Strigolactones (SLs) are a class of phytohormones and rhizosphere signaling compounds with high structural diversity. Three enzymes, carotenoid isomerase DWARF27 and carotenoid cleavage dioxygenases CCD7 and CCD8, were previously shown to convert all-trans-β-carotene to carlactone (CL), the SL precursor. However, how CL is metabolized to SLs has remained elusive. Here, by reconstituting the SL biosynthetic pathway in Nicotiana benthamiana, we show that a rice homolog of Arabidopsis More Axillary Growth 1 (MAX1), encodes a cytochrome P450 CYP711 subfamily member that acts as a CL oxidase to stereoselectively convert CL into ent-2'-epi-5-deoxystrigol (B-C lactone ring formation), the presumed precursor of rice SLs. A protein encoded by a second rice MAX1 homolog then catalyzes the conversion of ent-2'-epi-5-deoxystrigol to orobanchol. We therefore report that two members of CYP711 enzymes can catalyze two distinct steps in SL biosynthesis, identifying the first enzymes involved in B-C ring closure and a subsequent structural diversification step of SLs.

a b s t r a c tStrigolactones are phytohormones synthesized from carotenoids via a stereospecific pathway involving the carotenoid cleavage dioxygenases 7 (CCD7) and 8. CCD7 cleaves 9-cis-b-carotene to form a supposedly 9-cis-configured b-apo-10 0 -carotenal. CCD8 converts this intermediate through a combination of yet undetermined reactions into the strigolactone-like compound carlactone. Here, we investigated the substrate and stereo-specificity of the Arabidopsis and pea CCD7 and determined the stereo-configuration of the b-apo-10 0 -carotenal intermediate by using Nuclear Magnetic Resonance Spectroscopy. Our data unequivocally demonstrate the 9-cis-configuration of the intermediate. Both CCD7s cleave different 9-cis-carotenoids, yielding hydroxylated 9-cis-apo-10 0 -carotenals that may lead to hydroxylated carlactones, but show highest affinity for 9-cis-b-carotene.

In plants, metabolic homeostasis-the driving force of growth and development-is achieved through the dynamic behavior of a network of enzymes, many of which depend on coenzymes for activity. The circadian clock is established to influence coordination of supply and demand of metabolites. Metabolic oscillations independent of the circadian clock, particularly at the subcellular level is unexplored. Here, we reveal a metabolic rhythm of the essential coenzyme thiamine diphosphate (TDP) in the Arabidopsis nucleus. We show there is temporal separation of the clock control of cellular biosynthesis and transport of TDP at the transcriptional level. Taking advantage of the sole reported riboswitch metabolite sensor in plants, we show that TDP oscillates in the nucleus. This oscillation is a function of a light-dark cycle and is independent of circadian clock control. The findings are important to understand plant fitness in terms of metabolite rhythms.

A methodology for the synthesis of oligophosphate conjugates using phosphordiamidites is described. This strategy facilitates the straightforward preparation of C2-symmetric dinucleoside tri-, penta-, and heptaphosphates. Moreover, unsymmetric compounds such as thiamine adenosine triphosphate and thiamine cytidine triphosphate can be prepared. The material is used to study the inhibitory activity of thiaminylated nucleotides against adenosine diphosphate ribosyltransferases.

Vitamin B1 is a family of molecules, the most renowned member of which is diphosphorylated thiamine (TDP)—a coenzyme vital for the activity of key enzymes of energy metabolism. Triphosphorylated thiamine derivatives also exist within this family, specifically thiamine triphosphate (TTP) and adenosine thiamine triphosphate (ATTP). They have been investigated primarily in mammalian cells and are thought to act as metabolic messengers but have not received much attention in plants. In this study, we set out to examine for the presence of these triphosphorylated thiamine derivatives in Arabidopsis. We could find TTP in Arabidopsis under standard growth conditions, but we could not detect ATTP. Interestingly, TTP is found primarily in shoot tissue. Drivers of TTP synthesis are light intensity, the proton motive force, as well as TDP content. In plants, TTP accumulates in the organellar powerhouses, the plastids, and mitochondria. Furthermore, in contrast to other B1 vitamers, there are strong oscillations in tissue levels of TTP levels over diel periods peaking early during the light period. The elevation of TTP levels during the day appears to be coupled to a photosynthesis‐driven process. We propose that TTP may signify TDP sufficiency, particularly in the organellar powerhouses, and discuss our findings in relation to its role.

Cis-diammine dichloro platinum (II) (CDDP), is a highly potent antineoplastic agent that is used in the treatment of ovarian cancer. However, the clinical use of CDDP is restricted by its severe side effects. In order to reduce these side effects and to enhance its therapeutic efficacy, we developed specific immunoconjugates consisting of the murine monoclonal antibody OC125 and CDDP, using diethylene triamine pentaacetic acid (DTPA) as a linker. The coupling efficiencies of the different preparations synthesized, varied between 1.10 +/- 0.42 and 2.65 +/- 1.60 mol of CDDP per mol of antibody protein. Despite the chemical modification of the antibody molecule, specific binding activity of the OC125-CDDP conjugates toward the CA125 antigen was maintained as was demonstrated by means of immunohisto-/cytochemical staining of frozen sections of ovarian cancer tissue, amniotic epithelium, and the CA125 positive ovarian cancer cell line NIH:OVCAR 3. The antiproliferative activity of the immunoconjugates was tested against the human ovarian cancer cell lines NIH:OVCAR 3 and SKOV 3, applying a kinetic crystal violet microassay. Despite the promising results obtained with the specific immunostaining of the target cells, no significant antiproliferative activity of our immunoconjugates against the cell lines tested was observed. One possible explanation for the lack of antitumor activity could be the fact that CA125 is released in large amounts by the NIH:OVCAR 3 cells. This may have prevented an efficient immunotargeting of the cancer cells by the formation of soluble immune complexes.

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Triphosphate tunnel metalloenzymes (TTMs) are found in all biological kingdoms and have been characterized in microorganisms and animals. Members of the TTM family already characterized have divergent biological functions and act on a range of triphosphorylated substrates (RNA, thiamine tri-phosphate, inorganic polyphosphate). TTM proteins in plants have received considerably less atten-tion and are unique in that some homologs harbor additional domains including a P-loop kinase and transmembrane domain. Here we report on structural and functional aspects of the multimodular TTM1 and TTM2 of Arabidopsis thaliana. Tissue and cellular microscopy studies show that both AtTTM1 and AtTTM2 are expressed in actively dividing (meristem) tissue and are tail-anchored pro-teins at the outer mitochondrial membrane - mediated by the single transmembrane domain at the C-terminus, supporting earlier studies. Crystal structures of AtTTM1 in the presence and absence of a non-hydrolyzable ATP analog reveal a catalytically incompetent TTM tunnel domain tightly interact-ing with the P-loop kinase domain that is locked in an inactive conformation. Structural comparison reveals that a helical hairpin may facilitate movement of the TTM domain thereby activating the kinase. Genetic studies show that AtTTM2 is important for the developmental transition from the vegetative to the reproductive phase in Arabidopsis, whereas its closest paralog AtTTM1 is not. Rational design of mutations based on the 3D structure demonstrates that both the P-loop kinase and TTM tunnel mod-ules of AtTTM2 are required for the developmental switch.