Abstract
Phytochromobilin (PΦB) is a red/far-red light sensory pigment in plant phytochrome. PΦB synthase is a ferredoxin-dependent bilin reductase (FDBR) that catalyzes the site-specific reduction of bilins, which are sensory and photosynthesis pigments, and produces PΦB from biliverdin, a heme-derived linear tetrapyrrole pigment. Here, we determined the crystal structure of tomato PΦB synthase in complex with biliverdin at 1.95 Å resolution. The overall structure of tomato PΦB synthase was similar to those of other FDBRs, except for the addition of a long C-terminal loop and short helices. The structure further revealed that the C-terminal loop is part of the biliverdin-binding pocket and that two basic residues in the C-terminal loop form salt bridges with the propionate groups of biliverdin. This suggested that the C-terminal loop is involved in the interaction with ferredoxin and biliverdin. The configuration of biliverdin bound to tomato PΦB synthase differed from that of biliverdin bound to other FDBRs, and its orientation in PΦB synthase was inverted relative to its orientation in the other FDBRs. Structural and enzymatic analyses disclosed that two aspartic acid residues, Asp-123 and Asp-263, form hydrogen bonds with water molecules and are essential for the site-specific A-ring reduction of biliverdin. On the basis of these observations and enzymatic assays with a V121A PΦB synthase variant, we propose the following mechanistic product release mechanism: PΦB synthase-catalyzed stereospecific reduction produces 2(R)-PΦB, which when bound to PΦB synthase collides with the side chain of Val-121, releasing 2(R)-PΦB from the synthase.
Highlights
Phytochromobilin (P⌽B) is a red/far-red light sensory pigment in plant phytochrome
The chromophore of phytochrome for accepting red/far-red light is phytochromobilin (P⌽B),2 which is synthesized from a heme metabolite, biliverdin IX␣ (BV)
In cyanobacteria and red algae, phycobilins, which function in light harvesting for photosynthesis and in light sensing by cyanobacteriochrome, are produced from BV by ferredoxin-dependent bilin reductase (FDBR)
Summary
We examined the enzymatic activity of tomato monomeric P⌽B synthase using an assay reported for Arabidopsis P⌽B synthase [24]. In other FDBRs, an aspartic acid residue belonging to the central strand (S5), such as Asp-105 in Synechocystis PcyA [11], is located under the center of BV, and the carboxyl group of the aspartic acid side chain forms hydrogen bonds with the pyrrole nitrogen atoms of BV to stabilize the ZZZ-all-syn configuration This structural feature is conserved in all other FDBRs, excluding 15,16-DHBV– bound GtPEBB, in which the substrate conformation is similar to that of tomato P⌽B synthase (Fig. 2C). The light absorption shoulder observed at 740 nm in the BV–PcyA complex, which suggests that the formation of protonated BV (BVHϩ), was not observed in the BV–P⌽B synthase complex (Fig. 1A), suggesting that BV bound to P⌽B synthase exists in the neutral form and not as BVHϩ This is consistent with the structural feature of P⌽B synthase, in which the central Asp is substituted with Asn. The lactam oxygen atom of the D-ring of BV forms a hydrogen bond with a water molecule, which forms a hydrogen bond with Trp-258. Arg-207 and Lys-321 interact with the propionate group of BV via a water molecule
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