The mob locus of Escherichia coli K12 required for molybdenum cofactor biosynthesis is expressed at very low levels

Abstract

The mob locus of Escherichia coli encodes functions which catalyse the synthesis of active molybdenum cofactor, molybdopterin guanine dinucleotide, from molybdopterin and GTP. Reporter translational lac fusion mutations in the mobA gene have been constructed using IplacMu9 mutagenesis. The mob locus is expressed at very low levels under both aerobic and anaerobic growth conditions. Neither additions to the growth media (nitrate, tungstate or molybdate) nor secondary mutations at the moa, mob, mod, moe or mog loci affected the level of expression. Two transcription initiation sites and their associated promoter regions have been identified upstream of mobA. Both of the promoter regions show a poor match to the -35 and -10 consensus sequences for a70 promoters. A 2.2 kb chromosomal DNA fragment which complemented all available mob mutants has been sequenced. Two ORFs were identified, arranged as a single transcription unit. The encoded polypeptides have predicted molecular masses of 21 642 Da and 19362 Da, respectively. The DNA has been subcloned into a T7 overexpression system and the predicted products identified. The mobA gene encodes protein FA, which has been purified to homogeneity and brings about the activation of inactive molybdoenzymes in cell extracts of mob mutants. The mobB gene encodes a polypeptide with a putative nucleotide binding site. All available mob mutations which have been selected for by their ability to grow anaerobically in the presence of chlorate are located in the mobA gene.

Insight into the Role of Escherichia coli MobB in Molybdenum Cofactor Biosynthesis Based on the High Resolution Crystal Structure

“…The final step of this biosynthetic pathway requires the mob locus, which encodes functions that catalyze the synthesis of MGD from MPT and a GMP donor, most probably GTP (8). The mob locus in E. coli contains two genes, mobA and mobB, arranged as a single transcription unit (8,9). MobA is a well characterized monomeric enzyme capable of the synthesis of MGD from MPT and GMP, and it has been shown to activate the molybdoenzyme nitrate reductase (10).…”

“…It has been shown that the moaA and moaC gene products are required for the biosynthesis of precursor Z from the guanine nucleotide (3); that MPT is synthesized from precursor Z by the gene products of moaD, moaE, and moeB (4); that moeA and mogA are necessary for the incorporation of molybdenum (5,6); and that modABC are required for the acquisition and transport of molybdenum into the cell (7). The final step of this biosynthetic pathway requires the mob locus, which encodes functions that catalyze the synthesis of MGD from MPT and a GMP donor, most probably GTP (8). The mob locus in E. coli contains two genes, mobA and mobB, arranged as a single transcription unit (8,9).…”

The Product of the Molybdenum Cofactor Gene mobB of Escherichia Coli is a GTP‐Binding Protein

“…We have shown previously that the expression of the mob locus is low and constitutive, with the mob gene products failing to be significantly overexpressed even when cloned into a transcription overexpression vector (Iobbi-Nivol et al, 1995). DNA sequence analysis indicated that this may be the result of poor translational initiation.…”

“…A computer-assisted analysis of the MobB protein sequence revealed the motifs typical of a guanine-nucleotide-binding protein (Iobbi-Nivol et al, 1995). The guanine-nucleotide-binding properties of MobB were investigated by equilibrium dialysis using [3H]GTP as the ligand.…”

“…The mob locus contain two genes, arranged as a single transcription unit (Plunkett 111 et al, 1993;Iobbi-Nivol et al, 1995). Protein FA, the product of mobA, has previously been purified to homogeneity in small quantities and shown to bring about the activation of nitrate reductase in crude extracts of mob mutants (Palmer et al, 1994).…”