Chlorinated auxins–how does Arabidopsis thaliana deal with them?

Plant hormones have various functions in plants and play crucial roles in all developmental and differentiation stages. Auxins constitute one of the most important groups with the major representative indole-3-acetic acid (IAA). A halogenated derivate of IAA, 4-chloro-indole-3-acetic acid (4-Cl-IAA), has previously been identified in Pisum sativum and other legumes. While the enzymes responsible for the halogenation of compounds in bacteria and fungi are well studied, the metabolic pathways leading to the production of 4-Cl-IAA in plants, especially the halogenating reaction, are still unknown. Therefore, bacterial flavin-dependent tryptophan-halogenase genes were transformed into the model organism Arabidopsis thaliana. The type of chlorinated indole derivatives that could be expected was determined by incubating wild type A. thaliana with different Cl-tryptophan derivatives. We showed that, in addition to chlorinated IAA, chlorinated IAA conjugates were synthesized. Concomitantly, we found that an auxin conjugate synthetase (GH3.3 protein) from A. thaliana was able to convert chlorinated IAAs to amino acid conjugates in vitro. In addition, we showed that the production of halogenated tryptophan (Trp), indole-3-acetonitrile (IAN) and IAA is possible in transgenic A. thaliana in planta with the help of the bacterial halogenating enzymes. Furthermore, it was investigated if there is an effect (i) of exogenously applied Cl-IAA and Cl-Trp and (ii) of endogenously chlorinated substances on the growth phenotype of the plants

Dissecting the low catalytic capability of flavin-dependent halogenases

Although flavin-dependent halogenases (FDHs) are attractive biocatalysts, their practical applications are limited because of their low catalytic efficiency. Here, we investigated the reaction mechanisms and structures of tryptophan 6-halogenase (Thal) from Streptomyces albogriseolus using stopped-flow, rapid-quench flow, quantum/mechanics molecular mechanics calculations, crystallography, and detection of intermediate (hypohalous acid [HOX]) liberation. We found that the key flavin intermediate, C4a-hydroperoxyflavin (C4aOOH-FAD), formed by Thal and other FDHs (tryptophan 7-halogenase [PrnA] and tryptophan 5-halogenase [PyrH]), can react with I-, Br-, and Cl- but not F- to form C4a-hydroxyflavin and HOX. Our experiments revealed that I- reacts with C4aOOH-FAD the fastest with the lowest energy barrier and have shown for the first time that a significant amount of the HOX formed leaks out as free HOX. This leakage is probably a major cause of low product coupling ratios in all FDHs. Site-saturation mutagenesis of Lys79 showed that changing Lys79 to any other amino acid resulted in an inactive enzyme. However, the levels of liberated HOX of these variants are all similar, implying that Lys79 probably does not form a chloramine or bromamine intermediate as previously proposed. Computational calculations revealed that Lys79 has an abnormally lower pKa compared with other Lys residues, implying that the catalytic Lys may act as a proton donor in catalysis. Analysis of new X-ray structures of Thal also explains why premixing of FDHs with reduced flavin adenine dinucleotide generally results in abolishment of C4aOOH-FAD formation. These findings reveal the hidden factors restricting FDHs capability which should be useful for future development of FDHs applications.</p

An unusual flavin-dependent halogenase from the metagenome of the marine sponge Theonella swinhoei WA

The authors thank EU BlueGenics (Seventh Framework Programme, Collaborative project “BlueGenics”, Grant no. 311848 RJMG and JP), the SNF (Grant no.205321_165695 to JP), the Helmut Horten Foundation (JP), and ERAIB (Grant no. 031A338A KHVP and RJMG) for funding.Uncultured bacteria from sponges have been demonstrated to be responsible for the generation of many potent, bioactive natural products including halogenated metabolites.1 The identification of gene clusters from the metagenomes of such bacterial communities enables the discovery of enzymes that mediate new and useful chemistries and allows insight to be gained into the biogenesis of potentially pharmacologically important natural products. Here we report a new pathway to the keramamides (krm); the first functional evidence for the existence of a distinct producer in the Theonella swinhoei WA chemotype is revealed, and a key enzyme on the pathway, a unique flavin dependent halogenase with a broad substrate specificity, and with potential as a useful new biocatalytic tool is described.PostprintPeer reviewe

