Research Fellow
Ph. D., Department of Botany, Imperial College of Sci. and Tech., University of London (1972)
Tel¡G+886-2-2787-1170 (Office)
¡@ ¡@+886-2-2787-1049 (Lab)
E-mail: bohsing@gate.sinica.edu.tw
BIOCHEMICAL CHARACTERIZATIONS OF NITRATE REDUCTASE IN THE CYANOBACTERIA OF CYANOTHECE RF-1 AND SYNECHOCOCCUS PCC 7942
Cyanobacteria are ecologically and geographically diverse photosynthetic prokaryotes that are principal components of the food chain in many aquatic ecosystems. They perform oxygenic photosynthesis to assimilate inorganic carbon and use primarily inorganic nitrogen sources to fulfill their nitrogen requirement. Nitrate assimilation is one of the major processes of nitrogen acquisition in cyanobacteria. The transport and reduction of nitrate are importantly regulated steps in the nitrate assimilation processes. In cyanobacteria, the genes involving nitrate uptake and reduction are negatively regulated by ammonium and usually clustered in a large operon as nirAnrtABCD-narB, or organized into two transcription units of nrtABCD-narB and nirA. However, in Cyanothece sp. RF-1 (formerly Synechococcus sp. RF-1) the regulation of nitrate reduction is different from nitrate uptake at the protein and transcript levels. NarB proteins and in situ NR activities of Cyanothece sp. RF-1 are not repressed by ammonium but nitrate uptake and genes of nrtABCD are negatively regulated by ammonium. These suggested narB gene of Cyanothece sp. RF-1 appears not to cluster with the nitrate transporter genes in an operon.
Two types of nitrate reductase (NR) kinetics, mono-phase and bi-phase, have been characterized in cyanobacteria. The typical enzyme kinetics (mono-phase) of NRs was strictly found in unicellular non-diazotrophic cyanobacteria including the NR of Synechococcus sp. PCC 7942 and a biphasic kinetics NRs was usually found in diazotrophic cyanobacteria. Cyanothece sp. RF-1 is a unicellular diazotrophic cyanobacterium. The gene of narB encoding NR has been identified in some strains of cyanobacteria. We isolated and cloned the narB gene from Cyanothece sp. RF-1, and transferred narB gene into the E. coli cells for heteroexpressing recombinant NarB proteins (rNarB). We have provided evidence to suggest that monomeric rNarB is a dual-affinity nitrate reductase when methyl viologen was used as the electron donor in NR activity assay. Moreover, we found the functional importance of the rNarB N-terminal structure in that of kinetics.
We characterized the biochemical properties of the hetero-expressed rNarB of Cyanothece sp. RF-1. The monomeric rNarB exhibits dualaffinity for nitrate and the two kinetic components (low- and high-affinity) reflect the similar thermostability. When rNarB was incubated at 70°C for 30 min and then returned to room temperature, about 90% of untreated NR activities were recovered. However, some organic solvents (methanol, ethanol, acetone and isopropanol etc) had no significant effect on the high-affinity kinetics of NR activity, but had significant negative or positive effect on the lowaffinity of NR activity. KCN and NaN3 are strong inhibitors of most metalloenzymes. The rNarB of Cyanothece sp. RF-1 is very sensitive to KCN (1-10µM) and exhibits a competitive inhibition in both of low- and high-affinity kinetics. However, NaN3 has dramatic effect on the kinetics of rNarB of Cyanothece sp. RF-1. NaN3 at 1-2 mM is competitive inhibition in high-affinity kinetics (0.04-0.16 mM of nitrate) of rNarB and 4-7 mM of NaN3 exhibits a mix-type inhibition in lowaffinity (2-14 mM of nitrate). In the presence of 1 mM NaN3 seems enough to inhibit the highaffinity component of rNarB, but no significant effect on the low-affinity component. The hyperbolic enzyme kinetics of rNarB indicate that two distinct regulation components may harbor in the monomeric rNarB of Cyanothece sp. RF-1.
O2 PHOTOREDUCTION IN SYNECHOCOCCUS RF-1
Synechococcus sp. RF-1, a unicellular N2-fixing cyanobacterium, can grow photosynthetically and diazotrophically in continuous light. How the organism protects its nitrogenase from damage by oxygen is unclear. In this work, a PAM-101 fluorometer and an O2 electrode are used simultaneously to study the chlorophyll a fluorescence and to monitor O2 exchanges in Synechococcus sp. RF-1 cells. The results found that in addition to CO2, O2 can also act as electron acceptor to receive electrons derived from QA-, i.e. O2 photoreduction. Studies with various inhibitors of the electron transport chain demonstrated that 3-(3,4- dichlorophenyl)-1,1-dimethylurea (DCMU), 2,5-dibromo-3-methyl-6- isopropyl-p-benzo-quinone (DBMIB) and salicylhydroxamic acid (SHAM) inhibited the photoreduction of O2, while glycolaldehyde, disalicylidenepropanediamine (DSPD), methyl viologen (MV) and KCN did not. Addition of N, N, N', N'-tetramethyl-p-phenylenediamine (TMPD) bypassed the inhibition of DBMIB. The soluble proteins extracted from Synechococcus sp. RF-1 cells revealed cytochrome c-dependent, KCN-resistant and SHAM-sensitive O2 uptake activities. These results imply that a KCNresistant and SHAM-sensitive oxidase transfers electrons generated from photosystem II to O2 between cytochrome b6f complex and photosystem I. The oxidase can use reduced cytochrome c as substrate and results in the photoreduction of O2. Besides Synechococcus sp. RF-1, we used the same system to detect the chlorophyll a fluorescence of other cyanobacteria. Studies with non-nitorgen fixation species revealed no O2 photoreduction acticity in Synechococcus PCC 7942 and only low activity in Synechocystis PCC 6803, while nitrogen-fixing species, Cyanothece ATCC 51142 and Gloeothece sp. displayed high activities. The results suggest that photoreduction of O2 may be associated with protection of nitrogenase. In Synechococcus sp. RF-1 cells, which use a KCNresistant and SHAM-sensitive oxidase to perform this function. When SHAM blocked this alternative electron transport pathway, it decreased 83 % of dinitrogen-fixing activity in continuous light growing cells, but only decreased 38 % of the activity in 12 light/12 dark regimen-growing cells. We conclude that a KCNresistant and SHAM-sensitive oxidase dependent O2 photoreduction is an important mechanism to reduce the oxidative stress of photosystem and to protect the nitrogenase from the damage of oxygen for Synechococcus sp. RF-1 cells.
Wang, T. H., Fu, H., and Shieh, Y. J. 2003. Monomeric NarB is a dual-affinity nitrate reductase, and its activity is regulated differently from that of nitrate uptake in the unicellular diazotrophic cyanobacterium Synechococcus sp. strain RF-1. J. Bacteriol. 185: 5838-5846.
Weng, J. H., and Shieh, Y. J. 2004. Salicylhydroxamic acid (SHAM) inhibits O2 photoreduction which protects nitrogenase acitivty in the cyanobacterium Synechococcus sp. RF-1. Photosynthesis Research 82: 151-164.1.
