ABSTRACT
Oxygenic photosynthetic organisms often suffer from excessive irradiance, which cause harmful effects to the chloroplast proteins and lipids. Photoprotection and the photosystem II repair processes are the mechanisms that plants deploy to counteract the drastic effects from irradiance stress. Although the protective and repair mechanisms seemed to be similar in most plants, many species do confer different level of tolerance toward high light. Such diversity may originate from differences at the molecular level, i.e., perception of the light stress, signal transduction and expression of stress responsive genes. Comprehensive analysis of overall changes in the total pool of proteins in an organism can be performed using a proteomic approach. In this study, we employed 2-DE/LC-MS/MS-based comparative proteomic approach to analyze total proteins of the light sensitive model unicellular green alga Chlamydomonas reinhardtii in response to excessive irradiance. Results showed that among all the differentially expressed proteins, several heat-shock proteins and molecular chaperones were surprisingly down-regulated after 3-6 h of high light exposure. Discussions were made on the possible involvement of such down regulation and the light sensitive nature of this model alga.
Subject(s)
Chlamydomonas reinhardtii/metabolism , Chlamydomonas reinhardtii/radiation effects , Heat-Shock Proteins/metabolism , Light , Plant Proteins/metabolism , Proteomics/methods , Electrophoresis, Gel, Two-Dimensional , Signal Transduction/radiation effects , Tandem Mass SpectrometryABSTRACT
The luxG gene is part of the lux operon of marine luminous bacteria. luxG has been proposed to be a flavin reductase that supplies reduced flavin mononucleotide (FMN) for bacterial luminescence. However, this role has never been established because the gene product has not been successfully expressed and characterized. In this study, luxG from Photobacterium leiognathi TH1 was cloned and expressed in Escherichia coli in both native and C-terminal His6-tagged forms. Sequence analysis indicates that the protein consists of 237 amino acids, corresponding to a subunit molecular mass of 26.3 kDa. Both expressed forms of LuxG were purified to homogeneity, and their biochemical properties were characterized. Purified LuxG is homodimeric and has no bound prosthetic group. The enzyme can catalyze oxidation of NADH in the presence of free flavin, indicating that it can function as a flavin reductase in luminous bacteria. NADPH can also be used as a reducing substrate for the LuxG reaction, but with much less efficiency than NADH. With NADH and FMN as substrates, a Lineweaver-Burk plot revealed a series of convergent lines characteristic of a ternary-complex kinetic model. From steady-state kinetics data at 4 degrees C pH 8.0, Km for NADH, Km for FMN, and kcat were calculated to be 15.1 microM, 2.7 microM, and 1.7 s(-1), respectively. Coupled assays between LuxG and luciferases from P. leiognathi TH1 and Vibrio campbellii also showed that LuxG could supply FMNH- for light emission in vitro. A luxG gene knockout mutant of P. leiognathi TH1 exhibited a much dimmer luminescent phenotype compared to the native P. leiognathi TH1, implying that LuxG is the most significant source of FMNH- for the luminescence reaction in vivo.
Subject(s)
Bacterial Proteins/genetics , FMN Reductase/genetics , Flavins/metabolism , Luminescence , Oxidoreductases/genetics , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , FMN Reductase/chemistry , FMN Reductase/metabolism , Gene Deletion , Hydrogen-Ion Concentration , Molecular Sequence Data , NAD/metabolism , NADP/metabolism , Oxidation-Reduction , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Photobacterium/genetics , Photobacterium/metabolism , Plasmids/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , TemperatureABSTRACT
The genes encoding for the reductase and oxygenase components of p-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii were cloned and expressed in an E. coli system. The recombinant enzymes were purified and shown to have the same catalytic properties as the native enzyme. Sequence analysis and biochemical studies indicate that the enzyme represents a novel prototype of enzyme in the two-protein component class of aromatic hydroxylases. The C2 component shows little similarity to other oxygenases in the same class, correlating with its uniquely broad flavin specificity. Analysis of the C1 reductase sequence indicates that the binding sites of flavin and NADH mainly reside in the N-terminal half while the C-terminal half may be responsible for HPA-stimulation of NADH oxidation.
