Abstract
Utilizing an in vitro coupled assay system, we show that isolated plastids from cucumber cotyledons convert the linear tetrapyrrole biliverdin IX alpha to the free phytochrome chromophore, phytochromobilin, which assembles with oat apophytochrome to yield photoactive holoprotein. The spectral properties of this synthetic phytochrome are indistinguishable from those of the natural photoreceptor. The plastid-dependent biliverdin conversion activity is strongly stimulated by both NADPH and ATP. Substitution of the nonnatural XIII alpha isomer of biliverdin for the IX alpha isomer affords a synthetic holophytochrome adduct with blue-shifted difference spectra. These results, together with experiments using boiled plastids, indicate that phytochromobilin synthesis from biliverdin is enzyme-mediated. Experiments where NADPH (and ATP) levels in intact developing chloroplasts are manipulated by feeding the metabolites 3-phosphoglycerate, dihydroxyacetone phosphate, and glucose 6-phosphate or by illumination with white light, support the hypothesis that the enzyme that accomplishes this conversion, phytochromobilin synthase, is plastid-localized. It is therefore likely that all of the enzymes of the phytochrome chromophore biosynthetic pathway reside in the plastid.
Highlights
Utilizing an in vitro coupled assay system, we show What enzymes or cofactors areneeded for holoproteinassemthat isolated plastids from cucumber cotyledons convert the linear tetrapyrrole biliverdin I X a to the free phytochrome chromophore, phytochromobilin, which assembles with oat apophytochrome to yield photoactive holoprotein
biliverdin IXa (BV) Is Enzymatically Converted to the Free Phytochrome Chromophore in Organello-In previous studies, we showed that BV itself cannot covalently attach toapophytochrome in vitro but must first be metabolized to the free phytochrome chromophore prior to holoprotein assembly [6, 7]
When BV IXa is incubated with a plastid preparationfrom cucumber cotyledons, it is enzymatically converted to a bilin h
Summary
Utilizing an in vitro coupled assay system, we show What enzymes or cofactors areneeded for holoproteinassemthat isolated plastids from cucumber cotyledons convert the linear tetrapyrrole biliverdin I X a to the free phytochrome chromophore, phytochromobilin, which assembles with oat apophytochrome to yield photoactive holoprotein. Our studies and similar studies obinostyhnethesis of PCB in the red alga Cyanidium caldarium [9] support the model for the biosynthetic pathwayof the phytochrome chromophore shown in Scheme1 This manuscriptfocuses on phytochromobilin synthase, the penultimate enzyme of the phytochrome chromophobrieosynthetic pathway. Phytochrome synthesis tochromobilin synthesis from BV is mediated by a plastidtherefore involves the convergence of two biosynthetic path- localized enzyme system and that thenewly synthesized holways: onefor the apoprotein and another for the chromo- ophytochromeispectrophotometricallyindistinguishable phore.Phytochromehasbeen shown toregulateits own from the natural photoreceptor Knowledge of this biochemical pathway is necessary to fully Biliverdin I X a and XIIIa were prepared as described in Elich et appreciate how the level of potentially active phytochrome al. Knowledge of this biochemical pathway is necessary to fully Biliverdin I X a and XIIIa were prepared as described in Elich et appreciate how the level of potentially active phytochrome al. [6]. 4-Amino-5-hexynoic acid (AHA) was a gift from Merrell Dow (holophytochrome) is regulated within thceell
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