Characterization of a periplasmic nitrate reductase in complex with its biosynthetic chaperone

Abstract

Escherichia coli is a Gram‐negative bacterium that can use nitrate during anaerobic respiration. The catalytic subunit of the periplasmic nitrate reductase NapA contains two types of redox cofactor and is exported across the cytoplasmic membrane by the twin‐arginine protein transport pathway. NapD is a small cytoplasmic protein that is essential for the activity of the periplasmic nitrate reductase and binds tightly to the twin‐arginine signal peptide of NapA. Here we show, using spin labelling and EPR, that the isolated twin‐arginine signal peptide of NapA is structured in its unbound form and undergoes a small but significant conformational change upon interaction with NapD. In addition, a complex comprising the full‐length NapA protein and NapD could be isolated by engineering an affinity tag onto NapD only. Analytical ultracentrifugation demonstrated that the two proteins in the NapDA complex were present in a 1 : 1 molar ratio, and small angle X‐ray scattering analysis of the complex indicated that NapA was at least partially folded when bound by its NapD partner. A NapDA complex could not be isolated in the absence of the NapA Tat signal peptide. Taken together, this work indicates that the NapD chaperone binds primarily at the NapA signal peptide in this system and points towards a role for NapD in the insertion of the molybdenum cofactor.Structured digital abstract NapD and NapA bind by x ray scattering (View interaction)NapA and NapD physically interact by molecular sieving (View interaction)NapA and NapD bind by electron paramagnetic resonance (View interaction)

Cross-Species Functional Conservation and Possible Origin of the N-Terminal Specificity Domain of Mitochondrial Presequences

“…These biochemical results are consistent with the GFP patterns in the protoplasts (Figures 2B, C). To corroborate this finding, we evaluated another TAT signal sequence of NapA (nitrate reductase) (Dow et al, 2014). The N-terminal 34 amino acids of the FA presequence were replaced with NapA[1-34] to generate NapA[1-34]/FA (Supplementary Figure S1A).…”

A Post-Genomic View of the Ecophysiology, Catabolism and Biotechnological Relevance of Sulphate-Reducing Prokaryotes

“…NapA contains one molybdopterin cofactor and one [4Fe-4S] cluster (González, Correia, Moura, Brondino, & Moura, 2006;Romão, Dias, & Moura, 2001). In D. desulfuricans, the nap operon includes the napCMADGH genes (Marietou, Richardson, Cole, & Mohan, 2005), where NapGH are membrane-associated ironsulphur proteins that form a quinol dehydrogenase module (Brondijk, Fiegen, Richardson, & Cole, 2002;Kern & Simon, 2008); NapC is a membrane-associated tetrahaem cytochrome of the NapC/NrfH family, which acts as a quinol dehydrogenase (Rodrigues, Pereira, & Archer, 2011;Simon, 2002); NapD is a cytoplasmic maturation protein that may be involved in the insertion of the molybdenum cofactor into NapA (Dow et al, 2014); and NapM is a tetrahaem c-type cytochrome that is the probable direct electron donor to NapA, a role played by NapB in other bacteria (Marietou et al, 2005). The exact order of the electron transfer chain between the menaquinol and nitrate has not been established, nor if the NapC and NapGH systems are both essential.…”

Ferric Citrate Regulator FecR Is Translocated across the Bacterial Inner Membrane via a Unique Twin-Arginine Transport-Dependent Mechanism

“…Interestingly, some complex Tat substrates have signal peptides that contain greatly extended n-regions prior to the twin-arginine motif (39). Such extensions are almost invariably found on substrates that bind redox cofactors and/or partner proteins prior to export, and they appear to serve as binding sites for dedicated chaperones that co-ordinate folding and assembly (40)(41)(42)(43)(44). FecR is distinct from these Tat substrates since it does not contain any redox cofactor, and its signal sequence n-region is considerably longer than other Tat signal peptide n-regions.…”