Phytobilins are linear tetrapyrrole precursors of the light-harvesting prosthetic groups of the phytochrome photoreceptors of plants and the phycobiliprotein photosynthetic antennae of cyanobacteria, red algae, and cryptomonads. Previous biochemical studies have established that phytobilins are synthesized from heme via the intermediacy of biliverdin IX alpha (BV), which is reduced subsequently by ferredoxin-dependent bilin reductases with different double-bond specificities. By exploiting the sequence of phytochromobilin synthase (HY2) of Arabidopsis, an enzyme that catalyzes the ferredoxin-dependent conversion of BV to the phytochrome chromophore precursor phytochromobilin, genes encoding putative bilin reductases were identified in the genomes of various cyanobacteria, oxyphotobacteria, and plants. Phylogenetic analyses resolved four classes of HY2-related genes, one of which encodes red chlorophyll catabolite reductases, which are bilin reductases involved in chlorophyll catabolism in plants. To test the catalytic activities of these putative enzymes, representative HY2-related genes from each class were amplified by the polymerase chain reaction and expressed in Escherichia coli. Using a coupled apophytochrome assembly assay and HPLC analysis, we examined the ability of the recombinant proteins to catalyze the ferredoxin-dependent reduction of BV to phytobilins. These investigations defined three new classes of bilin reductases with distinct substrate/product specificities that are involved in the biosynthesis of the phycobiliprotein chromophore precursors phycoerythrobilin and phycocyanobilin. Implications of these results are discussed with regard to the pathways of phytobilin biosynthesis and their evolution.

Light perception by the plant photoreceptor phytochrome requires the tetrapyrrole chromophore phytochromobilin (P Phi B), which is covalently attached to a large apoprotein. Arabidopsis mutants hy1 and hy2, which are defective in P Phi B biosynthesis, display altered responses to light due to a deficiency in photoactive phytochrome. Here, we describe the isolation of the HY2 gene by map-based cloning. hy2 mutant alleles possess alterations within this locus, some of which affect the expression of the HY2 transcript. HY2 encodes a soluble protein precursor of 38 kD with a putative N-terminal plastid transit peptide. The HY2 transit peptide is sufficient to localize the reporter green fluorescent protein to plastids. Purified mature recombinant HY2 protein exhibits P Phi B synthase activity (i.e., ferredoxin-dependent reduction of biliverdin IX alpha to P Phi B), as confirmed by HPLC and by the ability of the bilin reaction products to combine with apophytochrome to yield photoactive holophytochrome. Database searches and hybridization studies suggest that HY2 is a unique gene in the Arabidopsis genome that is related to a family of proteins found in oxygenic photosynthetic bacteria.

In cyanobacteria, the biosynthesis of the phycobiliprotein and phytochrome chromophore precursor phycocyanobilin is catalyzed by the ferredoxin-dependent enzyme phycocyanobilin:ferredoxin oxidoreductase (PcyA), which mediates an atypical four-electron reduction of biliverdin IX␣. Here we describe the expression, affinity purification, and biochemical characterization of recombinant PcyA from Anabaena sp. PCC 7120. A monomeric protein with a native M r of 30,400 ؎ 5,000, recombinant PcyA forms a tight and stable stoichiometric complex with its substrate biliverdin IX␣. 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 18 1 ,18 2 -dihydrobiliverdin, indicating that exovinyl reduction precedes A-ring (endovinyl) 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 biliverdin IX␣ reduction by phycocyanobilin:ferredoxin oxidoreductase is envisaged.

Light perception by the plant photoreceptor phytochrome requires the tetrapyrrole chromophore phytochromobilin (P ⌽ B), which is covalently attached to a large apoprotein. Arabidopsis mutants hy1 and hy2 , which are defective in P ⌽ B biosynthesis, display altered responses to light due to a deficiency in photoactive phytochrome. Here, we describe the isolation of the HY2 gene by map-based cloning. hy2 mutant alleles possess alterations within this locus, some of which affect the expression of the HY2 transcript. HY2 encodes a soluble protein precursor of 38 kD with a putative N-terminal plastid transit peptide. The HY2 transit peptide is sufficient to localize the reporter green fluorescent protein to plastids. Purified mature recombinant HY2 protein exhibits P ⌽ B synthase activity (i.e., ferredoxin-dependent reduction of biliverdin IX ␣ to P ⌽ B), as confirmed by HPLC and by the ability of the bilin reaction products to combine with apophytochrome to yield photoactive holophytochrome. Database searches and hybridization studies suggest that HY2 is a unique gene in the Arabidopsis genome that is related to a family of proteins found in oxygenic photosynthetic bacteria. INTRODUCTIONPlants are exquisitely sensitive to their environment. Because they are sessile and use light as the energy source for photosynthesis, plants have developed well-refined photoreception and signaling systems to modulate their growth and development. The family of phytochromes, which are sensory photoreceptors for red and far-red lights, plays a key role in mediating responses to light quality, quantity, direction, and duration throughout plant development (Kendrick and Kronenberg, 1994;Quail et al., 1995;Furuya and Schäfer, 1996;Neff et al., 2000). Plant phytochromes are homodimers composed of ‫ف‬ 125-kD subunits each with a thioether-linked phytochromobilin (P ⌽ B) prosthetic group (Lagarias and Rapoport, 1980). Phytochrome action depends on its ability to photointerconvert between the red light-absorbing form and the far-red-light-absorbing form, a property conferred by covalently bound P ⌽ B in holophytochrome.Two pathways are involved in the biosynthesis of holophytochrome, one for the apoprotein, which is encoded by a small multigene family (e.g., PHYA-E in Arabidopsis) (Sharrock and Quail, 1989;Clack et al., 1994), and another for the synthesis of the P ⌽ B (Terry et al., 1993). Apophytochrome is synthesized in the cytosol, whereas P ⌽ B is synthesized entirely within the plastid compartment, followed by its release to the cytosol, where holophytochrome assembly occurs ( Figure 1). Based on spectroscopic studies of purified phytochromes, in vitro bilin assembly studies with recombinant apophytochromes, and physiological analyses of chromophore-deficient mutants, P ⌽ B appears to be the immediate chromophore precursor of all higher plant and cryptophyte phytochromes (Terry et al., 1993;Terry, 1997). P ⌽ B is synthesized from 5-aminolevulinic acid and shares many intermediates with the pathways of chlorophyll and heme biosynthesis...

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