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Background: Phytochrome photoreceptors are activated by light-induced isomerization of the chromophore cofactor. Results: Photoactivation of Synechococcus OS-BЈ phytochrome breaks an unusual chromophore D-ring hydrogen bond, whereas only subtle changes occur at the A-ring linkage to the protein. Conclusion:Activation arises from a photoflip of a strongly tilted D-ring. Significance: The hypothesis that the A-ring rotates upon photon absorption is wrong.

The coding sequence of the cyanobacterium Synechocystis sp. strain PCC 6803 slr0095 gene was cloned and functionally expressed in Escherichia coli. The corresponding enzyme was classified as a cation-and S-adenosyl-L-methionine-dependent O-methyltransferase (SynOMT), consistent with considerable amino acid sequence identities to eukaryotic O-methyltransferases (OMTs). The substrate specificity of SynOMT was similar with those of plant and mammalian CCoAOMT-like proteins accepting a variety of hydroxycinnamic acids and flavonoids as substrates. In contrast to the known mammalian and plant enzymes, which exclusively methylate the meta-hydroxyl position of aromatic di-and trihydroxy systems, Syn-OMT also methylates the para-position of hydroxycinnamic acids like 5-hydroxyferulic and 3,4,5-trihydroxycinnamic acid, resulting in the formation of novel compounds. The x-ray structure of SynOMT indicates that the active site allows for two alternative orientations of the hydroxylated substrates in comparison to the active sites of animal and plant enzymes, consistent with the observed preferred para-methylation and position promiscuity. Lys 3 close to the N terminus of the recombinant protein appears to play a key role in the activity of the enzyme. The possible implications of these results with respect to modifications of precursors of polymers like lignin are discussed.2 (EC 2.1.1) is a common modification in secondary product biosynthesis (1). Sitespecific O-methylation modulates the physiological properties and the chemical reactivity of phenolic compounds and renders them more hydrophobic. Cation-dependent OMTs constitute a small group of low molecular mass (23 to 27 kDa) enzymes (2). In mammals, these enzymes play important roles in the modification of catechol neurotransmitters in the brain or may inactivate potentially bioactive metabolites like quercetin in the liver and kidney (3, 4). They are therefore referred to as catechol OMTs (COMT) and have been investigated as potential targets to cure degenerate brain diseases (5). In plants, caffeoyl-coenzyme A O-methyltransferases (CCoAOMTs), named after their preferred in vitro substrate, in conjunction with a second group of cation-independent caffeic acid OMTs, are crucial for determining the structural integrity of lignin in plant vascular tissues (6, 7). Specific subtypes of CCoAOMT-like proteins also methylate, besides caffeoyl-CoA, other phenylpropanoids, preferentially flavonoids, with vicinal dihydroxy groups (2). The threedimensional structures of eukaryotic animal and plant OMTs known so far are quite similar despite their otherwise low sequence identities, irrespective of the involvement of bivalent cations and substrates. Structural data obtained so far reveal a conserved AdoMet-binding site in all OMTs from the animal and plant kingdom (8 -10). The methyl transfer mechanism proceeds via an S n 2-like transition state and a cation-facilitated deprotonation of one of the two hydroxyl groups.Atomic structures of the cation-dependent catechol OMT from rat ...

SUMMARYThe study of glucosinolates and their regulation has provided a powerful framework for the exploration of fundamental questions about the function, evolution, and ecological significance of plant natural products, but uncertainties about their metabolism remain. Previous work has identified one thiohydroximate S-glucosyltransferase, UGT74B1, with an important role in the core pathway, but also made clear that this enzyme functions redundantly and cannot be the sole UDP-glucose dependent glucosyltransferase (UGT) in glucosinolate synthesis. Here, we present the results of a nearly comprehensive in vitro activity screen of recombinant Arabidopsis Family 1 UGTs, which implicate other members of the UGT74 clade as candidate glucosinolate biosynthetic enzymes. Systematic genetic analysis of this clade indicates that UGT74C1 plays a special role in the synthesis of aliphatic glucosinolates, a conclusion strongly supported by phylogenetic and gene expression analyses. Finally, the ability of UGT74C1 to complement phenotypes and chemotypes of the ugt74b1-2 knockout mutant and to express thiohydroximate UGT activity in planta provides conclusive evidence for UGT74C1 being an accessory enzyme in glucosinolate biosynthesis with a potential function during plant adaptation to environmental challenge.

We present structural information for oat phyA3 in the far-red-light-absorbing (Pfr) signaling state, to our knowledge the first three-dimensional (3D) information for a plant phytochrome as Pfr. Solid-state magic-angle spinning (MAS) NMR was used to detect interatomic contacts in the complete photosensory module [residues 1–595, including the NTE (N-terminal extension), PAS (Per/Arnt/Sim), GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA) and PHY (phytochrome-specific) domains but with the C-terminal PAS repeat and transmitter-like module deleted] auto-assembled in vitro with 13C- and 15N-labeled phycocyanobilin (PCB) chromophore. Thereafter, quantum mechanics/molecular mechanics (QM/MM) enabled us to refine 3D structural models constrained by the NMR data. We provide definitive atomic assignments for all carbon and nitrogen atoms of the chromophore, showing the Pfr chromophore geometry to be periplanar ZZEssa with the D-ring in a β-facial disposition incompatible with many earlier notions regarding photoconversion yet supporting circular dichroism (CD) data. The Y268 side chain is shifted radically relative to published Pfr crystal structures in order to accommodate the β-facial ring D. Our findings support a photoconversion sequence beginning with Pr photoactivation via an anticlockwise D-ring Za→Ea photoflip followed by significant shifts at the coupling of ring A to the protein, a B-ring propionate partner swap from R317 to R287, changes in the C-ring propionate hydrogen-bonding network, breakage of the D272–R552 salt bridge accompanied by sheet-to-helix refolding of the tongue region stabilized by Y326–D272–S554 hydrogen bonding, and binding of the NTE to the hydrophobic side of ring A. We discuss phyA photoconversion, including the possible roles of mesoscopic phase transitions and protonation dynamics in the chromophore pocket. We also discuss possible associations between structural changes and translocation and signaling processes within the cell.