Katsuhiko Inomata

Green/red cyanobacteriochromes regulate complementary chromatic acclimation via a protochromic photocycle

Cyanobacteriochromes (CBCRs) are cyanobacterial members of the phytochrome superfamily of photosensors. Like phytochromes, CBCRs convert between two photostates by photoisomerization of a covalently bound linear tetrapyrrole (bilin) chromophore. Although phytochromes are red/far-red sensors, CBCRs exhibit diverse photocycles spanning the visible spectrum and the near-UV (330–680 nm). Two CBCR subfamilies detect near-UV to blue light (330–450 nm) via a “two-Cys photocycle” that couples bilin 15Z/15E photoisomerization with formation or elimination of a second bilin–cysteine adduct. On the other hand, mechanisms for tuning the absorption between the green and red regions of the spectrum have not been elucidated as of yet. CcaS and RcaE are members of a CBCR subfamily that regulates complementary chromatic acclimation, in which cyanobacteria optimize light-harvesting antennae in response to green or red ambient light. CcaS has been shown to undergo a green/red photocycle: reversible photoconversion between a green-absorbing 15Z state (15ZPg) and a red-absorbing 15E state (15EPr). We demonstrate that RcaE from Fremyella diplosiphon undergoes the same photocycle and exhibits light-regulated kinase activity. In both RcaE and CcaS, the bilin chromophore is deprotonated as 15ZPg but protonated as 15EPr. This change of bilin protonation state is modulated by three key residues that are conserved in green/red CBCRs. We therefore designate the photocycle of green/red CBCRs a “protochromic photocycle,” in which the dramatic change from green to red absorption is not induced by initial bilin photoisomerization but by a subsequent change in bilin protonation state.

Biliverdin Binds Covalently to Agrobacterium Phytochrome Agp1 via Its Ring A Vinyl Side Chain

The widely distributed phytochrome photoreceptors carry a bilin chromophore, which is covalently attached to the protein during a lyase reaction. In plant phytochromes, the natural chromophore is coupled by a thioether bond between its ring A ethylidene side chain and a conserved cysteine residue within the so-called GAF domain of the protein. Many bacterial phytochromes carry biliverdin as natural chromophore, which is coupled in a different manner to the protein. In phytochrome Agp1 of Agrobacterium tumefaciens, biliverdin is covalently attached to a cysteine residue close to the N terminus (position 20). By testing different natural and synthetic biliverdin derivatives, it was found that the ring A vinyl side chain is used for chromophore attachment. Only those bilins that have ring A vinyl side chain were covalently attached, whereas bilins with an ethylidene or ethyl side chain were bound in a noncovalent manner. Phycocyanobilin, which belongs to the latter group, was however covalently attached to a mutant in which a cysteine was introduced into the GAF domain of Agp1 (position 249). It is proposed that the regions around positions 20 and 249 are in close contact and contribute both to the chromophore pocket. In competition experiments it was found that phycocyanobilin and biliverdin bind with similar strength to the wild type protein. However, in the V249C mutant, phycocyanobilin bound much more strongly than biliverdin. This finding could explain why during phytochrome evolution in cyanobacteria, the chromophore-binding site swapped from the N terminus into the GAF domain.

The cyanobacteriochrome, TePixJ, isomerizes its own chromophore by converting phycocyanobilin to phycoviolobilin.

