Progress 09/15/01 to 09/30/04
Outputs
Cyanobacterial phycocyanobilin:ferredoxin oxidoreductase (PcyA) catalyzes the four electron reduction of biliverdin IX-alpha (BV) to phycocyanobilin, a key step in the biosynthesis of the linear tetrapyrrole (bilin) prosthetic groups of cyanobacterial phytochromes and the light-harvesting phycobiliproteins. Using an anaerobic assay protocol, optically detected bilin-protein intermediates, produced during the PcyA catalytic cycle, were shown to correlate well with the appearance and decay of an isotropic organic radical EPR signal measured at low temperature (TU et al, 2004). Absorption spectral simulations of biliverdin XIII-alpha reduction support a mechanism involving direct electron transfers from ferredoxin to protonated bilin-PcyA complexes.
Impacts
Phytochrome is a pigment-protein complex that enables plants to modify their growth and development in response to the changing light environment. An understanding of the chemical mechanism of the enzymes that contribute to the synthesis of the phytobilin pigment prosthetic group of phytochromes should lead to development of novel inhibitors and altered substrates that could have profound effects on light-mediated plant growth and development.
Publications
- TU, S-L., GUNN, A., TONEY, M.D., BRITT, R.D. AND LAGARIAS, J.C. (2004) Biliverdin reduction by cyanobacterial phycocyanobilin:ferredoxin oxidoreductase (PcyA) proceeds via linear tetrapyrrole radical intermediates. J. Am. Chem. Soc. 126, 8682-8693.
|
Progress 01/01/03 to 12/31/03
Outputs
The biosynthesis of phytobilin pigments is critical for plant photomorphogenesis and for survival of phycobiliprotein-containing organisms that live in light-limiting environments. Two recent collaborative studies underscore the importance of the biosynthesis of the natural phytochromobilin prosthetic group to light-mediated plant growth and devlopment. In one study (FRANKLIN et al, 2003), we show that some of the 'photomorphogenetic-deficient' phenotypes of transgenic plants expressing mammalian biliverdin reductase arise from direct misregulation of chlorophyll biosynthesis. Since chlorophyll biosynthesis in plants is light-dependent, these results document the interplay between phytochrome-mediated gene expression and chlorophyll biosynthesis in plants which must be tightly coordinated to avoid photodamage especially during early seedling development. In the second study (KAMI et al, in press), we show that the expression of the cyanobacterial bilin reductase PcyA
restores normal photomorphogenesis to phytochrome chromophore-deficient hy2 mutant plants. Of note is the observation that complementation of the FR high irradiance phytochrome A-mediated response is notably shifted to shorter wavelengths. These studies show that the substitution of phycocyanobilin, the chromophore precursor of phytochromes from algae and cyanobacteria, for phytochromobilin yields functional plant phytochromes. We speculate that the evolution of a longer wavelength absorbing photoreceptor may have adaptive significance to land plant evolution.
Impacts
Phytochrome is a pigment-protein complex that enables plants to modify their growth and development in response to the changing light environment. Our studies seek to develop new methods to alter yield-reducing responses of plants to light through modification of the synthesis and/or assembly of the light sensing pigment component of phytochrome. Such knowledge will facilitate design of experimental systems for generating inactive, hyperactive and/or spectrally shifted species of phytochromes and phycobiliproteins in vivo. Targeted expression of bilin reductases in plants also will enable us to better define sites of photoperception for selective photomorphogenetic responses and ultimately, to alter individual phytochrome-mediated responses in agronomically important plant species.
