The multifunctional folic acid sythesis fas gene of Pneumocystis carinii appears to encode dihydropteroate synthase and hydroxymethyldihydropterin pyrophosphokinase

Volpe, Filippo; Dyer, Martin; Scaife, John; Darby, G.; Stammers, D.K.; Delves, C. J.

doi:10.1016/0378-1119(92)90378-3

Cited by 71 publications

(64 citation statements)

References 13 publications

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“…They were based upon the known H,Pte synthase sequences of Bacillus subtilis [29], Streptococcus pneumoniae [30] and F! carinii [25]. A frag-…”

Section: Resultsmentioning

confidence: 99%

“…(z)-GGNGG-3', based on the conserved amino acid motif TPDSFFDGG, equivalent to residues 480-488 of Pneumocystis carinii H,Pte synthase [25], with an EcoRl site added (underlined) to facilitate cloning. C(z)AT(g)TC-3', based on the complementary strand sequence encoding the motif DIGGQSTRP, equivalent to residues 511-519, with an Xbal site added (underlined).…”

Section: Polymerase Chain Reactionsmentioning

confidence: 99%

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Sequence Variation of the Hydroxymethyldihydropterin Pyrophosphokinase: Dihydropteroate Synthase Gene in Lines of the Human Malaria Parasite, Plasmodium falciparum, with Differing Resistance to Sulfadoxine

Brooks

Wang

Read

et al. 1994

European Journal of Biochemistry

336

276

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Dihydropteroate synthase (H2Pte synthase) is the target of the sulfur‐based antimalarial drugs, which are frequently used in synergistic combination with inhibitors of dihydrofolate reductase (H2folate reductase) to combat chloroquine‐resistant malaria. We have isolated the H2Pte synthase coding sequence of the most pathogenic human parasite Plasmodium falciparum. It forms part of a longer coding sequence, located on chromosome 8, that also specifies 6‐hydroxymethyl‐7,8‐dihydropterin pyrophosphokinase (CH2OH‐H2pterinPP kinase) at its 5′ proximal end. This domain is unusually large, with two long insertions relative to other CH2OH‐H2pterinPP kinase molecules. To investigate a possible genetic basis for clinical resistance to sulfa drugs, we sequenced the complete H2Pte synthase domains from eleven isolates of P. falciparum with diverse geographical origins and levels of sulfadoxine resistance. Overall, point mutations in five positions were observed, affecting four codons. Parasite lines exhibiting high‐level resistance were found to carry either a double mutation, altering both Ser436 and Ala613, or a single mutation affecting Ala581. The mutations at positions 436 and 581 have the same location relative to each of two degenerate repeated amino acid motifs that are conserved across all other known H2Pte synthase molecules. The amino acid alteration at residue 613 is identically positioned relative to a different conserved motif. The fourth amino acid residue (437) affected by mutation, though adjacent to the apparently crucial residue 436, shows no obvious correlation with resistance. Although these mutations have no exact counterparts in any other organism, that at position 581 falls within a region of three amino acids where H2Pte synthase is modified in various ways in a number of sulfonamide‐resistant pathogenic bacteria. Copy‐number analysis indicated that there was no amplification of the H2Pte synthase domain in resistant parasite lines of P. falciparum, compared to sensitive lines.

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“…They were based upon the known H,Pte synthase sequences of Bacillus subtilis [29], Streptococcus pneumoniae [30] and F! carinii [25]. A frag-…”

Section: Resultsmentioning

confidence: 99%

Section: Polymerase Chain Reactionsmentioning

confidence: 99%

Sequence Variation of the Hydroxymethyldihydropterin Pyrophosphokinase: Dihydropteroate Synthase Gene in Lines of the Human Malaria Parasite, Plasmodium falciparum, with Differing Resistance to Sulfadoxine

Brooks

Wang

Read

et al. 1994

European Journal of Biochemistry

336

276

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“…The insertion in BT054 was also verified by size determinations : the mutated region was PCR amplified with primers NM6 (Ra/ dstro$ m et al, 1992) and NM10 (5h-GCAGGCATCGCACCGCAACG-3h) and the amplimer was run on a 8 % polyacrylamide gel, which could separate the mutated 110 bp fragment from the original 104 bp fragment. Mutated folP genes were cloned in pUC18 and pUC19 vectors (Yannisch-Perron et al, 1985) and transformed into DH5α by electroporation (Dower et al, 1988).…”

