In vitro system for molybdopterin biosynthesis.
A high-Mr fraction present in chl+ and chlA1 strains of Escherichia coli synthesizes molybdopterin (MPT) from the low-Mr fraction of several MPT-deficient mutants. Using this in vitro complementation as an assay, we have partially characterized the high-Mr fraction as a protein, termed MPT converting factor, of Mr 45,000, distinguishable from the Mo cofactor carrier protein of similar Mr by its absolute requirement for the low-Mr fraction of a non-chlA1 mutant in the nit-1 reconstitution assay. MPT converting factor was rapidly inactivated in the absence of a reduced sulfhydryl compound. Anaerobic incubation of MPT converting factor with trypsin destroyed its activity. High-performance liquid chromatographic analysis of alkaline KMnO4 oxidation products demonstrated that the factor did not contain any bound pterin. Since mutants lacking MPT converting factor are not auxotrophs for folate or riboflavin, the factor appears to be distinct from known pteridine biosynthetic enzymes in E. coli. We have partially purified and characterized the low-Mr fractions as probable MPT precursors. Several distinct precursors were separable by high-performance liquid chromatography. Like MPT activity, precursor activity was oxygen sensitive. Precursor activity was not correlated with levels of L-threo-neopterin, a major pterin of unknown function in E. coli. Precursor activity was correlated with levels of a new 6-alkylpterin, compound Z, produced by acidic iodine oxidation. Compound Z has the properties expected of an oxidized MPT precursor.
D002851:Chromatography, High Pressure Liquid; D003067:Coenzymes; D004926:Escherichia coli / Q000201:enzymology; D066298:In Vitro Techniques; D008667:Metalloproteins; D008970:Molecular Weight; D008982:Molybdenum / Q000096:biosynthesis*; D000090006:Molybdenum Cofactors; D010100:Oxygen; D011498:Protein Precursors / Q000378:metabolism; D011621:Pteridines / Q000096:biosynthesis*; D011622:Pterins / Q000378:metabolism
D003067:Coenzymes; D008667:Metalloproteins; D008982:Molybdenum; D000090006:Molybdenum Cofactors; D010100:Oxygen; D011498:Protein Precursors; D011621:Pteridines; D011622:Pterins; C022261:molybdopterin