442634-22-2Relevant academic research and scientific papers
An enzymatic pathway for the biosynthesis of the formylhydroxyornithine required for rhodochelin iron coordination
Bosello, Mattia,Mielcarek, Andreas,Giessen, Tobias W.,Marahiel, Mohamed A.
experimental part, p. 3059 - 3066 (2012/07/14)
Rhodochelin, a mixed catecholate-hydroxamate type siderophore isolated from Rhodococcus jostii RHA1, holds two l-δ-N-formyl-δ-N- hydroxyornithine (l-fhOrn) moieties essential for proper iron coordination. Previously, bioinformatic and genetic analysis proposed rmo and rft as the genes required for the tailoring of the l-ornithine (l-Orn) precursor [Bosello, M. (2011) J. Am. Chem. Soc.133, 4587-4595]. In order to investigate if both Rmo and Rft constitute a pathway for l-fhOrn biosynthesis, the enzymes were heterologously produced and assayed in vitro. In the presence of molecular oxygen, NADPH and FAD, Rmo monooxygenase was able to convert l-Orn into l-δ-N-hydroxyornithine (l-hOrn). As confirmed in a coupled reaction assay, this hydroxylated intermediate serves as a substrate for the subsequent N 10-formyl-tetrahydrofolate-dependent (N10-fH4F) Rtf-catalyzed formylation reaction, establishing a route for the l-fhOrn biosynthesis, prior to its incorporation by the NRPS assembly line. It is of particular interest that a major improvement to this study has been reached with the use of an alternative approach to the chemoenzymatic FolD-dependent N 10-fH4F conversion, also rescuing the previously inactive CchA, the Rft-homologue in coelichelin assembly line [Buchenau, B. (2004) Arch. Microbiol.182, 313-325; Pohlmann, V. (2008) Org. Biomol. Chem.6, 1843-1848].
Purification and characterization of 5,10-Methenyltetrahydrofolate synthetase from chicken liver
Cho, Yong Kweon
experimental part, p. 567 - 572 (2011/01/12)
5,10-Methenyltetrahydrofolate synthetase from chicken liver was purified through 30-70% ammonium sulfate fractionation, Q Sepharose Fast Flow anion exchange and Source 15Phe hydrophobic interaction chromatography. Specific activities of cell extract, ammonium sulfate, Q Sepharose Fast Flow and Source 15Phe were 0.0085, 0.031, 0.80 and 1.27 U/mg, respectively. Purification fold activities of cell extract, ammonium sulfate, Q Sepharose Fast Flow and Source 15Phe were 1, 3.7, 94.1 and 149.4, respectively. HPLC gel permeation chromatography and SDS-polyacrylamide electrophoresis experiments indicated that the enzyme is a monomeric protein with a molecular weight of 22.8 kDa. Km for 5-methyl THF and Mg-ATP were 7.1 μM and 63 μM, respectively. Optimum temperature and pH were 30 °C and 6.0, respectively. The data for metal ion specificity and stoichiometry showed that the maximum activity was obtained with a 1:1. ratio of Mg2+. The ATP and Km values increased in the order of MgATP, MgCTP, MgUTP and MgGTP, and the maximum activities also decreased in the same order, indicating MgATP as the most efficient substrate. The enzyme was chemically modified only by tetranitrometane and 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide, indicating that tyrosine and carboxylate are present in the active site.
Stereochemistry of Reduction of the Vitamin Folic acid by Dihydrofolate Reductase
Charlton, Peter A.,Young, Douglas W.,Birdsall, Berry,Feeney, James,Roberts, Gordon C. K.
, p. 1349 - 1354 (2007/10/02)
Reduction of the vitamin folic acid (6) to the coenzyme 5,6,7,8-tetrahydrofolic acid (1) by the enzyme dihydrofolate reductase is shown to involve transfer of the 4-pro R hydrogen of NADPH to the same face at both C-6 and C-7 of the pteridine system (the re face at C-6 and the si face at C-7).The orientations of the pteridine system of folic acid (6) and of dihydrofolic acid (5) when bound to the enzyme are different from the orientation of the pteridine ring of the anti-cancer drug methotrexate (11) when bound to this enzyme.