Section: Sulphonamide Resistance In Neisseria Meningitidismentioning

confidence: 99%

Additive effects of a two-amino-acid insertion and a single-amino-acid substitution in dihydropteroate synthase for the development of sulphonamide-resistant Neisseria meningitidis

Qvarnström

Swedberg

2000

Microbiology

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Sulphonamide resistance in some clinical isolates of Neisseria meningitidis is associated with an insertion in the chromosomal folP gene leading to the addition of two amino acids, serine and glycine, in the drug target enzyme dihydropteroate synthase (DHPS). Removal of the insertion resulted in a markedly higher K m for the substrate p-aminobenzoic acid and a markedly lower K m for 2-amino-4-hydroxy-6-(hydroxymethyl)-7,8-dihydropteridine pyrophosphate. In the same isolates an additional important difference, compared to wild-type enzymes, was found at amino acid position 68, which is a proline in most DHPS enzymes, but is serine in one and leucine in another clinical isolate of sulphonamide-resistant N. meningitidis. The alteration at position 68 was found to affect mainly the level of sulphonamide resistance and had only a minor effect on the K m for the substrates. Introduction of the serine-glycine dipeptide at position 194 and a proline to serine substitution at position 68 in DHPS from normal, susceptible N. meningitidis failed to produce a functional sulphonamide-resistant enzyme. The conclusion of this study is that it is not possibile to change a normal chromosomally encoded DHPS of N. meningitidis to a sulphonamide-resistant one simply by an insertion of serine and glycine as seen in clinical isolates. It is likely that the resistance gene found in clinical isolates has evolved in another bacterial species where a combination of other amino acid changes may have contributed to produce a functionally resistant enzyme. This new resistance gene may have then been introduced into N. meningitidis by natural transformation.

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“…There is a polypyrimidine tract near the 3' end of the intron and a sequence (YTRAT) that has been identified as a putative lariat forming motif in other P. carinii intervening sequences ) is present. The occurrence of small introns has been reported in other P. carinii genes (Edman et al, 1989b;Volpe et al, 1992).…”

Section: Sequencing Of the P Carinii Arom Locusmentioning

confidence: 60%

“…An example of this phenomenon has recently been discovered in P. carinii by Volpe et al (1992), who cloned the folic acid synthesis gene (fas) which has two identifiable domains, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase and dihydropteroate synthase. However, it was shown by Edman et al (1989 b) that the dihydrofolate reductase and thymidylate synthase genes of P. carinii were unfused, which differs from the protozoon arrangement but is in accord with the fungi.…”

Section: Multijimctional Enzymesmentioning

confidence: 99%

The cloning and characterization of the arom gene of Pneumocystis carinii

Banerji

Wakefield²,

Allen³

et al. 1993

Journal of General Microbiology

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The avom gene, encoding a single polypeptide that catalyses five consecutive steps of the pre-chorismate aromatic amino acid biosynthetic pathway, has been cloned from the opportunistic pathogen Pneumocystis cavinii. There is a single open reading frame of 4788 bp which includes an intron of 45 bp that does not introduce a stop codon into the sequence. Thus, the derived amino acid sequence consists of 1581 residues, which is highly homologous to all fungal AROM proteins studied to date. These data support the view that P. cavinii is a fungus and imply that its aromatic amino acid biosynthesis is conventionally organized.

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The multifunctional folic acid sythesis fas gene of Pneumocystis carinii appears to encode dihydropteroate synthase and hydroxymethyldihydropterin pyrophosphokinase

Cited by 71 publications

References 13 publications

Sequence Variation of the Hydroxymethyldihydropterin Pyrophosphokinase: Dihydropteroate Synthase Gene in Lines of the Human Malaria Parasite, Plasmodium falciparum, with Differing Resistance to Sulfadoxine

Sequence Variation of the Hydroxymethyldihydropterin Pyrophosphokinase: Dihydropteroate Synthase Gene in Lines of the Human Malaria Parasite, Plasmodium falciparum, with Differing Resistance to Sulfadoxine

Additive effects of a two-amino-acid insertion and a single-amino-acid substitution in dihydropteroate synthase for the development of sulphonamide-resistant Neisseria meningitidis

The cloning and characterization of the arom gene of Pneumocystis carinii

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