Progress 11/15/04 to 11/14/07
Outputs
Major activities associated with this project include mentoring/training of key personnel associated with this project. These include one postdoctoral fellow, Dr. Shih-Long Tu, two graduate students, Wesley Sughrue and Lixia Shang, and two undergraduates, Kelechi Chikere and Keenan Taylor. Patents: John Clark Lagarias, Takayuki Kohchi, Nicole Frankenberg, Gregory A. Gambetta, Beronda L. Mongomery HY2 Family of Bilin Reductases US Patent No. 7,033,806, Issued April 25, 2006. Collaborations fostered by project with the laboratories of Dr. Andrew J. Fisher (UCD Chemistry), Dr. R. David Britt (UCD Chemistry) and Dr. Keichi Fukuyama (Osaka University, Japan) Major dissemination events associated with this project include participation at the following meetings/symposia. Plant Biology 2005. American Society of Plant Biology Annual Meeting, July 16-20, 2005, Seattle, WA. "Radical enzymology of cyanobacterial phycocyanobilin:ferredoxin oxidoreductase (PcyA)" (S.-L Tu, W.
Sughrue, R. D. Britt and J. C. Lagarias, poster) Genes to Products USDA Awardee Workshop, Airlie Center, Virginia, February 27-28, 2006 "Mechanistic Biochemical Analysis of Bilin:Ferredoxin Oxidoreductases" (S.-L. Tu, W. Sughrue, A. J. Fisher, R. D. Britt and J. C. Lagarias, poster presentation by W. Sughrue) Gordon Research Conference on the Chemistry and Biology of Tetrapyrroles, Salve Regina University, Newport RI, July 23-26, 2006. "Structure and Mechanism of Phycocyanobilin:Ferredoxin Oxidoreductase (PcyA) from Anabaena sp PCC7120." (S.-L. Tu, W. Sughrue, N. C. Rockwell, A. J. Fisher, R. D. Britt, and J. C. Lagarias, poster presentation) 232nd American Chemical Society National Meeting, San Francisco, CA (2006) "Synthesis of Biliverdin Amides and Substrate Specificity for Bacteriophytochromes and Bilin Reductases (L. Shang and J. C Lagarias, poster presentation) Genes to Products USDA Awardee Workshop, Bethesda Marriott Hotel, March 12-14, 2007 "Mechanistic Biochemical Analysis
of Bilin:Ferredoxin Oxidoreductases" (W. Sughrue, S.-L. Tu, A. F. Fisher, J. Clark Lagarias and R. David Britt, poster presentation) Annual Meeting of Crystallographic Society of Japan, Tokyo, Japan December 1-2, 2007. Crystal structure of PcyA in complex with 181,182-dihydrobiliverdin. (Y. Hagiwara, M. Sugishima, H. Khawn, H. Kinoshita, K. Inomata, L. Shang, J. C. Lagarias, Y.Takahashi, K. Fukuyama, poster) 7th International Conference on Tetrapyrrole Photoreceptors in Photosynthetic Organisms, Kyoto, Japan, December 9-14, 2007. Mechanistic and Structural Analyses of Ferredoxin-Dependent Bilin Reductases. (J. C. Lagarias, S.-L. Tu, Nathan C. Rockwell, Lixia Shang, Wesley Sughrue, Alexander Gunn, M. Brynda, C. Aznar, F. Allen-Piccolo, A. C. Kohler, A. J. Fisher, and R. D. Britt), JCL invited speaker
Impacts
This project has successfully addressed basic science objectives designed to elucidate the structural basis of the catalytic specificity of a family of bilin reductase enzymes critical to the synthesis of linear tetrapyrrole pigments involved in light harvesting and light sensing in a wide variety of oxygenic photosynthetic organisms (i.e. plants, cyanobacteria and algae). These organisms are among the most important on earth for the fixation and sequestration of atmospheric carbon dioxide as biomass. This project focused on phycocyanobilin:ferredoxin oxidoreductase (PcyA), one member of the HY2 family of ferredoxin-dependent bilin reductases (FDBRs) that mediates conversion of biliverdin (BV) to the immediate precursor of the phytochrome and phycobiliprotein chromophores. Through biochemical analysis of recombinant FDBRs, we sought to elucidate both the chemical mechanism and molecular basis of their unique substrate/product specificities, as well as to synthesize
FDBRs with novel biochemical activities. Investigations were initially undertaken to identify catalytic residues in PcyA from the cyanobacterium Nostoc sp. PCC7120 through site-specific chemical modification and mutagenesis of candidate proton-donating residues. Our studies identified a conserved histidine-aspartate pair that is essential for the catalytic activity of PcyA. In collaboration with the laboratory of R. David Britt, spectroscopic evidence for the formation of stable enzyme-bound BV radicals for selected missense mutants of both residues supported their role as a 'coupled' proton donating pair during the reduction of the substrate's exo-vinyl group. Companion studies with the laboratory of our collaborator Dr. Andy Fisher led to the determination of the three dimensional structure of PcyA from Nostoc sp. PCC7120 at 2.5 angstrom resolution. Comparative analysis of this structure with those recently reported for substrate-bound and substrate free forms of PcyA from the
cyanobacterium Synechocystis sp. PCC6803 has provided a compelling picture of substrate-induced changes in the PcyA enzyme and the chemical basis of PcyA's catalytic activity. More recently, we have determined the crystal structures at 1.3 angstrom resolution of the two missense mutants of Synechocystis PcyA complexed with BV. Both of these mutants reveal a well-ordered water molecule that sits above the center of the tetrapyrrole. Both mutants could be reduced in the crystal to stable radical complexes using dithionite - an observation that has enabled structural analysis to 1.5 angstrom resolution! Based on these structures and biochemical analysis of site-directed mutants enabled us to propose a revised mechanism for PcyA-mediated catalysis. The mechanistic implications of these studies, along with homology modeling, have provided compelling insight into the broader catalytic mechanisms of other members of the ferredoxin-dependent bilin reductase family.
Publications
- Tu, S-L., Sughrue, W. L., Britt, R.D. and Lagarias, J.C. (2006) A catalytic histidine-aspartate Pair in exo-vinyl reduction of biliverdin by phycocyanobilin:ferredoxin oxidoreductase (PcyA). J. Biol. Chem. 281, 3127-3136.
- Kohler, A. C., Gae, D. D., Allen-Piccolo, F., Tu, S.-L., Lagarias, J. C., and Fisher, A. J. (2008). Crystal structures of Pycocyanobilin:Ferredoxin Oxidoreductase (PcyA) mutants in the resting and radical state. in preparation.
|