title: Enzyme kinetics, inhibitors, mutagenesis and electron paramagnetic resonance analysis of dual-affinity nitrate reductase in unicellular N(2)-fixing cyanobacterium Cyanothece sp. PCC 8801.
abstract: The assimilatory nitrate reductase (NarB) of N(2)-fixing cyanobacterium Cyanothece sp. PCC 8801 is a monomeric enzyme with dual affinity for substrate nitrate. We purified the recombinant NarB of Cyanothece sp. PCC 8801 and further investigated it by enzyme kinetics analysis, site-directed mutagenesis, inhibitor kinetics analysis, and electron paramagnetic resonance (EPR) spectroscopy. The NarB showed 2 kinetic regimes at pH 10.5 or 8 and electron-donor conditions methyl viologen or ferredoxin (Fd). Fd-dependent NR assay revealed NarB with very high affinity for nitrate (K(m)1, ∼1μM; K(m)2, ∼270μM). Metal analysis and EPR results showed that NarB contains a Mo cofactor and a [4Fe-4S] cluster. In addition, the R352A mutation on the proposed nitrate-binding site of NarB greatly altered both high- and low-affinity kinetic components. Furthermore, the effect of azide on the NarB of Cyanothece sp. PCC 8801 was more complex than that on the NarB of Synechococcus sp. PCC 7942 with its single kinetic regime. With 1mM azide, the kinetics of the wild-type NarB was transformed from 2 kinetic regimes to hyperbolic kinetics, and its activity was enhanced significantly under medium nitrate concentrations. Moreover, EPR results also suggested a structural difference between the two NarBs. Taken together, our results show that the NarB of Cyanothece sp. PCC 8801 contains only a single Mo-catalytic center, and we rule out that the enzyme has 2 independent, distinct catalytic sites. In addition, the NarB of Cyanothece sp. PCC 8801 may have a regulatory nitrate-binding site.
authors.affiliation: Institute of Plant and Microbial Biology, Academia Sinica, Taipei 11529, Taiwan.
authors.forename: Tung-Hei
authors.initials: TH
authors.lastname: Wang
authors.forename: Yung-Han
authors.initials: YH
authors.lastname: Chen
authors.forename: Jine-Yung
authors.initials: JY
authors.lastname: Huang
authors.forename: Kang-Cheng
authors.initials: KC
authors.lastname: Liu
authors.forename: Shyue-Chu
authors.initials: SC
authors.lastname: Ke
authors.forename: Hsiu-An
authors.initials: HA
authors.lastname: Chu
mesh_terms: D000595:Amino Acid Sequence; D001386:Azides / Q000494:pharmacology; D001426:Bacterial Proteins / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001665:Binding Sites; D055162:Biocatalysis; D020134:Catalytic Domain; D003067:Coenzymes; D046889:Cyanothece / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005288:Ferredoxins / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D050901:Nitrate Reductase / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009566:Nitrates / Q000378:metabolism*; D009586:Nitrogen Fixation; D010084:Oxidation-Reduction; D010269:Paraquat / Q000378:metabolism; D011994:Recombinant Proteins; D017422:Sequence Analysis, DNA
chemical_list: D001386:Azides; D001426:Bacterial Proteins; D003067:Coenzymes; D004791:Enzyme Inhibitors; D005288:Ferredoxins; D009566:Nitrates; D011994:Recombinant Proteins; D050901:Nitrate Reductase; D010269:Paraquat