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

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 (Blasco, F., Dos Santos, J. P., Magalon, A., Frixon, C., Guigliarelli, B., Santini, C. L., and Giordano, G. (1998) Mol. Microbiol. 28, 435-447). 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.

Protein Crystallography Reveals a Role for the FS0 Cluster of Escherichia coli Nitrate Reductase A (NarGHI) in Enzyme Maturation

“…The estimated intracellular concentration of GDP is ϳ0.68 mM (51), a concentration that hypothetically could competitively inhibit Mo-bisPGD insertion. The final steps of cofactor insertion are orchestrated by the MobAB, MoeA, MogA, and NarJ proteins (52), and it is likely that these steps prevent inhibition by cytoplasmic GDP.…”

“…Other possible routes include the interaction between the carbonyl oxygen of NarG-Cys 53 and N-10 of the P-pterin. Finally, a conserved Asn (NarG-Asn 52 ) provides an interaction between its carboximide amine side chain and two dithiolene sulfurs, one from each pterin. Fig.…”

“…hydrogen bonds between the amide nitrogen of NarG-Asn 52 and dithiolene sulfurs of both pterins and a hydrogen bond between the carbonyl oxygen of NarG-Cys 53 and the N-10 secondary amine of the P-pterin. Finally, the carbonyl oxygen of the NarG-Gly 50 amide is ϳ3.4 Å from the N-7 of the purine bicycle of the guanine nucleotide attached to the Q-pterin.…”

Assembly pathway of a bacterial complex iron sulfur molybdoenzyme

“…TF and DnaK are promiscuous chaperones that assist in protein folding and have a combined substrate pool of over 1000 cellular proteins (48). Studies have demonstrated that TF and DnaK interact with Tat system substrates, and both chaperones have an apparently similar substrate specificity: a stretch of hydrophobic amino acid residues flanked on either end by positively charges residues (38,49,50).…”

“…As bisPGD is not stable in its free form, it is immediately bound by Mocobinding chaperones, which insert the cofactor specifically into its target enzymes. The system specific chaperone of Nitrate reductase A, NarJ, has been shown to interact with Moco biosynthesic machinery and to facilitate final complex assembly prior to Tat translocation (48,115,116). Moreover, TorD, induces a conformational change of apoTorA to allow competency for Moco insertion, as well as interaction with the MobA protein involved in bisMGD formation (49).…”