Mutational analysis of the pea phytochrome A chromophore pocket: Chromophore assembly with apophytochrome A and photoreversibility

Deforce, Lily; Furuya, Masaki; Song, Pill Soon

doi:10.1021/bi00214a014

Cited by 43 publications

(47 citation statements)

References 34 publications

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“…Hence, characterization of the molecular interaction between the chromophore and the apoprotein is crucial for understanding the functional mechanism(s) of phytochromes. Several studies have addressed this interaction by using in vitro assembly of PHYA (5,28,29,47), PHYB (35,37,48), PHYC and PHYE (43), or apoprotein mutants (30)(31)(32). However, in contrast to the vertebrate photoreceptor rhodopsin (49), in the phytochrome field, little has been done to examine the relationships among chromophore structure, its assembly to apoprotein, and photochromism of the holoprotein, because of the difficulty of synthesizing the chromophore and its structural analogs.…”

Section: Discussionmentioning

confidence: 99%

“…In vitro assembly was carried out as described (30). After disruption of the cells, the extract was clarified (12,000 ϫ g for 30 min at 4°C).…”

Section: Methodsmentioning

confidence: 99%

“…Holophytochromes assembled with PCB showed photoreversible spectral change, although its peaks were slightly blue shifted when compared with the native phytochrome (27)(28)(29). Other strategies, including systematic N-and C-terminal truncations and site-directed mutagenesis of the apoprotein, have been used to study the structural requirements of the chromophore-apoprotein interaction in terms of photochromism (30)(31)(32).…”

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confidence: 99%

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In vitro assembly of phytochrome B apoprotein with synthetic analogs of the phytochrome chromophore

Hanzawa

Inomata

Kinoshita

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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Phytochrome B (PhyB), one of the major photosensory chromoproteins in plants, mediates a variety of light-responsive developmental processes in a photoreversible manner. To analyze the structural requirements of the chromophore for the spectral properties of PhyB, we have designed and chemically synthesized 20 analogs of the linear tetrapyrrole (bilin) chromophore and reconstituted them with PhyB apoprotein (PHYB). The A-ring acts mainly as the anchor for ligation to PHYB, because the modification of the side chains at the C2 and C3 positions did not significantly influence the formation or difference spectra of adducts. In contrast, the side chains of the B-and C-rings are crucial to position the chromophore properly in the chromophore pocket of PHYB and for photoreversible spectral changes. The side-chain structure of the D-ring is required for the photoreversible spectral change of the adducts. When methyl and ethyl groups at the C17 and C18 positions are replaced with an n-propyl, n-pentyl, or n-octyl group, respectively, the photoreversible spectral change of the adducts depends on the length of the side chains. From these studies, we conclude that each pyrrole ring of the linear tetrapyrrole chromophore plays a different role in chromophore assembly and the photochromic properties of PhyB.

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Section: Discussionmentioning

confidence: 99%

“…In vitro assembly was carried out as described (30). After disruption of the cells, the extract was clarified (12,000 ϫ g for 30 min at 4°C).…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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In vitro assembly of phytochrome B apoprotein with synthetic analogs of the phytochrome chromophore

Hanzawa

Inomata

Kinoshita

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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“…One of the two GAF a5 sites, R231F, is two residues away from the eid4 mutant, which is hypersensitive to FR and has increased stability in light (Dieterle et al, 2005). Three residues map to the GAF loop, two of which when mutated in pea or oat, lead to a blue shift in the absorption maximum or to reduced rate of chromophore ligation, respectively ( Figure 3C; Deforce et al, 1993;Kim et al, 2007). A positively selected site in the PHY domain (K528G on the A branch) is in b17 of the tongue region that closes the chromophore pocket and stabilizes Pfr (Essen et al, 2008).…”

Section: Changes On the Na And A Branchesmentioning

confidence: 99%

Evolutionary Studies Illuminate the Structural-Functional Model of Plant Phytochromes

Mathews

2010

The Plant Cell

107

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A synthesis of insights from functional and evolutionary studies reveals how the phytochrome photoreceptor system has evolved to impart both stability and flexibility. Phytochromes in seed plants diverged into three major forms, phyA, phyB, and phyC, very early in the history of seed plants. Two additional forms, phyE and phyD, are restricted to flowering plants and Brassicaceae, respectively. While phyC, D, and E are absent from at least some taxa, phyA and phyB are present in all sampled seed plants and are the principal mediators of red/far-red-induced responses. Conversely, phyC-E apparently function in concert with phyB and, where present, expand the repertoire of phyB activities. Despite major advances, aspects of the structural-functional models for these photoreceptors remain elusive. Comparative sequence analyses expand the array of locus-specific mutant alleles for analysis by revealing historic mutations that occurred during gene lineage splitting and divergence. With insights from crystallographic data, a subset of these mutants can be chosen for functional studies to test their importance and determine the molecular mechanism by which they might impact light perception and signaling. In

