Functional Genomic Analysis of the HY2 Family of Ferredoxin-Dependent Bilin Reductases from Oxygenic Photosynthetic Organisms

Abstract

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.

Phycocyanobilin:Ferredoxin Oxidoreductase ofAnabaena sp. PCC 7120

“…PCC 7120 pcyA was cloned into the Escherichia coli expression vector pGEX-6-P1 (Amersham Biosciences) to produce pGEXpcyA (4). E. coli strain DH5␣ containing pGEXpcyA was induced to express glutathione S-transferase-PcyA, which was purified according to instructions supplied by the manufacturer and protocols described earlier (10).…”

“…Standard assays contained 1.5 M PcyA, 4.8 M ferredoxin, and 5 M BV IX␣ in 25 mM TES-KOH, pH 7.5 (assay buffer), and were incubated for 30 min at 28°C under green safe light unless otherwise specified. Following catalysis, bilins were isolated using a C18 Sep-Pak column (Waters) and evaporated to dryness in vacuo (4).…”

“…The metabolic fate of BV IX␣ differs in mammals, cyanobacteria, and plants, where BV IX␣ is metabolized by different reductases with unique double bond specificities. In contrast with the mammalian NAD(P)H-dependent BV IX␣ reductase, cyanobacteria and red algae possess ferredoxin-dependent bilin reductases primarily for the synthesis of the linear tetrapyrrole precursors of their phycobiliprotein light-harvesting antennae complexes, whereas evolutionarily related ferredoxin-dependent bilin reductases are found in higher plants for the synthesis of the phytochrome chromophore precursor phytochromobilin (P⌽B) (3,4).…”

“…Designated phycocyanobilin:ferredoxin oxidoreductases (EC 1.3.7.5), PcyA enzymes are atypical bilin reductases, because they catalyze a four-electron reduction; all others catalyze two-electron reductions (4). Formally two-electron reductions of vinyl substituents on the pyrrole A-and D-rings of BV IX␣, the sequence of reductions mediated by PcyA, is presently unknown (see Fig.…”

“…1). Neither of the two likely dihydrobiliverdin (DHBV) intermediates, (3Z)-P⌽B or 18 1 ,18 2 -DHBV IX␣, have yet been detected (4). Since (3Z)-P⌽B is an intermediate in the biosynthesis of PCB, the precursor of the phytochrome chromophore in the green alga Mesotaenium caldariorum (5), its intermediacy in the PcyA-mediated biosynthesis of PCB in cyanobacteria is therefore a reasonable possibility.…”