In Vitro Molybdenum Ligation to Molybdopterin Using Purified Components

Jason D Nichols,K.V Rajagopalan

doi:10.1074/jbc.m413783200

Abstract

We have previously shown that Escherichia coli MoeA and MogA are required in vivo for the final step of molybdenum cofactor biosynthesis, the addition of the molybdenum atom to the dithiolene of molybdopterin. MoeA was also shown to facilitate the addition of molybdenum in an assay using crude extracts from E. coli moeA(-) cells. The experiments detailed in this report utilized an in vitro assay for MoeA-mediated molybdenum ligation to de novo synthesized molybdopterin using only purified components and monitoring the reconstitution of human aposulfite oxidase. In this assay, maximum activation was achieved by delaying the addition of aposulfite oxidase to allow for adequate molybdenum coordination to occur. Tungsten, which substitutes for molybdenum in hyperthermophilic organisms, could also be ligated to molybdopterin using this system, though not as efficiently as molybdenum. Addition of thiol compounds to the assay inhibited activity. Addition of MogA also inhibited the reaction. However, in the presence of ATP and magnesium, addition of MogA to the assay increased the level of aposulfite oxidase reconstitution beyond that observed with MoeA alone. This effect was not observed in the absence of MoeA. The results presented here demonstrate that MoeA is responsible for mediating molybdenum ligation to molybdopterin, whereas MogA stimulates this activity in an ATP-dependent manner.

Highlights

In all molybdenum-containing enzymes, with the exception of nitrogenase, the molybdenum cofactor (Moco)1 consists of a mononuclear Mo atom coordinated to the cis-dithiolene moiety of molybdopterin (MPT)
In Vitro Mo Ligation Using Purified Components—We have previously demonstrated that purified E. coli MoeA is able to reconstitute sulfite oxidase (SO) activity in a moeAϪ cell lysate in the presence of Na2MoO4 (9)
To better examine the process of Mo ligation, a fully defined assay for this step of Moco biosynthesis was developed utilizing purified E. coli proteins and precursor Z to make Moco, the formation of which was monitored by the activation of MPT-free recombinant human SO

Summary

Introduction

In all molybdenum-containing enzymes, with the exception of nitrogenase, the molybdenum cofactor (Moco) consists of a mononuclear Mo atom coordinated to the cis-dithiolene moiety of molybdopterin (MPT). The moeAϪ crude extract assay demonstrated that MoeA can facilitate Mo incorporation into MPT-containing SO, it did not define the mechanism of Mo ligation to de novo formed MPT. The in vitro synthesis of MPT-guanine dinucleotide using only MobA, Mg2ϩ, GTP, and Moco from denatured SO is quantitated by monitoring the activation of Rhodobacter sphaeroides apo-DMSO reductase (12), whereas de novo synthesized MPT is quantitated by the reconstitution of aposulfite oxidase (apoSO) or by conversion to the stable, fluorescent derivative Form A (6, 13). The results presented in this report delineate the development and optimization of a fully defined in vitro assay for MoeA-mediated Mo ligation to de novo synthesized MPT. The assay contains only purified components (activated MPT synthase, precursor Z, molybdate, MoeA, and apoSO) and monitors the activation of MPT-free human apoSO. MoeA was required for this stimulatory effect as apoSO reconstitution was not observed in its absence

Methods

Results

Conclusion