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“…The only biliprotein lyases that had previously been characterized mechanistically in some detail are of the E/F-type (7,17,18). In the following discussion, these two types will be compared with each other and with the phytochromes and the related cyano(bacterio)chromes that bind bilin chromophores autocatalytically (24,(45)(46)(47).…”

Section: Discussionmentioning

confidence: 99%

Catalytic Mechanism of S-type Phycobiliprotein Lyase

Kupka¹,

Zhang

et al. 2009

Journal of Biological Chemistry

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We now show that CpcS1 binds PCB and PEB rapidly with bi-exponential kinetics (38/119 and 12/8300 ms, respectively). Chromophore binding to the lyase is reversible and much faster than the spontaneous, but low fidelity chromophore addition to the apo-protein in the absence of the lyase. This indicates kinetic control by the enzyme, which then transfers the chromophore to the apo-protein in a slow (tens of minutes) but stereo-and regioselectively corrects the reaction. This mode of action is reminiscent of chaperones but does not require ATP. The amino acid residues Arg-18 and Arg-149 of the lyase are essential for chromophore attachment in vitro and in Escherichia coli, mutations of His-21, His-22, Trp-75, Trp-140, and Arg-147 result in reduced activity (<30% of wild type in vitro). Mutants R147Q and W69M were active but had reduced capacity for PCB binding; additionally, with W69M there was loss of fidelity in chromophore attachment. Imidazole is a noncompetitive inhibitor, supporting a bilin-binding function of histidine. Evidence was obtained that CpcS1 also catalyzes exchange of C-␤84-bound PCB in biliproteins by PEB.Phycobiliproteins are a homologous family of light-harvesting proteins present in cyanobacteria, red algae, and cryptophytes that absorb light in the spectral region between the chlorophyll absorption maxima at ϳ430 and ϳ680 nm: they contain linear tetrapyrroles (phycobilins) of which 1-4 are covalently attached to the subunits by thioether bonds to conserved cysteines (1-4). The chromophores are derived from heme by oxidative ring-opening and subsequent reduction (5) and then attached post-translationally. The correct attachment of most chromophores is catalyzed by lyases. Three phylogenetically unrelated types of lyases have been characterized in cyanobacteria (6). They are specific for certain binding sites and chromophores (7-16), but the reaction mechanisms are still unclear. A chaperone-like action has been proposed for E/Ftype lyases that catalyze chromophore attachment to Cys-84 of ␤-subunits of phyco(erythro)cyanins (17, 18). A more highly evolved function is suggested, however, by concomitant isomerizing reactions catalyzed by certain lyases (12,19,20), and by the finding that most lyases can bind the chromophore and then transfer it to the acceptor protein (15,21).Chromophore binding is particularly pronounced with the S-type lyases. CpcS1 from Nostoc PCC7120 6 rapidly forms an adduct with phycocyanobilin (PCB), 7 and the latter can be transferred in a much slower reaction to cysteine 84 of the ␤-subunits of phycocyanin (CpcB) or phycoerythrocyanin (PecB), suggesting that PCB-CpcS1 is an intermediate of the enzymatic reaction (21). Model reactions of PCB with nucleophiles resulted in the formation of addition products in which the chromophore is isomerized and which are also substrates for a CpcS1-catalzed chromophore transfer to the apo-proteins (20). We now report chromophore binding kinetics obtained by stopped-flow techniques, and identification of functional amino acid residues f...

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Mutational analysis of the pea phytochrome A chromophore pocket: Chromophore assembly with apophytochrome A and photoreversibility

Cited by 43 publications

References 34 publications

In vitro assembly of phytochrome B apoprotein with synthetic analogs of the phytochrome chromophore

In vitro assembly of phytochrome B apoprotein with synthetic analogs of the phytochrome chromophore

Evolutionary Studies Illuminate the Structural-Functional Model of Plant Phytochromes

Catalytic Mechanism of S-type Phycobiliprotein Lyase

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