmentioning

confidence: 97%

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The Enzymic Synthesis of Dihydropteroate and Dihydrofolate by Plasmodium berghei

Ferone

1973

The Journal of Protozoology

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“…1 are catalyzed by enzymes of the tetrahydrofolate pathway and all were documented in the literature (4,6,(25)(26)(27)(28)(29)(30). The details of removal of the triphosphate motif under physiological conditions in the biosynthetic pathway (Fig.…”

Section: Resultsmentioning

confidence: 99%

Biosynthesis of Tetrahydrofolate

Illarionova¹,

Eisenreich²,

Fischer

et al. 2002

Journal of Biological Chemistry

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7,8-Dihydroneopterin aldolase catalyzes the formation of the tetrahydrofolate precursor, 6-hydroxymethyl-7,8-dihydropterin, and is a potential target for antimicrobial and anti-parasite chemotherapy. The last step of the enzyme-catalyzed reaction is believed to involve the protonation of an enol type intermediate. In order to study the stereochemical course of that reaction step, [1,2,3,6,7-13 C 5 ]dihydroneopterin was treated with aldolase in deuterated buffer. The resulting, partially deuterated [6␣,6,7-13 C 3 ]6-hydroxymethyl-7,8-dihydropterin was converted to partially deuterated 6-(R)-[6,7,9,11-13 C 4 ]5,10-methylenetetrahydropteroate by a sequence of three enzyme-catalyzed reactions followed by treatment with [13 C]formaldehyde. The product was analyzed by multinuclear NMR spectroscopy. The data show that the carbinol group of enzymatically formed 6-hydroxymethyl-dihydropterin contained 2 H predominantly in the pro-S position.Tetrahydrofolate and its derivatives are essential cofactors of one-carbon metabolism. Although plants and many microorganisms obtain folate coenzymes by de novo synthesis, vertebrates are absolutely dependent on nutritional sources (1). Insufficient supply of the vitamin is conducive to anemia in adults and to neural tube malformation in human embryos (2).The biosynthesis of tetrahydrofolate has been studied in some detail (for review see Ref.3). The first committed step catalyzed by GTP cyclohydrolase I converts GTP into dihydroneopterin triphosphate (1, Fig. 1). The triphosphate motif is removed by a previously unknown process, and the resulting 7,8-dihydro-D-neopterin (2) is converted to 6-hydroxymethyldihydropterin (3) by dihydroneopterin aldolase (FolB) (4). The consecutive action of FolK, FolP, FolC, and FolA enzymes finally affords 6-(S)-tetrahydrofolate via the intermediates 4 -6.The folate biosynthetic pathway is a well established drug target for antimicrobial as well as antiparasite therapy. Sulfonamides, the first synthetic antimicrobial and antiparasitic drugs with a broad action spectrum, act via inhibition of dihydropteroate synthase, the penultimate enzyme of the dihydrofolate biosynthetic pathway (Fig. 1, step E) (5-7), and trimethoprim acts against a variety of bacterial pathogens via inhibition of dihydrofolate reductase (Fig. 1, step G) (8 -10).The rapid development of microbial resistance against all antibiotics in current use has generated an urgent requirement for novel anti-infective agents. Because the folate pathway is already a well established drug target, it appears worthwhile to explore other folate biosynthetic enzymes besides dihydropteroate synthase and dihydrofolate reductase. This paper describes studies on the mechanism of dihydroneopterin aldolase. C 5 ]GTP was prepared as described (11). All other reagents used were of the highest purity available. A Nucleosil C18 HPLC 1 column (4 ϫ 250 mm) was from Schambeck, Bad Honnef, Germany. Superdex 75, Superdex 200, Q-Sepharose Fast Flow, and DEAE-Sepharose Fast Flow were purchased from Amersham Bioscienc...

