135-16-0Relevant articles and documents
Cloning and characterization of a novel, plasmid-encoded trimethoprim- resistant dihydrofolate reductase from Staphylococcus haemolyticus MUR313
Dale,Langen,Page,Then,Stuber
, p. 1920 - 1924 (1995)
In recent years resistance to the antibacterial agent trimethoprim (Tmp) has become more widespread, and several trimethoprim-resistant (Tmp(r)) dihydrofolate reductases (DHFRs) have been described from gram-negative bacteria. In staphylococci, only one Tmp(r) DHFR has been described, the type S1 DHFR, which is encoded by the dfrA gene found on transposon Tn4003. In order to investigate the coincidence of high-level Tmp resistance and the presence of dfrA, we analyzed the DNAs from various Tmp(r) staphylococci for the presence of dfrA sequences by PCR with primers specific for the thyE- dfrA genes from Tn4003. We found that 30 of 33 isolates highly resistant to Tmp (MICs, ≥512 μg/ml) contained dfrA sequences, whereas among the Tmp(r) (MICs, ≤256 μg/ml) and Tmp(r) isolates only the Staphylococcus epidermidis isolates (both Tmp(r) and Tmp(r)) seemed to contain the dfrA gene. Furthermore, we have cloned and characterized a novel, plasmid-encoded Tmp(r) DHFR from Staphylococcus haemolyticus MUR313. The dfrD gene of plasmid pABU17 is preceded by two putative Shine-Dalgarno sequences potentially allowing for the start of translation at two triplets separated by nine nucleotides. The predicted protein of 166 amino acids, designated S2DHFR, encoded by the longer open reading frame was overproduced in Escherichia coli, purified, and characterized. The molecular size of the recombinant S2DHFR was determined by ion spray mass spectrometry to be 19,821.2 ± 2 Da, which is in agreement with the theoretical value of 19,822 Da. In addition, the recombinant S2DHFR was shown to exhibit DHFR activity and to be highly resistant to Tmp.
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Blakley
, p. 231 (1960)
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Screening of inhibitors using enzymes entrapped in sol-gel-derived materials
Besanger, Travis R.,Chen, Yang,Deisingh, Anil K.,Hodgson, Richard,Jin, Wen,Mayer, Stanislas,Brook, Michael A.,Brennan, John D.
, p. 2382 - 2391 (2003)
In recent years, a number of new methods have been reported that make use of immobilized enzymes either on microarrays or in bioaffinity columns for high-throughput screening of compound libraries. A key question that arises in such methods is whether immobilization may alter the intrinsic catalytic and inhibition constants of the enzyme. Herein, we examine how immobilization within sol-gel-derived materials affects the catalytic constant (kcat), Michaelis constant (KM), and inhibition constant (KI) of the clinically relevant enzymes Factor Xa, dihydro-folate reductase, cyclooxygenase-2, and γ-glutamyl transpeptidase. These enzymes were encapsulated into solgel-derived glasses produced from either tetraethyl orthosilicate (TEOS) or the newly developed silica precursor diglyceryl silane (DGS). It was found that the catalytic efficiency and long-term stability of all enzymes were improved upon entrapment into DGS-derived materials relative to entrapment in TEOS-based glasses, likely owing to the liberation of the biocompatible reagent glycerol from DGS. The KM values of enzymes entrapped in DGS-derived materials were typically higher than those in solution, whereas upon entrapment, kcat values were generally lowered by a factor of 1.5-7 relative to the value in solution, indicating that substrate turnover was limited by partitioning effects or diffusion through the silica matrix. Nonetheless, the apparent KI value for the entrapped enzyme was in most cases within error of the value in solution, and even in the worst case, the values differed by no more than a factor of 3. The implications of these findings for high-throughput screening are discussed.
Trimethoprim resistance of dihydrofolate reductase variants from clinical isolates of Pneumocystis jirovecii
Queener,Cody,Pace,Torkelson,Gangjee
, p. 4990 - 4998 (2013)
Pneumocystis jirovecii is an opportunistic pathogen that causes serious pneumonia in immunosuppressed patients. Standard therapy and prophylaxis include trimethoprim (TMP)-sulfamethoxazole; trimethoprim in this combination targets dihydrofolate reductase (DHFR). Fourteen clinically observed variants of P. jirovecii DHFR were produced recombinantly to allow exploration of the causes of clinically observed failure of therapy and prophylaxis that includes trimethoprim. Six DHFR variants (S31F, F36C, L65P, A67V, V79I, and I158V) showed resistance to inhibition by trimethoprim, with Ki values for trimethoprim 4-fold to 100-fold higher than those for the wild-type P. jirovecii DHFR. An experimental antifolate with more conformational flexibility than trimethoprim showed strong activity against one trimethoprim-resistant variant. The two variants that were most resistant to trimethoprim (F36C and L65P) also had increased Km values for dihydrofolic acid (DHFA). The catalytic rate constant (kcat) was unchanged for most variant forms of P. jirovecii DHFR but was significantly lowered in F36C protein; one naturally occurring variant with two amino acid substitutions (S106P and E127G) showed a doubling of kcat, as well as a Km for NADPH half that of the wild type. The strongest resistance to trimethoprim occurred with amino acid changes in the binding pocket for DHFA or trimethoprim, and the strongest effect on binding of NADPH was linked to a mutation involved in binding the phosphate group of the cofactor. This study marks the first confirmation that naturally occurring mutations in the gene for DHFR from P. jirovecii produce variant forms of DHFR that are resistant to trimethoprim and may contribute to clinically observed failures of standard therapy or prophylaxis. Copyright
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O'Dell et al.
, p. 250,252 (1947)
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Aqueous diastereoselective hydrogenation of folic acid to tetrahydrofolic acid in the presence of water-soluble Rh and Ir diphosphine complexes
Pugin, Benoit,Groehn, Viola,Moser, Rudolf,Blaser, Hans-Ulrich
, p. 544 - 549 (2006)
Rhodium and iridium catalysts with chiral, water soluble diphosphine ligands, were used for the diastereoselective hydrogenation of folic acid disodium salt in water. Using a modified Rh/Josiphos type at 30 °C, l-tetrahydrofolic acid, a relevant pharmaceutical intermediate, was obtained with a selectivity of up to 49% de; at 70 °C turnover numbers of up to 2800 were achieved, albeit with lower selectivity. These results define the state of the art for this reaction.
Process for preparing L-5 - methyl tetrahydrofolinate
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Paragraph 0050-0051; 0055-0056; 0058-0059; 0061-0062, (2021/11/06)
The invention relates to a preparation process of L-5 - methyl tetrahydrofolic acid, which adopts asymmetric catalytic hydrogenation to convert folic acid into (6S) - tetrahydro folic acid, and has high folic acid conversion rate, and (6S) - tetrahydrofolic acid diastereomeric excess degree. By salt formation and crystallization, the (6S) - tetrahydrofolic acid intermediate with extremely high diastereomeric excess can be easily enriched, and thus the yield and purity of L-5 - methyltetrahydrofolinate finally obtained are high. The catalytic hydrogenation process is very mature in industrial application and convenient to operate. The subsequent methylation and salt formation steps are easy to implement, and the preparation process has high economic value and practical value under the condition that the yield and purity of the product are improved.