In VivoInteractions between Gene Products Involved in the Final Stages of Molybdenum Cofactor Biosynthesis inEscherichia coli

Axel Magalon,Chantal Frixon,Jeanine Pommier,Gérard Giordano,Francis Blasco

doi:10.1074/jbc.m205806200

Abstract

The final stages of bacterial molybdenum cofactor (Moco) biosynthesis correspond to molybdenum chelation and nucleotide attachment onto an unique and ubiquitous structure, the molybdopterin. Using a bacterial two-hybrid approach, here we report on the in vivo interactions between MogA, MoeA, MobA, and MobB implicated in several distinct although linked steps in Escherichia coli. Numerous interactions among these proteins have been identified. Somewhat surprisingly, MobB, a GTPase with a yet unclear function, interacts with MogA, MoeA, and MobA. Probing the effects of various mo. mutations on the interaction map allowed us (i) to distinguish Moco-sensitive interactants from insensitive ones involving MobB and (ii) to demonstrate that molybdopterin is a key molecule triggering or facilitating MogA-MoeA and MoeA-MobA interactions. These results suggest that, in vivo, molybdenum cofactor biosynthesis occurs on protein complexes rather than by the separate action of molybdenum cofactor biosynthetic proteins.

Highlights

Molybdenum is an essential trace element for most living systems including microorganisms, plants, and animals
Probing the effects of various mo. mutations on the interaction map allowed us (i) to distinguish Moco-sensitive interactants from insensitive ones involving MobB and (ii) to demonstrate that molybdopterin is a key molecule triggering or facilitating MogA-MoeA and MoeA-MobA interactions. These results suggest that, in vivo, molybdenum cofactor biosynthesis occurs on protein complexes rather than by the separate action of molybdenum cofactor biosynthetic proteins
With the exception of nitrogenase, molybdenum is incorporated into proteins as the molybdenum cofactor (Moco),1 an ubiquitous basic structure which contains a mononuclear Mo atom coordinated to an organic cofactor named molybdopterin (MPT) [2]

Summary

The abbreviations used are

Molybdenum cofactor; MPT, molybdopterin; MGD, molybdopterin guanine dinucleotide. Molybdoenzymes, most bacterial enzymes require a modification of this basic structure to be functionally active This modification involves attachment of a nucleotide moiety, GMP, AMP, IMP, or CMP, onto the terminal phosphate group of the MPT side chain (reviewed in Rajagopalan and Johnson [2]). Similar studies performed on MGDenzymes such as Me2SO reductase from Rhodobacter sphaeroides have shown that both MobB and a chaperone protein are not absolutely required for MGD insertion [15]. Such observations have led us to consider alternative approaches to decipher the functions played by the mo. A comprehensive model is presented for the protein interaction network existing during the course of Moco biosynthesis in E. coli

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION