The research interest in our lab is mainly on the molecular mechanism of chromophore biosynthesis. We seek to elucidate both the chemical mechanism and molecular basis of ferredoxin-dependent bilin reductases (FDBRs) as well as to evolve novel biochemical activities of these enzymes.
Biosynthesis of phytobilin chromophore
Light perception by phytochromes and phycobiliproteins in plant, algae and cyanobacteria depend upon the presence of the linear tetrapyrrole chromophores, phytobilins. Phytobilins are derived from heme, which is first converted into the linear tetrapyrrole biliverdin IXa (BV), an intermediate in the biosynthesis pathway. BV is metabolized in plants, cyanobacteria and red algae into different phytobilins by different FDBRs with distinct double-bond specificities (Figure 1). In plants, the enzyme PFB synthase (HY2) catalyzes double bond reduction at the A-ring of BV to yield PFB, the immediate precursor of the phytochrome chromophore. In cyanobacteria and red algae, BV is converted into chromophore precursors of light-harvesting phycobiliproteins for photosynthesis. Two enzymes PebA and PebB catalyze double bond reductions on the 15,16 carbons and A-ring vinyl group of BV to yield PEB. By contrast, a single enzyme PcyA catalyzes two double bond reductions on D-ring and A-ring vinyl groups respectively to yield PCB, which also serves as the chromophore precursor of the cyanobacterial phytochromes. Figure 1. Biosynthesis of phytobilins. The heme-derived phytobilin precursors of phytochrome and phycobiliprotein chromophores of plants, algae, and cyanobacteria, i.e. PFB, PCB and PEB, share the common intermediate biliverdin IXa. BV is metabolized by the FDBR family of enzymes which includes HY2, phytochromobilin synthase; PcyA, phycocyanobilin: ferredoxin oxidoreductase; PebA, 15,16-dihydrobiliverdin: ferredoxin oxidoreductase and PebB, phycoerythrobilin: ferredoxin oxidoreductase. Holophytochrome assembly is autocatalytic, while phycobiliprotein assembly requires bilin lyases - CpcEF, PecEF and CpeYZ.
Accomplishments and future direction
PcyA catalyzes four electron reduction of BV to PCB via the two-electron reduced intermediate 181,182-DHBV, indicating that the D-ring (exo-vinyl) reduction proceeds A-ring (endo-vinyl) reduction (Figure 1). The catalytic mechanism was proposed to involve sequential electron-coupled proton transfers from reduced ferredoxin and PcyA to the bound BV substrate. With a newly developed anaerobic assay system, two radical intermediates have been identified in the PcyA reaction
spectrally (Tu et al., 2004b). We proposed a more detail mechanism that there are several proton donating residues on PcyA involved in the catalysis steps. By using chemical modification and site-directed mutagenesis, a histidine-aspartate pair critical for PcyA-catalyzed reaction has been identified (Tu et al., 2006). The crystal structure of PcyA supports that this critical histidine-aspartate pair is located in the substrate binding pocket (Tu et al., 2007) (Figure 2B). These structural and functional studies on PcyA raise a number of questions. Do the other FDBRs have a similar mechanism? If so, what residues on each enzyme account for their distinct catalytic activities? Can one member of FBDR be engineered into another? Is there any specific inhibitor for each enzyme?
Figure 2. Active site of PcyA.
A.Overall structure of PcyA. PcyA folds into a single-domain three-layer a-b-a sandwich. The ribbon representation is rainbow-colored with the N-terminus colored in blue ending with red at the C-terminus.
B.Active site of PcyA. Catalytic residues are colored in gold, including the His85-Asp102 pair and Glu73 that have been shown critical for BV reduction. All residues are in proximity to the BV substrate (green color) that potentially hydrogen bond to carbonyl oxygens and D-ring vinyl group. Red balls indicate the location of water molecules in the active site.
The immediate precursor of the phytochrome chromophore, PFB, is synthesized by plastid-localized PFB synthase in higher plants. PFB synthase is encoded by the HY2 gene in Arabidopsis, catalyzes the ferredoxin-dependent reaction of PFB formation. Mutations in HY2 have been shown to severely affect light-mediated plant growth and development due to the loss of all functional phytochrome.
My research seeks to determine the structure and function of HY2. By understanding of enzymatic mechanism of HY2, new catalytic activities can be generated by both site-directed mutagenesis and directed evolution. Mutant HY2 with new activities can be introduced into hy2 null mutant plants to regulate the activity of phytochromes in vivo and thereof modulate photomorphogenesis. The identification of potential inhibitors for HY2 based on the structural and functional information can be applied to herbicide design to control plant growth and development.
Tu, S. L., Chen, H. C., and Ku, L. W. (2008). Mechanistic studies of the phytochromobilin synthase HY2 from Arabidopsis. J. Biol. Chem. In press.
Tu, S. L., Rockwell, N. C., Fisher, A. J., and Lagarias, J. C. (2007). Insight into the radical mechanism of phycocyanobilin:ferredoxin oxidoreductase (PcyA) revealed by X-ray crystallography and biochemical measurements. Biochemistry. 46, 1484-1494.
Tu, S. L. , Sughrue, W., Britt, R. D., and Lagarias, J. C. (2006). A conserved histidine-aspartate pair is required for exo-vinyl reduction of biliverdin by a cyanobacterial phycocyanobilin:ferredoxin oxidoreductase. J Biol. Chem. 281, 3127-3136.
Tu, S. L., and Lagarias, J. C. (2005). The phytochromes. In Handbook of Photosensory Receptors, W. R. Briggs, and J. A. Spudich, eds. (Weinheim, Germany, Wiley-VCH).
Tu, S. L., Chen, L. J., Smith, M. D., Su, Y. S., Schnell, D. J., and Li, H. M. (2004a). Import pathways of chloroplast interior proteins and the outer-membrane protein OEP14 converge at Toc75. The Plant Cell. 16, 2078-2088.
Tu, S. L., Gunn, A., Toney, M. D., Britt, R. D., and Lagarias, J. C. (2004b). Biliverdin reduction by cyanobacterial phycocyanobilin:ferredoxin oxidoreductase (PcyA) proceeds via linear tetrapyrrole radical intermediates. J Am. Chem. Soc. 126, 8682-8693.
Chou, M. L., Fitzpatrick, L. M., Tu, S. L., Budziszewski, G., Potter-Lewis, S., Akita, M., Levin, J. Z., Keegstra, K., and Li, H. M. (2003). Tic40, a membrane-anchored co-chaperone homolog in the chloroplast protein translocon. EMBO J. 22, 2970-2980.
Sun, Y. J., Forouhar, F., H.M., L., Tu, S. L., Yeh, Y. H., Kao, S., Shr, H. L., Chou, C. C., Chen, C., and Hsiao, C. D. (2002). Crystal structure of pea Toc34, a novel GTPase of the chloroplast protein translocon. Nat. Struct. Biol. 9, 95-100.
Tu, S. L., and Li, H. M. (2000). Insertion of OEP14 into the outer envelope membrane is mediated by proteinaceous components of chloroplasts. The Plant Cell. 12, 1951-1960.