The cyanobacterial phototaxis regulator protein, TePixJ, is a member of the subfamily of cyanobacteriochromes that binds phycoviolobilin (PVB) as a chromophore and exhibits reversible photoconversion between blue light-absorbing (Pb) and green light-absorbing (Pg) forms. We reconstituted the PVB-binding photoactive holocomplex in vivo and in vitro. Coexpression of the apoprotein and phycocyanobilin (PCB) in Escherichia coli (in vivo reconstitution) produced a mixture of the PCB-bound and PVB-bound holoproteins. Reconstitution in vitro of the apoprotein and synthetic PCB quickly generated a photoactive complex, which covalently bound PCB and exhibited partially reversible photoconversion between two species by UV-vis spectroscopy (with a λ(max) values of 430 and 545 nm). Further incubation produced slow isomerization of PCB to PVB with concomitant improvement of photoreactivity. Site-directed mutagenesis confirmed that Cys522, and a second conserved Cys (Cys494), are both essential for the assembly of the photoactive complex. Fourier transform infrared (FTIR) spectroscopy revealed green light-induced cross-linking, and blue light-induced release, of a thiol group, possibly that of Cys494. These results suggest that the Pb/Pg-type cyanobacteriochrome TePixJ is assembled in at least three steps: (i) rapid and stable chromophorylation of PCB, (ii) additional photoreversible chromophorylation, and (iii) subsequent slow isomerization of PCB to PVB. In addition to its known autolyase activity with Cys522 and photoreversible isomerase activity (of the Z and E isomers at C15 and C16 of PCB), the GAF domain of TePixJ therefore appears to have other roles: as an isomerase (converting PCB to PVB) and as a photoreversible autolyase with a second conserved Cys residue.

Assembly of Synthetic Locked Chromophores with Agrobacterium Phytochromes Agp1 and Agp2

Phytochromes are photoreceptors with a bilin chromophore in which light triggers the conversion between the red-absorbing form Pr and the far-red-absorbing form Pfr. Agrobacterium tumefaciens has two phytochromes, Agp1 and Agp2, with antagonistic properties: in darkness, Agp1 converts slowly from Pfr to Pr, whereas Agp2 converts slowly from Pr to Pfr. In a previous study, we have assembled Agp1 with synthetic locked chromophores 15Za, 15Zs, 15Ea, and 15Es in which the C15=C16 double bond is fixed in either the E or Z configuration and the C14–C15 single bond is fixed in either the syn (s) or anti (a) conformation. In the present study, the locked chromophores 5Za and 5Zs were used for assembly with Agp1; in these chromophores, the C4=C5 double bond is fixed in the Z configuration, and the C5–C6 single bond is fixed in either the syn or anti conformation. All locked chromophores were also assembled with Agp2. The data showed that in both phytochromes the Pr chromophore adopts a C4=C5 Z C5–C6 syn C15=C16 Z C14–C15 anti stereochemistry and that in the Pfr chromophore the C15=C16 double bond has isomerized to the E configuration, whereas the C14–C15 single bond remains in the anti conformation. Photoconversion shifted the absorption maxima of the 5Zs adducts to shorter wavelengths, whereas the 5Za adducts were shifted to longer wavelengths. Thus, the C5–C6 single bond of the Pfr chromophore is rather in an anti conformation, supporting the previous suggestion that during photoconversion of phytochromes, a rotation around the ring A-B connecting single bond occurs.

Structural requirement of bilin chromophore for the photosensory specificity of phytochromes A and B

Phytochromes are an important class of chromoproteins that regulate many cellular and developmental responses to light in plants. The model plant species Arabidopsis thaliana possesses five phytochromes, which mediate distinct and overlapping responses to light. Photobiological analyses have established that, under continuous irradiation, phytochrome A is primarily responsible for plants sensitivity to far-red light, whereas the other phytochromes respond mainly to red light. The present study reports that the far-red light sensitivity of phytochrome A depends on the structure of the linear tetrapyrrole (bilin) prosthetic group. By reconstitution of holophytochrome in vivo through feeding various synthetic bilins to chromophore-deficient mutants of Arabidopsis, the requirement for a double bond on the bilin D-ring for rescuing phytochrome A function has been established. In contrast, we show that phytochrome B function can be rescued with various bilin analogs with saturated D-ring substituents.