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“…While we have demonstrated that AbgT imports PABA-GLU, and PGH cleaves it, the physiological significance of this is not clear. PABA-GLU is not a physiologically relevant substrate for the biosynthetic pathway as the kinetics of dihydropteroate synthase are not favorable [29]. It is possible, however, that this operon participates as a recycling or salvage pathway for p -aminobenzoate (PABA) which is an intermediate in biosynthesis of folate.…”

Section: Discussionmentioning

confidence: 99%

Comparison of Substrate Specificity of <em>Escherichia Coli</em> p-Aminobenzoyl-Glutamate Hy-drolase with <em>Pseudomonas</em> Carboxypeptidase G

Larimer¹,

Slavnic²,

Pitstick³

et al. 2014

AER

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Reduced folic acid derivatives support biosynthesis of DNA, RNA and amino acids in bacteria as well as in eukaryotes, including humans. While the genes and steps for bacterial folic acid synthesis are known, those associated with folic acid catabolism are not well understood. A folate catabolite found in both humans and bacteria is p-aminobenzoyl-glutamate (PABA-GLU). The enzyme p-aminobenzoyl-glutamate hydrolase (PGH) breaks down PABA-GLU and is part of an apparent operon, the abg region, in E. coli. The subunits of PGH possess sequence and catalytic similarities to carboxypeptidase enzymes from Pseudomonas species. A comparison of the subunit sequences and activity of PGH, relative to carboxypeptidase enzymes, may lead to a better understanding of bacterial physiology and pathway evolution. We first compared the amino acid sequences of AbgA, AbgB and carboxypeptidase G2 from Pseudomonas sp. RS-16, which has been crystallized. Then we compared the enzyme activities of E. coli PGH and commercially available Pseudomonas carboxypeptidase G using spectrophotometric assays measuring cleavage of PABA-GLU, folate, aminopterin, methotrexate, 5-formyltetrahydrofolate, and 5-methyltetrahydrofolate. The Km and Vmax values for the folate and anti-folate substrates of PGH could not be determined, because the instrument reached its limit before the enzyme was saturated. Therefore, activity of PGH was compared to the activity of CPG, or normalized to PABA-GLU (nmole/min/µg). Relative to its activity with 10 µM PABA-GLU (100%), PGH cleaved glutamate from methotrexate (48%), aminopterin (45%) and folate (9%). Reduced folates leucovorin (5-formyltetrahydrofolate) and 5-methyltetrahydrofolate were not cleaved by PGH. Our data suggest that E. coli PGH is specific for PABA-GLU as its activity with natural folates (folate, 5-methyltetrahydrofolate, and leucovorin) was very poor. It does, however, have some ability to cleave anti-folates which may have clinical applications in treatment of chemotherapy overdose.

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A comparison of the effectiveness with which p-aminobenzoic acid and p-aminobenzoylglutamic acid are used as substrate by dihydropteroate synthetase from Escherichia coli

Cited by 12 publications

References 12 publications

The Enzymic Synthesis of Dihydropteroate and Dihydrofolate by Plasmodium berghei

The Enzymic Synthesis of Dihydropteroate and Dihydrofolate by Plasmodium berghei

Biosynthesis of Tetrahydrofolate

Comparison of Substrate Specificity of <em>Escherichia Coli</em> p-Aminobenzoyl-Glutamate Hy-drolase with <em>Pseudomonas</em> Carboxypeptidase G

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A comparison of the effectiveness with which p-aminobenzoic acid and p-aminobenzoylglutamic acid are used as substrate by dihydropteroate synthetase from Escherichia coli

Cited by 12 publications

References 12 publications

The Enzymic Synthesis of Dihydropteroate and Dihydrofolate by Plasmodium berghei

The Enzymic Synthesis of Dihydropteroate and Dihydrofolate by Plasmodium berghei

Biosynthesis of Tetrahydrofolate

Comparison of Substrate Specificity of &lt;em&gt;Escherichia Coli&lt;/em&gt; p-Aminobenzoyl-Glutamate Hy-drolase with &lt;em&gt;Pseudomonas&lt;/em&gt; Carboxypeptidase G

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Comparison of Substrate Specificity of <em>Escherichia Coli</em> p-Aminobenzoyl-Glutamate Hy-drolase with <em>Pseudomonas</em> Carboxypeptidase G