In vitro system for molybdopterin biosynthesis

Abstract

A high-Mr fraction present in chl+ and chLIA 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 characteirzed 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-chLIA mutant in the nit-i reconstitution assay. MPT converting factor was rapidly inactivated in the absence of a reduced sulfhydryl compound. Anaerobic incubation of MPT Previous experiments have shown that the pleiotropic mutants Escherichia coli chlAl (12) and Neurospora crassa nit-i (13) lack molybdopterin (MPT), the organic moiety of the molybdenum cofactor. It has also been demonstrated that a high-molecular-weight fraction in E. coli chlAl can generate authentic MPT when incubated with a lowmolecular-weight fraction from N. crassa nit-i (12). In this paper we present a partial characterization of the highmolecular-weight factor in chlAl extracts and discuss the nature of the compounds supplied by the nit-i lowmolecular-weight fraction. Our experiments were designed to test the hypothesis that the chlAl factor is an enzyme in the MPT biosynthetic pathway and that reconstitution of nit-i apo-nitrate reductase occurs by the action of the converting factor on MPT precursor(s) in nit-i to generate true MPT. MATERIALS AND METHODSMaterials and methods (including terminology) were as described in the accompanying paper (12), with the following additions.Chemicals. Protein molecular weight standards for sizeexclusion high-pressure liquid chromatography (HPLC) were from U.S. Biochemical Corp. N-Bromobimane was from Calbiochem. Neopterin was from B. Schirks. Matrex Red gel was from Amicon Corp. Preswollen microgranular DE-52 cellulose was from Whatman, Inc. All other biochemicals were from Sigma Chemical Co. All inorganic reagents were reagent grade. Pterin-6-carlboxylic-7-sulfonic acid was synthesized from deoxyurothione as described previously (10). Sulfite oxidase was purified from chicken liver and quantitated as described previously (11).Bacterial strains. All strains are described in the accompanying paper (12), except that MJ351 (chlAI) was constructed by P1 MJ345 (gal' chlAI) crossed with spontaneous * Corresponding author.2-deoxygalactose-resistant MJ17 (gal chl+) to yield the Gal' phenotype. HPLC. For size-exclusion HPLC, a Kratos TSK3000SW column was used with the equipment described in the accompanying paper (12). Crude extracts were filtered through a Gelman Metricel 0.2-,um filter before injection. Reverse-phase (C18) HPLC was as described in the accompanying paper (12).Enzyme assays. MPT converting factor activity is defined as the ability to reconstitute nitrate reductase in crude nit-] extract, but not in the G-25-excluded fraction of nit-i extract. MPT converting factor in chlAI extract was assayed by incubating the extract with nit-i crude extract in t...

Mechanistic and Mutational Studies of Escherichia coli Molybdopterin Synthase Clarify the Final Step of Molybdopterin Biosynthesis

“…Mutations in either MPT synthase subunit or in MoeB result in accumulation of precursor Z within the affected organism. This has been observed in bacteria (18), humans (19), and Neurospora (20), and such mutants can serve as experimental sources of precursor Z (21).…”

Thiocarboxylation of Molybdopterin Synthase Provides Evidence for the Mechanism of Dithiolene Formation in Metal-binding Pterins

“…The N. crassa mutant nit-1 is defective in the second step of Moco biosynthesis and accumulates precursor Z that can be converted to MPT by MPT synthase, which is exogenously added to the crude nit-1 protein extract (20). In the presence of 1 mM molybdate, the resulting MPT is non-catalytically converted to Moco that reconstitutes N. crassa apo-nitrate reductase (20,22).…”

“…In the second step, two sulfur atoms are incorporated into the precursor Z thereby forming MPT as structural moiety of the cofactor. In Escherichia coli, three gene products are essential for the synthesis of MPT from precursor Z. MoaD (small subunit) and MoaE (large subunit) form the MPT synthase (20) that stoichiometrically converts precursor Z to MPT (21,22). MPT synthase is activated (sulfurated) by MoeB (MPT synthase sulfurase).…”

Molybdenum uptake in Escherichia coli K12

“…The activity of these enzymes is pleiotropically lost in several types of chlorate-resistant mutants (Puig & Azoulay, 1967;Adhya et al, 1968;Venables & Guest, 1968;Casse, 1970;Dagert & Puig, 1976;Johnson & Rajagopalan, 1987a). These chZD mutants were the first to be suspected to have an altered metabolism of *Author for correspondence.…”