Research Interests: The Lagarias Laboratory
Ferredoxin Dependent Bilin Reductases
Linear tetrapyrrole (bilin) biosynthesis shares common intermediates with the chlorophyll and heme biosynthetic pathways up to the level of protoporphyrin IX after which the pathways diverge. The major classes of bilins are derived from heme, via its conversion to biliverdin IXα (BV) - the first committed intermediate in bilin biosynthesis. The metabolic fate of BV differs in mammals, cyanobacteria, and plants, with BV being metabolized by different reductases with unique double bond specificities (Scheme 1). Plants and most oxygenic photosynthetic organisms possess phytobilins that arise via the activity of members of the ferredoxin dependent bilin reductase (FDBR) family of enzymes. In contrast with NADPH-dependent BV reductases BVR and BvdR, FDBRs catalyze the ferredoxin-dependent 2- or 4-electron reduction of BV to yield the phytobilins, phytochromobilin (PΦB), phycocyanobilin (PCB) and phycoerythrobilin (PEB). These metal-free enzymes possess high-affinity bilin binding sites that stereoselectively transfer protons via single electron transfers from reduced ferredoxin. Catalysis is accompanied by formation of bilin radical intermediates that are not released from the enzyme. Structural analyses have shown that FDBRs adopt a protein fold similar to that of oxygen-dependent coproporphyrinogen oxidase, suggesting that FDBRs have evolved from enzymes that play a core role in tetrapyrrole metabolism in all aerobic organisms (Tu et al, 2007).
Scheme 1. Committed steps of phytobilin biosynthesis in cyanobacteria and plants. Heme oxygenases (HO1/HY1) catalyze the ferredoxin-dependent production of biliverdin IXα (BV) in plants and cyanobacteria. BV reduction by one of several ferredoxin-dependent bilin reductases, HY2, PcyA, PebAB, or the NADPH-dependent BV reductase BvdR with distinct regiospecificities yield a wide variety of linear tetrapyrrole precursors of the chromophores of phycobiliproteins, phytochromes and phytochrome-related proteins. Bilin attachment to apophytochromes is autocatalytic while bilin lyases, CpcEF, CpeYZ or PecEF, are required to catalyze bilin attachment/isomerization to apophycobiliproteins
FDBRs also play a central role in the catabolism of chlorophyll, a process critical for minimizing photooxidative damage from released chlorophyll metabolites that accompany pathogen infection, misregulation of tetrapyrrole metabolism and/or other environmental stresses. The FDBR red chlorophyll catabolite reductase (RCCR) is critical for chlorophyll breakdown that occurs during leaf sensence in the fall (see fall colors in Kyoto). Recent studies in our lab indicate that these enzymes mediate bilin reduction via radical intermediates. Through biochemical analysis, spectroscopic and x-ray crystallography of recombinant FDBRs, we seek to elucidate both the chemical mechanism and molecular basis of their unique substrate/product specificities, as well as to engineer FDBRs with novel biochemical activities. This mechanistic understanding will facilitate design of inhibitors of the FDBR family, which may prove to be potent herbicides, algicides and/or regulators of light-mediated plant growth and development. Ultimately, this insight will open new avenues for generation of inactive, hyperactive and/or spectrally shifted phytochromes in vivo for tailoring photomorphogenesis of agronomically important species.
A long-term goal of these investigations is to rationally alter the natural responses of plants to their light environment. In this regard, we have demonstrated that constitutive expression of the mammalian enzyme biliverdin reductase (BVR) in transgenic plants alters a broad range of photomorphogenetic responses throughout their life cycle. These include reduced light-mediated seed germination, inability to de-etiolate under red or far red light, constitutive shade avoidance, and early flowering phenotypes, all of which can readily be ascribed to a loss of photoactive holophytochrome. In collaboration with Takayuki Kohchi at Kyoto University, the feasibility of altering the spectral sensitivity of plants was demonstrated by complementation of phytochromobilin synthase-deficient mutants with plastid-targeted PcyA (Kami et al, 2002). Ongoing studies to assess the feasibility of selective inhibition of phytochrome-mediated responses by subcellular targeted and tissue-specific expression of bilin-metabolizing enzymes in transgenic plants are underway in our laboratory and that of Beronda Montgomery-Kaguri at Michigan State University (Warnasooriya and Montgomery, 2009). Through selective expression of bilin reductases in transgenic plants, we hope to better define sites of photoperception for selective photomorphogenetic responses and ultimately to alter individual phytochrome-mediated responses in agronomically important plant species.