Janine Pommier

TorD, A Cytoplasmic Chaperone That Interacts with the Unfolded Trimethylamine N-Oxide Reductase Enzyme (TorA) in Escherichia coli

Reduction of trimethylamine N-oxide (TMAO) in Escherichia coli involves the terminal molybdoreductase TorA, located in the periplasm, and the membrane anchored c type cytochrome TorC. In this study, the role of the TorD protein, encoded by the third gene of torCADoperon, is investigated. Construction of a mutant, in which thetorD gene is interrupted, showed that the absence of TorD protein leads to a two times decrease of the final amount of TorA enzyme. However, specific activity and biochemical properties of TorA enzyme were similar to those of the enzyme produced in the wild type. Excess of TorD protein restores the normal level of TorA enzyme, and also, leads to the appearance of a new cytoplasmic form of TorA on SDS-polyacrylamide gel electrophoresis using gentle conditions. This probably indicates a new folding state of the cytoplasmic TorA protein when TorD is overexpressed. BIAcore techniques demonstrated direct specific interaction between the TorA and TorD proteins. This interaction was enhanced when TorA was previously unfolded by heating. Finally, as TorA is a molybdoenzyme, we demonstrated that TorD can interact with TorA before the molybdenum cofactor has been inserted. As TorD homologue encoding genes are found in various TMAO reductase loci, we propose that TorD is a chaperone protein specific for the TorA enzyme. It belongs to a family of TorD-like chaperones present in several bacteria, and, probably, involved in TMAO reductase folding.

Involvement of the narJ or narW gene product in the formation of active nitrate reductase in Escherichia coli.

Two membrane‐bound nitrate reductases, NRA and NRZ, exist in Escherichia coil. Both isoenzymes are composed of three structural subunits, α, β and γ encoded by narG/narZ, narH/narY and narl/narV, respectively. The genes are in transcription units which also contain a fourth gene encoding a polypeptide, δ, which is not part of the final enzyme. A strain which is devoid of, or does not express, the nar genes, was used to investigate the role of the δ and γ polypeptides in the formation and/or processing of the nitrate reductase. When only the α and γ polypeptides are produced, an (αβ) complex exists which is inactive and soluble. When the α, β and δ and polypeptides are produced, the (αβ) complex is active with artificial donors such as benzyl viologen but is soluble. When the α, β, and δ polypeptides are produced, the (αβ) complex is inactive but partially binds the membrane. It was concluded that the γ polypeptide is involved in the binding of the (αβ) complex to the membrane while the δ polypeptide is indispensable for the (αβ) nitrate reductase activity. The activation by the δ polypeptide does not seem to involve the insertion of the redox centres of the enzyme since the purified inactive (αβ) complex was shown to contain the four iron–sulphur centres and the molybdenum cofactor, which are normally present in the native purified enzyme. The extreme sensitivity of this inactive complex to thermal denaturation or tryptic treatment favours the idea that the δ polypeptide promotes the correct assembly of the α and β subunits. Although this corresponds to the definition of a chaperone protein this possibility has been rejected. In this study we have also demonstrated that the δ or γ polypeptide encoded by one nar operon can be substituted succesfully for by its respective counterpart from the other nar operon to give an active membrane bound heterologous nitrate reductase enzyme.

The inducible trimethylamine-N-oxide reductase of Escherichia coli K12: biochemical and immunological studies