Diketopiperazine-mediated peptide formation in aqueous solution

Though diketopiperazines (DKP) are formed in most experiments concerning the prebiotic peptide formation, the molecules have not been paid attention in the studies of chemical evolution. We have found that triglycine, tetraglycine or pentaglycine are formed in aqueous solution of glycine anhydride (DKP) and glycine, diglycine or triglycine, respectively. A reaction of alanine with DKP resulted in the formation of glycylglycylalanine under the same conditions. These results indicate that the formation of the peptide bonds proceeds through the nucleophilic attack of an amino group of the amino acids or the oligoglycines on the DKP accompanied by the ring-opening.The formation of glycine anhydride, di-, tri- and tetraglycine was also observed in a mixed aqueous solution of urea and glycine in an open system to allow the evaporation of ammonia. A probable pathway is proposed for prebiotic peptide formation through diketopiperazine on the primitive Earth.

O-silylative Passerini reaction: a new one-pot synthesis of α-siloxyamides.

A new method for the highly effective synthesis of α-siloxyamides is described. The addition of isocyanide to aldehyde proceeded smoothly in the presence of silanol to give the corresponding α-siloxyamides in high yields. A wide range of aldehydes and isocyanides are applicable in this reaction.

Assembly of Agrobacterium phytochromes Agp1 and Agp2 with doubly locked bilin chromophores.

The natural chromophore of most bacterial and fungal phytochromes is biliverdin (BV), which is incorporated in a covalent manner into the protein. Upon photoconversion between the red light-absorbing form Pr and the far-red light-absorbing form Pfr, the stereochemistry of the chromophore around the C15 methine bridge changes from Z anti to E anti. Recombinant phytochromes Agp1 and Agp2 from Agrobacterium tumefaciens were assembled with a set of synthetic chromophores, including 2,18-Et-BV, 3,18-Et-BV, and the doubly locked 5Ea15Ea-BV, 5Es15Ea-BV, 5Za15Ea-BV, and 5Zs15Ea-BV. In all chromophores, covalent bond formation is restricted. As shown by spectral changes and desalting column separation, all chromophores are bound to Agp1 and Agp2. Adducts with 2,18-Et-BV and 3,18-Et-BV undergo normal photoconversion between Pr and Pfr. As opposed to typical phytochromes, the BV-Agp2 adduct converts from Pr to Pfr in darkness. However, the 2,18-Et-BV-Agp2 and 3,18-Et-BV-Agp2 adducts can undergo dark conversion from Pr to Pfr and Pfr to Pr, showing that ring A of the chromophore has a direct impact on the direction of dark conversion. The doubly locked chromophores were designed to probe for the stereochemistry of the C5 methine bridge in the Pfr form. The adducts with 5Es15Ea-BV and 5Zs15Ea-BV absorbed in the blue spectral range only. Therefore, the C5 E syn and Z syn stereochemistries are unlikely for the Pfr chromophore of Agp1 and Agp2. According to our spectra, the Agp2 chromophore most likely adopts an E anti stereochemistry at its C5 methine bridge. Thus, during Pr to Pfr conversion, the C5 methine bridge of the chromophore might undergo a Hula-twist isomerization. In Agp1, the Pfr chromophore is most likely in the C5 Z anti stereochemistry. We propose that the stereochemistry of the C5 methine bridge might differ between different phytochromes, most particularly in the Pfr form.

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

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.

Light-Induced Conformational Changes of the Chromophore and the Protein in Phytochromes: Bacterial Phytochromes as Model Systems

Recombinant phytochromes Agp1 and Agp2 from Agrobacterium tumefaciens are used as model phytochromes for biochemical and biophysical studies. In biliverdin binding phytochromes the site for covalent attachment of the chromophore lies in the N-terminal region of the protein, different from plant phytochromes. The issue which stereochemistry the chromophore adopts in the so-called Pr and Pfr forms is addressed by using a series of locked chromophores which form spectrally characteristic adducts with Agp1 and Agp2. Studies on light-induced conformational changes of Agp1 give an insight into how the intrinsic histidine kinase is modulated by light. Comparison of the crystal structure of an Agp1 fragment with other phytochrome crystal structures supports the idea that a light induced rearrangement of subunits within the homodimer modulates the activity of the kinase.