The Thioesterase Bhp is Involved in the Formation of β-Hydroxytyrosine during Balhimycin Biosynthesis in Amycolatopsis balhimycina

The putative hydrolase gene bhp from the balhimycin biosynthetic gene cluster has been cloned and overexpressed in Escherichia coli. The corresponding enzyme Bhp was purified to homogeneity by nickel-chelating chromatography and characterized. Although Bhp has sequence similarities to hydrolases with “haloperoxidase”/perhydrolase activity, it did not show any enzymatic activity with standard “haloperoxidase”/perhydrolase substrates (e.g., monochlorodimedone and phenol red), nonspecific esterase substrates (such as p-nitrophenyl acetate, p-nitrophenyl phosphate and S-thiophenyl acetate) or the model lactonase substrate dihydrocoumarin. However, Bhp could be shown to catalyse the hydrolysis of S-β-hydroxytyrosyl-N-acetyl cysteamine thioester (β-OH-Tyr-SNAC) with 15 times the efficiency of S-L-tyrosyl- N-acetyl cysteamine thioester (L-Tyr-SNAC). This is in agreement with the suggestion that Bhp is involved in balhimycin biosynthesis, during which it was supposed to catalyse the hydrolysis of β-OH-Tyr-S-PCP (PCP=peptidyl carrier protein) to free β-hydroxytyrosine (β-OH-Tyr) and strongly suggests that Bhp is a thioesterase with high substrate specificity for PCP-bound β-OH-Tyr and not a “haloperoxidase”/perhydrolase or nonspecific esterase

Assessing the Conditions for Multilateral Interventions or Non-Interventions: Intervention and Non-Intervention in the Asia Pacific Region

The focus of this thesis has been on the identification of the primary conditions that attract or deter multilateral interventions into internal conflicts in the Asia Pacific region. This thesis develops a framework which is applied to four cases of internal conflict to see what roles twenty-two structural and perceptual conditions have played in determining why multilateral intervention was initiated in two of the cases, and why multilateral intervention failed to be initiated in the other two cases. The research found that multilateral organizations will accept risks and costs associated with intervention if certain structural and perceptual conditions make intervention an attractive option. These conditions are, a favourable or significant international environment or international event(s), the consent of a sovereign state (even if it is induced), sustained and critical regional and international media coverage, a complete collapse of the state in conflict tainting it with the term 'failed state', a high probability of success, potential economic benefits, a humanitarian crisis (in respect of Unregulated Population Movements and genocide/politicide), the possibility of a clear exit strategy, and a self-interested Member State who can greatly subsidize an intervention. Multilateral non-interventions, on the contrary, are driven by a combination of a lack of sustained and critically analyzed media coverage on conflict issues and consequences, generally positive tactics and strategies adopted by disputants, conflicts of a long duration, the international environment, economic factors unfavourable to intervention, resistance levels to intervention or a failure to call for intervention, lack of any clear exit points, and an escalation phase. The importance of these conditions suggest that multilateral organizations are reluctant to take risks and costs when political will, for the collective and self, are not provoked. Consequently, particular structural and perceptual conditions trigger or influence political will. The analysis of four case studies (East Timor, Solomon Islands, Philippines (Moros), and West Papua) concludes that multilateral interventions will be the exception to the rule in the foreseeable future given the obvious selection bias evident in these policies, and the project questions the ad hoc determinants of current multilateral intervention policies

A role for the cell-wall protein silacidin in cell size of the diatom Thalassiosira pseudonana

Diatoms contribute 20% of global primary production and form the basis of many marine food webs. Although their species diversity correlates with broad diversity in cell size, there is also an intraspecific cell-size plasticity due to sexual reproduction and varying environmental conditions. However, despite the ecological significance of the diatom cell size for food-web structure and global biogeochemical cycles, our knowledge about genes underpinning the size of diatom cells remains elusive. Here, a combination of reverse genetics, experimental evolution and comparative RNA8 sequencing analyses enabled us to identify a previously unknown genetic control of cell size in the diatom Thalassiosira pseudonana. In particular, the targeted deregulation of the expression of the cell-wall protein silacidin caused a significant increase in valve diameter. Remarkably, the natural downregulation of the silacidin gene transcript due to experimental evolution under low temperature also correlated with cell-size increase. Our data give first evidence for a genetically controlled regulation of cell size in Thalassiosira pseudonana and possibly other centric diatoms as they also encode the silacidin gene in their genomes