ABSTRACT
NafY participates in the final steps of nitrogenase maturation, having a dual role as iron-molybdenum cofactor (FeMo-co) carrier and as chaperone to the FeMo-co-deficient apo-NifDK (apo-dinitrogenase). NafY contains an N-terminal domain of unknown function (n-NafY) and a C-terminal domain (core-NafY) necessary for FeMo-co binding. We show here that n-NafY and core-NafY have very weak interactions in intact NafY. The NMR structure of n-NafY reveals that it belongs to the sterile α-motif (SAM) family of domains, which are frequently involved in protein-protein interactions. The presence of a SAM domain in NafY was unexpected and could not be inferred from its amino acid sequence. Although SAM domains are very commonly found in eukaryotic proteins, they have rarely been identified in prokaryotes. The n-NafY SAM domain binds apo-NifDK. As opposed to full-length NafY, n-NafY impaired FeMo-co insertion when present in molar excess relative to FeMo-co and apo-NifDK. The implications of these observations are discussed to offer a plausible mechanism of FeMo-co insertion. NafY domain structure, molecular tumbling, and interdomain motion, as well as NafY interaction with apo-NifDK are consistent with the function of NafY in FeMo-co delivery to apo-NifDK.
Subject(s)
Azotobacter vinelandii/enzymology , Bacterial Proteins/chemistry , Coenzymes/chemistry , Iron/chemistry , Molecular Chaperones/chemistry , Molybdenum/chemistry , Nitrogenase/chemistry , Amino Acid Motifs , Apoenzymes/chemistry , Apoenzymes/genetics , Apoenzymes/metabolism , Azotobacter vinelandii/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Coenzymes/metabolism , Iron/metabolism , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Molybdenum/metabolism , Nitrogenase/genetics , Nitrogenase/metabolism , Nuclear Magnetic Resonance, Biomolecular , Protein Structure, Quaternary , Protein Structure, TertiaryABSTRACT
Heme nitric oxide/oxygen (H-NOX) proteins are found in eukaryotes where they are typically part of a larger protein such as soluble guanylate cyclase and in prokaryotes where they are often found in operons with a histidine kinase, suggesting that H-NOX proteins serve as sensors for NO and O(2) in signaling pathways. The Fe(II)-NO complex of the H-NOX protein from Shewanella oneidensis inhibits the autophosphorylation of the operon-associated histidine kinase, whereas the ligand-free H-NOX has no effect on the kinase. NMR spectroscopy was used to determine the structures of the Fe(II)-CO complex of the S. oneidensis H-NOX and the Fe(II)-CO complex of the H103G H-NOX mutant as a mimic of the ligand-free and kinase-inhibitory Fe(II)-NO H-NOX, respectively. The results provide a molecular glimpse into the ligand-induced conformational changes that may underlie kinase inhibition and the subsequent control of downstream signaling.
Subject(s)
Heme/metabolism , Nitric Oxide/metabolism , Protein Conformation , Protein Kinases/chemistry , Protein Kinases/metabolism , Shewanella/enzymology , Signal Transduction/physiology , Animals , Carbon Monoxide/chemistry , Carbon Monoxide/metabolism , Heme/chemistry , Histidine Kinase , Molecular Structure , Nitric Oxide/chemistry , Nuclear Magnetic Resonance, Biomolecular , Phosphorylation , Protein Kinases/geneticsABSTRACT
The xenognostic mechanisms of two multi-host pathogens, the causative agent of crown gall tumors Agrobacterium tumefaciens and the parasitic plant Striga asiatica, are compared. Both organisms are general plant pathogens and require similar information prior to host commitment. Two mechanistic strategies, chemical perception and metabolic complementation, are used to ensure successful host commitment. The critical reactions at host-parasite contact are proton and electron transfer events. Such strategies may be common among multi-host pathogens.