Publications
- FRANKLIN, K.A., LINLEY, P.J., MONTGOMERY, B.L., LAGARIAS, J.C., THOMAS, B., JACKSON, S.D. and TERRY, M.J. (2003) Misregulation of the tetrapyrrole biosynthesis in transgenic tobacco expressing mammalian biliverdin reductase. Plant Journal 35, 717-728
- KAMI, C., MUKOUGAWA, K., MURAMOTO, T., YOKOTA, A., SHINOMURA, T., LAGARIAS, J.C. and KOHCHI, T. (2004) Functional Complementation of Phytochrome Chromophore-Deficient Arabidopsis Plants by Expression of Phycocyanobilin:Ferredoxin Oxidoreductase Proc. Natl. Acad. Sci (USA). In press.
|
Progress 01/01/02 to 12/31/02
Outputs
The biosynthesis of phytobilin pigments is critical for plant photomorphogenesis and for survival of phycobiliprotein-containing organisms that live in light-limiting environments (see review by FRANKENBERG, N. and LAGARIAS, J.C.). Our work focuses on the ferredoxin-dependent bilin reductases that mediate the reductive conversion of biliverdin (BV) to the bilin precursors of phytochrome and the phycobiliprotein chromophores. Studies during the past year have mainly focussed on recombinant phycocyanobilin:ferredoxin oxidoreductase (PcyA) which mediates an atypical four-electron reduction of BV (FRANKENBERG, N. and LAGARIAS, J.C., submitted). In this study, we describe the expression, affinity purification and biochemical characterization of recombinant PcyA from Anabaena sp. PCC7120. A monomeric protein with a native Mr of 30,400 plus or minus 5,000, recombinant PcyA forms a tight and stable stoichiometric complex with its bilin substrate. The enzyme exhibits a strong
preference for plant-type [2Fe-2S]-ferredoxins, however, flavodoxin can also serve as an electron donor. HPLC analyses establish that catalysis proceeds via the two electron-reduced intermediate 181,182-dihydrobiliverdin, indicating that exo-vinyl reduction preceeds A-ring (endo-vinyl) reduction. Substrate specificity studies indicate that the arrangement of the A- and D-ring substituents alters the positioning of the bilin substrate within the enzyme, profoundly influencing the course of catalysis. Based on these observations and the apparent lack of a metal or small molecule cofactor, a radical mechanism for BV reduction by phycocyanobilin:ferredoxin oxidoreductase is envisaged.
Impacts
Phytochrome is a pigment-protein complex that enables plants to modify their growth and development in response to the changing light environment. Our studies seek to develop new methods to alter yield-reducing responses of plants to light through modification of the synthesis and/or assembly of the light sensing pigment component of phytochrome. Such knowledge will facilitate design of experimental systems for generating inactive, hyperactive and/or spectrally shifted species of phytochromes and phycobiliproteins in vivo. Targeted expression of bilin reductases in plants also will enable us to better define sites of photoperception for selective photomorphogenetic responses and ultimately, to alter individual phytochrome-mediated responses in agronomically important plant species.
Publications
- No publications reported this period
|
Progress 01/01/01 to 12/31/01
Outputs
Phytobilins are linear tetrapyrroles that function as the immediate precursors of the light harvesting prosthetic groups of the plant photoreceptor phytochrome and the phycobiliprotein antennae of cyanobacteria, red algae and cryptomonads. The biosynthesis of these pigments is critical for plant photomorphogenesis and for survival of phycobiliprotein-containing organisms that live in light-limiting environments. This newly funded grant focuses on the ferredoxin-dependent bilin reductases that mediate conversion of biliverdin IXa to the bilin precursors of phytochrome and the phycobiliprotein chromophores. Genes for four distinct classes of bilin reductases from plants and oxygenic photosynthetic bacteria were identified during previous studies, enabling expression of catalytically active recombinant enzymes in bacteria for the first time. Through biochemical characterization of recombinant enzymes, we seek to define the molecular basis of their unique
substrate/product specificities about which nothing is presently known.
Impacts
Phytochrome is a pigment-protein complex that enables plants to modify their growth and development in response to the changing light environment. Our studies seek to develop new methods to alter yield-reducing responses of plants to light through modification of the synthesis and/or assembly of the light sensing pigment component of phytochrome. Such knowledge will facilitate design of experimental systems for generating inactive, hyperactive and/or spectrally shifted species of phytochromes and phycobiliproteins in vivo. Targeted expression of bilin reductases in plants also will enable us to better define sites of photoperception for selective photomorphogenetic responses and ultimately, to alter individual phytochrome-mediated responses in agronomically important plant species.
Publications
- No publications reported this period
|
|