The inducible trimethylamine-N-oxide reductase which migrates on non-denaturing polyacrylamide gels with an RF of 0.22, has been purified from the soluble fraction of wild-type E. coli K12. The molecular weight of the purified enzyme estimated by molecular-sieve chromatography is about 230,000. It is composed of two subunits of molecular weight 110,000. Antiserum specific for the enzyme has been produced. Gel filtration on Sephadex G-200 of the soluble fraction gave two peaks of trimethylamine-N-oxide reductase, one with an Mr of 230,000 and an RF of 0.22, and another with an Mr of 120,000 and an RF of 0.36. Since the anti-trimethylamine-N-oxide reductase serum recognises the two forms and shows a single subunit with an Mr of 110,000, we conclude that in E. coli there is a single inducible trimethylamine-N-oxide reductase which can exist as a dimer or a monomer. Other immunological studies with anti-trimethylamine-N-oxide reductase serum on crude extracts prepared from cells grown in the absence of inducer showed that the constitutive trimethylamine-N-oxide reductase was not recognised by the antiserum. The same analyses carried out on a tor mutant (defective in the structural gene of the inducible enzyme) confirmed without ambiguity that the constitutive enzyme is immunologically distinct from the inducible enzyme. In the same way, using the anti-trimethylamine-N-oxide reductase serum, rocket immunoelectrophoresis analyses were able to show that the inducible apoenzyme is not regulated by the fnr gene product and that molybdate does not seem necessary for the synthesis or stabilisation of this enzyme.

The inducible trimethylamine N-oxide reductase of Escherichia coli K12: its localization and inducers

We used an anti-trimethylamine-N-oxide reductase (EC 1.6.6.9) serum and different immunological techniques (Ouchterlony, rocket immunoelectrophoresis, immunoblotting) to show that dimethylsulphoxide (DMSO), tetrahydrothiophene 1-oxide (THTO) and pyridine N-oxide (PNO) were effective inducers of the inducible form of trimethylamine N-oxide reductase. We confirmed this genetically and biochemically using a strain in which phage MudII 1734 carrying lacZ was inserted into torA, the structural gene for inducible trimethylamine-N-oxide reductase. By subcellular fractionation and quantitation with rocket immunoelectrophoresis, we showed that the enzyme was principally localized in the periplasmic fraction. Constitutive trimethylamine-N-oxide reductase was localized in the membrane fraction and, like the inducible enzyme showed a broad specificity with respect to various compounds such as DMSO, THTO and PNO. Apart from their immunological properties, the two enzymes could be clearly differentiated by their temperature stability.

NarJ chaperone binds on two distinct sites of the aponitrate reductase of Escherichia coli to coordinate molybdenum cofactor insertion and assembly.

Understanding when and how metal cofactor insertion occurs into a multisubunit metalloenzyme is of fundamental importance. Molybdenum cofactor insertion is a tightly controlled process that involves specific interactions between the proteins that promote cofactor delivery, enzyme-specific chaperones, and the apoenzyme. In the assembly pathway of the multisubunit molybdoenzyme, membrane-bound nitrate reductase A from Escherichia coli, a NarJ-assisted molybdenum cofactor (Moco) insertion step, must precede membrane anchoring of the apoenzyme. Here, we have shown that the NarJ chaperone interacts at two distinct binding sites of the apoenzyme, one interfering with its membrane anchoring and another one being involved in molybdenum cofactor insertion. The presence of the two NarJ-binding sites within NarG is required to ensure productive formation of active nitrate reductase. Our findings supported the view that enzyme-specific chaperones play a central role in the biogenesis of multisubunit molybdoenzymes by coordinating subunits assembly and molybdenum cofactor insertion.

Cardiolipin-based respiratory complex activation in bacteria

Anionic lipids play a variety of key roles in membrane function, including functional and structural effects on respiratory complexes. However, little is known about the molecular basis of these lipid–protein interactions. In this study, NarGHI, an anaerobic respiratory complex of Escherichia coli, has been used to investigate the relations in between membrane-bound proteins with phospholipids. Activity of the NarGHI complex is enhanced by anionic phospholipids both in vivo and in vitro. The anionic cardiolipin tightly associates with the NarGHI complex and is the most effective phospholipid to restore functionality of a nearly inactive detergent-solubilized enzyme complex. A specific cardiolipin-binding site is identified on the basis of the available X-ray diffraction data and of site-directed mutagenesis experiment. One acyl chain of cardiolipin is in close proximity to the heme bD center and is responsible for structural adjustments of bD and of the adjacent quinol substrate binding site. Finally, cardiolipin binding tunes the interaction with the quinol substrate. Together, our results provide a molecular basis for the activation of a bacterial respiratory complex by cardiolipin.

