Involvement of the Molybdenum Cofactor Biosynthetic Machinery in the Maturation of the Escherichia coli Nitrate Reductase A

Alexandra Vergnes,Kamila Gouffi-Belhabich,Francis Blasco,Gérard Giordano,Axel Magalon

doi:10.1074/jbc.m407087200

Abstract

The maturation of Escherichia coli nitrate reductase A requires the incorporation of the Mo-(bis-MGD) cofactor to the apoprotein. For this process, the NarJ chaperone is strictly required. We report the first description of protein interactions between molybdenum cofactor biosynthetic proteins (MogA, MoeA, MobA, and MobB) and the aponitrate reductase (NarG) using a bacterial two-hybrid approach. Two conditions have to be satisfied to allow the visualization of the interactions, (i) the presence of an active and mature molybdenum cofactor and (ii) the presence of the NarJ chaperone and of the NarG structural partner subunit, NarH. Formation of tungsten-substituted cofactor prevents the interaction between NarG and the four biosynthetic proteins. Our results suggested that the final stages of molybdenum cofactor biosynthesis occur on a complex made up by MogA, MoeA, MobA, and MobB, which is also in charge with the delivery of the mature cofactor onto the aponitrate reductase A in a NarJ-assisted process.

Highlights

Molybdenum plays a critical role in the biogeochemistry of nitrogen and sulfur and, as such, has been found to be essential in most mammals as well as in plants
Interaction between Several Mo Proteins and NarG Requires Active molybdenum cofactor (Moco)—To evaluate whether the observed in vivo interaction network among Mo proteins involved in the final stages of Moco biosynthesis [19] might be involved in its incorporation within resident apomolybdoenzymes, interactions between these Mo proteins and the aponitrate reductase chosen as a model were tested by a bacterial two-hybrid approach [24]
How do the interactions among the Mo proteins involved in the final stages of Moco biosynthesis contribute to its subsequent incorporation into various apomolybdoenzymes? In this report, using a bacterial two-hybrid approach we have demonstrated the physical interaction of an apomolybdoenzyme exemplified by NarG, the Moco-containing subunit of the E. coli nitrate reductase A and four distinct Mo proteins (MogA, MoeA, MobA, and MobB)

Summary

TABLE I Bacterial strains and plasmids used in this study

Strain or plasmid DH5␣ BTH101 BTH101moa BTH101mod BTH101mob BTH101mobB BTH101narA,narZ BTH101narZ,narJ supE44, ⌬lacU169 (␾80 lacZ⌬M15), hsdR17, recA, endA1, gyrA96, thi-1, relA1 FЈ, cya-99, araD139, galE15, galK16, rpsL1(StrR), hsdR2, mcrA1, mcrB1 BTH101 moa254::Tn10, StrR, TcR. BTH101 ⌬(narЈU-narZЈ) ␻, ⌬nar25(narG-narH), SpcR, KmR, StrR BTH101 ⌬(narЈU-narZЈ) ␻, ⌬narJ, SpcR, StrR. Two-hybrid vectors pT18-Zip pT25-Zip pT18-MoeA pT18-MogA pT18-MobA pT18-MobB pT25-MoeA pT25-MogA pT25-MobA pT25-MobB pT25-NarG pT25-NarGH pT25-NarGJ pT25-NarGHJ pT18, Leucine zipper fused to T18 fragment (225–399 amino acids of CyaAa) pT25, Leucine zipper fused to T25 fragment (1–224 amino acids of CyaAa) pT18, MoeA-T18 fusion protein, AmpR pT18, MogA-T18 fusion protein, AmpR pT18, MobA-T18 fusion protein, AmpR pT18, MobB-T18 fusion protein, AmpR pT25, T25-MoeA fusion protein, CmR pT25, T25-MogA fusion protein, CmR pT25, T25-MobA fusion protein, CmR pT25, T25-MobB fusion protein, CmR pT25, T25-NarG fusion protein, CmR pT25, T25-NarG fusion protein, NarH CmR pT25, T25-NarG fusion protein, NarJ CmR pT25, T25-NarG fusion protein, NarHJ CmR a E. coli adenylate cyclase protein

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION