sulfur-shift mechanism catalytic mechanism, detailed overview. The mechanism is defined by a change in the Mo ion coordination, which involves a first-to-second shell displacement (shift) of the sulfur from the Cys, resulting in a free coordination position that is used by the enzyme to bind the substrate with a low energy cost, molybdenum coordinates an oxygen atom from the substrate, an oxygen atom from the substrate is transferred to the Mo ion, and later released as a water molecule. The reaction requires two electrons, which are provided by external reducing species, and two protons that are obtained from the solvent either directly or indirectly mediated by residues from the enzyme catalytic pocket
the periplasmic cytochrome c-linked nitrate reductase is encoded by the napFDAGHBC operon. The napF operon apparently encodes a low-substrate-induced reductase that is maximally expressed only at low levels of nitrate. Expression is suppressed under high-nitrate conditions. In contrast, the narGHJI operon is only weakly expressed at low nitrate levels but is maximally expressed when nitrate is elevated. The narGHJI operon is therefore a high-substrate-induced operon that somehow provides a second and distinct role in nitrate metabolism by the cell. Nitrite, the end product of each enzyme, has only a minor effect on the expression of either operon. Finally, nitrate, but not nitrite, is essential for repression of napF gene expression. These studies reveal that nitrate rather than nitrite is the primary signal that controls the expression of these two nitrate reductase operons in a differential and complementary fashion
the periplasmic cytochrome c-linked nitrate reductase is encoded by the napFDAGHBC operon. The napF operon apparently encodes a low-substrate-induced reductase that is maximally expressed only at low levels of nitrate. Expression is suppressed under high-nitrate conditions. In contrast, the narGHJI operon is only weakly expressed at low nitrate levels but is maximally expressed when nitrate is elevated. The narGHJI operon is therefore a high-substrate-induced operon that somehow provides a second and distinct role in nitrate metabolism by the cell. Nitrite, the end product of each enzyme, has only a minor effect on the expression of either operon. Finally, nitrate, but not nitrite, is essential for repression of napF gene expression. These studies reveal that nitrate rather than nitrite is the primary signal that controls the expression of these two nitrate reductase operons in a differential and complementary fashion
i.e. NapB, contains two c-type hemes and is assembled and secreted into the periplasm by the Ccm (cytochrome c maturation) machinery independently from enzyme NapA. Once in the periplasm they form the heterodimer NapAB
NapA contains a molybdo-bis(molybdopterin guanine dinucleotide) cofactor. The molybdenum ion coordination sphere of NapA includes two molybdopterin guanine dinucleotide dithiolenes, a protein-derived cysteinyl ligand and an oxygen atom. The Mo-O bond length is 2.6 A, which is indicative of a water ligand. In NapA or NapAB, the Mo5+ state can not be further reduced to Mo4+. A catalytic cycle for NapA is proposed in which nitrate binds to the Mo5+ ion and where a stable des-oxo Mo6+ species may participate
NapA contains a molybdo-bis(molybdopterin guanine dinucleotide) cofactor. The molybdenum ion coordination sphere of NapA includes two molybdopterin guanine dinucleotide dithiolenes, a protein-derived cysteinyl ligand and an oxygen atom. The Mo-O bond length is 2.6 A, which is indicative of a water ligand. In NapA or NapAB, the Mo5+ state can not be further reduced to Mo4+. A catalytic cycle for NapA is proposed in which nitrate binds to the Mo5+ ion and where a stable des-oxo Mo6+ species may participate
a small (9.3 kDa) cytoplasmic protein that is essential for Nap activity, role for NapD in the insertion of the molybdenum cofactor. The NapD cysteine residues (C8 and C32) are not conserved and a cysteine-free variant of NapD complements a DELTAnapD strain for restoration of NapA activity. A NapD C8S/C32A variant remains attached to the NapA signal peptide. Copurification of recombinant NapA complexed with N-terminally His-tagged NapD activator by nickel afinity chromatography
NapA is exported to the periplasm in a folded form by the twin-arginine protein transport (Tat) pathway. NapA is subject to Tat proofreading prior to export by the Tat pathway
the prokaryotic nitrate reductases can be subgrouped as respiratory nitrate reductases (Nar), assimilatory nitrate reductases (Nas), and periplasmic nitrate reductases (Nap). Periplasmic nitrate reductase (Nap) and formate dehydrogenase (Fdh), both belonging to the DMSO reductase family, subfamily I, have a very similar structure, but very different activities. The show key differences that tune them for completely different functions in living cells. Both enzymes share almost identical three-dimensional protein foldings and active sites, in terms of coordination number, geometry and nature of the ligands. The substrates of both enzymes (nitrate and formate) are polyatomic anions that also share similar charge and stereochemistry. In terms of the catalytic mechanism, both enzymes have a common activation mechanism (the sulfur-shift mechanism) that ensures a constant coordination number around the metal ion during the catalytic cycle. In spite of these similarities, they catalyze very different reactions: Nap abstracts an oxygen atom from nitrate releasing nitrite, whereas FdH catalyzes a hydrogen atom transfer from formate and releases carbon dioxide. Detailed comparison, overview. A key difference between the catalytic mechanisms of Nap and FdH is the fact that only Mo is used to reduce nitrate but in Fdhs both Mo and W are catalytically competent to oxidize formate to carbon dioxide
Escherichia coli is a Gram-negative bacterium that can use nitrate during anaerobic respiration. The catalytic subunit of the involved 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 twinarginine signal peptide of NapA. NapA is structured in its unbound form. The NapA signal peptide undergoes conformational rearrangement upon interaction with NapD. NapA is at least partially folded when bound by its NapD partner. 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
NapD is a small cytoplasmic protein that is essential for the activity of the periplasmic nitrate reductase and binds tightly to the twinarginine signal peptide of NapA. NapA is structured in its unbound form. The NapA signal peptide undergoes conformational rearrangement upon interaction with NapD. NapA is at least partially folded when bound by its NapD partner. 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
the enzyme shows a sulfur-shift mechanism catalytic mechanism, the active site is deeply buried and centered on the Mo atom, which is hexacoordinated to four sulfur atoms of two pyranopterin guanosine dinucleotides, one inorganic sulfur, and one S (Nap) atom from the side chain of a Cys, structure. Above the region of the metal center, the enzyme presents an arginine residue, that is proposed to be key for stabilization and substrate binding. The side chain of this residues probably interacts electrostatically with the substrates, compensating for the negative charge and favoring their interaction with the negatively charged active site. Comparisons of reaction mechanisms of members of the DMSO reductase family and structure analysis and modelling, overview. The NapA (product of the napA gene) is the catalytic subunit that contains the Mo-bisPGD and one 4Fe-4S center involved in electron transfer. Similar to other periplasmic Mo- and W-enzymes, immature NapA contains a signal peptide that is recognized by the TAT (twin arginine translocator) system. Prior to translocation, the two metallic cofactors are incorporated into NapA with the aid of the chaperone NapD, which accompanies the assembled metalloenzyme to the transporter, maturation mechanism of Mo. NapB contains two c-type hemes and is assembled and secreted into the periplasm by the Ccm (cytochrome c maturation) machinery independently from NapA. Once in the periplasm they form the heterodimer NapAB, except in the case of monomeric Naps. It is remarkable that napM is present only when the napB gene is absent. NapM is a tetrahemic c-type cytochrome. This cytochrome may mediate electron transfer to NapA in a similar way that NapB does in heterodimeric Naps. NapC is a membrane-anchored protein harboring four c-type hemes belonging to the NapC/NirT family. In bacteria like Desulfovibrio desulfuricans ATCC 27774 and Escherichia coli K12, where nitrate reduction catalyzed by Nap is coupled to an energy conserving process, the genes napG and napH are always present
1 * 17000 (NapB) + 1 * 90000 (NapA), the NapA holoenzyme associates with a di-heme c-type cytochrome redox partner (NapB). NapA and NapB proteins purify independently and not as a tight heterodimeric complex. Dissociation constants of 0.015 mM and 0.032 mM are determined for oxidized and reduced NapAB complexes, respectively
1 * 17000 (NapB) + 1 * 90000 (NapA), the NapA holoenzyme associates with a di-heme c-type cytochrome redox partner (NapB). NapA and NapB proteins purify independently and not as a tight heterodimeric complex. Dissociation constants of 0.015 mM and 0.032 mM are determined for oxidized and reduced NapAB complexes, respectively
1 * 17000 (NapB) + 1 * 90000 (NapA), the NapA holoenzyme associates with a di-heme c-type cytochrome redox partner (NapB). NapA and NapB proteins purify independently and not as a tight heterodimeric complex. Dissociation constants of 0.015 mM and 0.032 mM are determined for oxidized and reduced NapAB complexes, respectively
PELDOR analysis of recombinant MTSL-labelled MalE-NapASP fusion mutant S4C/S24C alone or in complex with NapD, comparison of bound, NMR-derived NapASP helix from PDB ID 2PQ4 versus free generated helix, positions of the spin labels in the two conformations of the signal peptide, overview
PELDOR analysis of recombinant MTSL-labelled MalE-NapASP fusion mutant S4C/S24C alone or in complex with NapD, comparison of bound, NMR-derived NapASP helix from PDB ID 2PQ4 versus free generated helix, positions of the spin labels in the two conformations of the signal peptide, overview
the catalytic subunit of Nap is usually encoded in a nap operon together with accessory proteins involved in its maturation (chaperones) and redox proteins that transfer reducing equivalents from the physiological electron donor (quinone pool) to the active site of the enzyme, nap enzyme domain structure analysis, overview
NapF plays a role in the post-translational modification of NapA prior to the export of folded NapA via the twin-arginine translocation pathway into the periplasm
site-directed mutagenesis, native, NapD results in a loss of some of the spin labels from the NapA signal peptide possibly due to the surface-exposed native cysteine residues of NapD. The NapD cysteine residues (C8 and C32) are not conserved and a cysteine-free variant of NapD complements a DELTAnapD strain for restoration of NapA activity. A NapD C8S/C32A variant remains attached to the NapA signal peptide
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
copurification of recombinant NapA complexed with N-terminally His-tagged NapD activator by nickel afinity chromatography from Escherichia coli strains MC4100 and BL21(DE3)
gene napA, enzyme NapA is encoded, along with its periplasmic di-heme c-type cytochrome redox partner NapB, in the seven gene nap operon, coexpression of NapA with His-tagged NapD activator in Escherichia coli strains MC4100 and BL21(DE3), recombinant expression of MTSL-labelled MalE-NapASP fusion mutant S4C/S24C in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain LCB2048
The NapF protein of the Escherichia coli periplasmic nitrate reductase system: demonstration of a cytoplasmic location and interaction with the catalytic subunit, NapA
The napF and narG nitrate reductase operons in Escherichia coli are differentially expressed in response to submicromolar concentrations of nitrate but not nitrite
Gonzalez, P.; Rivas, M.; Mota, C.; Brondino, C.; Moura, I.; Moura, J.
Periplasmic nitrate reductases and formate dehydrogenases biological control of the chemical properties of Mo and W for fine tuning of reactivity, substrate specificity and metabolic role