High substrate specificity and induction characteristics of trimethylamine-N-oxide reductase of Escherichia coli

Using a wide variety of N- and S-oxide compounds we have shown by kinetic analysis that only two N-oxides, trimethylamine-N-oxide and 4-methylmorpholine-N-oxide, can be considered good substrates for trimethylamine-N-oxide (TMAO) reductase on the basis of their kcat/Km ratio. This result demonstrates that TMAO reductase possesses a high substrate specificity. Induction of the torCAD operon using the same S- and N-oxide compounds was also analyzed. We demonstrate that there is no correlation between the ability for a compound to be reduced by TMAO reductase and to induce TMAO reductase synthesis.

Formation of active heterologous nitrate reductases between nitrate reductases A and Z of Escherichia coli

Two nitrate reductases, NRA and NRZ, are present in Escherichia coli. These isoenzymes have the same αβγ, subunits composition and have similar size and genetic organization. Corresponding subunits of the complexes share at least 75% identity. By subcloning the different genes and expressing them from separate transcriptional units, we have demonstrated (i) that the translation of the subunits and their assembly are not coupled processes, since subunits produced concomitantly but independently can meet efficiently and associate to form active enzymes, and (ii) that the α subunit of a given complex can be replaced by its counterpart from the other isoenzyme to yield an active membrane‐bound heterologous enzyme. One such heterologous enzyme, αAβZγZ, has been purified; it is less stable than the native enzymes, more susceptible to thermal denaturation, and shows increased sensitivity to proteolysis. It is also less stably bound to the membrane and, consequently, its activity with physiological electron donors is drastically reduced. The possibility that heterologous nitrate reductases could be formed in vivo is discussed with reference to the existence of porin heterotrimers of the outer membrane proteins OmpC, OmpF and PhoE.

A second phenazine methosulphate-linked formate dehydrogenase isoenzyme in Escherichia coli

A biochemical and immunological study has revealed a new formate dehydrogenase isoenzyme in Escherichia coli. The enzyme is an isoenzyme of the respiratory formate dehydrogenase (FDH-N) which forms part of the formate to nitrate respiratory pathway found in the organisms when it is grown anaerobically in the presence of nitrate. The new enzyme, termed FDH-Z, cross reacts with antibodies raised to FDH-N and possesses a similar polypeptide composition to FDH-N. FDH-Z catalyses the phenazine methosulphate-linked formate dehydrogenase activity present in the aerobically-grown bacterium. FDH-Z and FDH-N exhibit distinct regulation. Like formate dehydrogenase N, formate dehydrogenase Z is a membrane-bound molybdoenzyme. With nitrate reductase it can catalyse electron transfer between formate and nitrate. Quinones are required for the physiological electron transfer to nitrate. It seems likely that like FDH-N, FDH-Z functions physiologically as a formate: quinone oxidoreductase.

Membrane reconstitution in chl-r mutants of Escherichia coli K 12. IX. Part played by phospholipids in the complementation process.

The supernatant extracts of the chl A and chl B mutants of Escherichia coli K 12, the phospholipids of which are labeled by growth in 32 P or [2- 3H]glycerol media, contain 20 times more radioactivity than the supernatant extract of the wild-type strain grown under the same conditions. We have observed that, after complementation, 80% of the radioactivity previously contained by Extracts A and B is incorporated into reconstituted particles. The chromatography of 3H-labeled Extract B on DEAE-cellulose and followed by gel filtration of radioactive fractions on Sephadex G-200 has shown that the phospholipids of Extract B are only bound to soluble proteins and not to fragments of membranes; it can be assumed that they have been solubilized in the form of a lipid-protein complex by cell breakage. When Extracts A and B are treated by phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) before being mixed together, an inhibition of the reconstitution of nitrate reductase activity which is proportional to the phospholipase C concentration and the length of treatment is observed. The analysis of lipids and phospholipids of particles (Peak I, Peak II and Peak III) formed during complementation and reconstituted nitrate reductase shows that their phospholipid contents (phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and phosphatidylserine) and especially that of Peak II (d equals 1.18) are closely related to that of native particles from the wild-type strain. These results allow one to propose a hypothesis explaining the mechanism involved in complementation.