Two proteins encoded at the chlA locus constitute the converting factor of Escherichia coli chlA1

Abstract

Molybdopterin (MPT) is not produced by the Escherichia coli mutants chlA1, chlM, or chlN or by the Neurospora crassa mutant nit-1. Extracts of E. coli chlA1 contain an activity, the converting factor, which is functionally defined by its ability to convert a low-molecular-weight precursor present in crude extracts of N. crassa nit-1 into molybdopterin in vitro. In this study, it has been shown that the converting factor consists of two associative proteins (10 and 25 kilodaltons [kDa]) which can be separated by using either anion-exchange or gel filtration chromatography. Neither protein is able to complement extracts of nit-1 by itself. Analysis of chlA Mu insertion mutants has shown that the two proteins are distinct gene products encoded at the chlA locus. Twelve chlA Mu insertion strains which lacked converting factor activity were deficient in one or both of the proteins. Converting factor activity could be generated by mixing extracts from strains having the 25-kDa protein with those having the 10-kDa protein but not those lacking both proteins. Finally, it was shown that the chlM mutant lacks the 10-kDa protein while the chlN mutant, which contains both the 10- and 25-kDa proteins, lacks a function required to activate the 10-kDa protein.

The Sulfur Carrier Protein TusA Has a Pleiotropic Role in Escherichia coli That Also Affects Molybdenum Cofactor Biosynthesis*

“…Further, for the formation of MPT from cPMP, two sulfur atoms are incorporated into the C1Ј and C2Ј positions of cPMP, a reaction catalyzed by MPT synthase (20). The E. coli MPT synthase is a heterotetrameric enzyme consisting of two MoaE and two MoaD subunits (20,21). In its active form, MoaD contains a C-terminal thiocarboxylate group that acts as a direct sulfur donor for the synthesis of the dithiolene group of Moco (20,22,23).…”

In Vitro Molybdenum Ligation to Molybdopterin Using Purified Components

“…In Escherichia coli, biosynthesis of Moco begins with the conversion of GTP to a pterin intermediate termed precursor Z, catalyzed by the MoaA and MoaC proteins (1)(2)(3)(4). MPT is synthesized from precursor Z by the MoaD and MoaE proteins, which together compose the MPT synthase complex (5,6). The MoeB protein is involved in reactivating the MoaD subunit of MPT synthase between rounds of MPT synthesis (7,8).…”

Characterization of a molybdenum cofactor biosynthetic gene cluster in Rhodobacter capsulatus which is specific for the biogenesis of dimethylsulfoxide reductase

“…In vlzoeB and moaD mutants an MPT precursor which lacks sulfur atoms, known as precursor Z, has been identified (Pitterle & Rajagopalan, 1989;Pitterle et al, 1993). Precursor Z cannot be detected in a moaA mutant and this has led to the view that MoaA is involved in one of the earliest steps in MPT synthesis (Johnson & Rajagopalan, 1987a, b).…”

“…This protein is part of the converting factor, a complex involved in sulfur addition to precursor Z (Pitterle & Rajagopalan, 1989;Pitterle et al, 1993 …”

“…In oxomolybdenum enzymes from eukaryotes the organic component of Moco is a tricyclic pterin derivative known as molybdopterin (MPT) and its carbon side chain ends with a phosphate group. In almost all bacterial oxomolybdenum enzymes the M P T is in a dinucleotide form and this has been revealed in a spectacular fashion via the high resolution crystal structures of DMSO reductase (Schindelin et al, 1996;Schneider et al, 1996;McAlpine et al, 1997), aldehyde oxidoreductase (Romeo et al, 1995) and formate dehydrogenase (Boyington et moieties, since moeB and moaD mutants accumulate a sulfur-free molecule known as precursor Z , derived from the action of MoaABC (Pitterle & Rajagopalan, 1989). The enzymic step catalysed by MoaABC is still unclear.…”