Metabolite Damage and Damage Control in a Minimal Genome.

Analysis of the genes retained in the minimized Mycoplasma JCVI-Syn3A genome established that systems that repair or preempt metabolite damage are essential to life. Several genes known to have such functions were identified and experimentally validated, including 5-formyltetrahydrofolate cycloligase, coenzyme A (CoA) disulfide reductase, and certain hydrolases. Furthermore, we discovered that an enigmatic YqeK hydrolase domain fused to NadD has a novel proofreading function in NAD synthesis and could double as a MutT-like sanitizing enzyme for the nucleotide pool. Finally, we combined metabolomics and cheminformatics approaches to extend the core metabolic map of JCVI-Syn3A to include promiscuous enzymatic reactions and spontaneous side reactions. This extension revealed that several key metabolite damage control systems remain to be identified in JCVI-Syn3A, such as that for methylglyoxal. IMPORTANCE Metabolite damage and repair mechanisms are being increasingly recognized. We present here compelling genetic and biochemical evidence for the universal importance of these mechanisms by demonstrating that stripping a genome down to its barest essentials leaves metabolite damage control systems in place. Furthermore, our metabolomic and cheminformatic results point to the existence of a network of metabolite damage and damage control reactions that extends far beyond the corners of it that have been characterized so far. In sum, there can be little room left to doubt that metabolite damage and the systems that counter it are mainstream metabolic processes that cannot be separated from life itself.

Solution of the multistep pathway for assembly of corynanthean, strychnos, iboga, and aspidosperma monoterpenoid indole alkaloids from 19E-geissoschizine.

Monoterpenoid indole alkaloids (MIAs) possess a diversity of alkaloid skeletons whose biosynthesis is poorly understood. A bioinformatic search of candidate genes, combined with their virus-induced gene silencing, targeted MIA profiling and in vitro/in vivo pathway reconstitution identified and functionally characterized six genes as well as a seventh enzyme reaction required for the conversion of 19E-geissoschizine to tabersonine and catharanthine. The involvement of pathway intermediates in the formation of four MIA skeletons is described, and the role of stemmadenine-O-acetylation in providing necessary reactive substrates for the formation of iboga and aspidosperma MIAs is described. The results enable the assembly of complex dimeric MIAs used in cancer chemotherapy and open the way to production of many other biologically active MIAs that are not easily available from nature.

Geissoschizine synthase controls flux in the formation of monoterpenoid indole alkaloids in a Catharanthus roseus mutant.

MAIN CONCLUSION: A Catharanthus roseus mutant accumulates high levels of ajmalicine at the expense of catharanthine and vindoline. The altered chemistry depends on increased expression and biochemical activities of strictosidine ß-glucosidase and ajmalicine synthase activities and reduced expression and biochemical activity of geissoschizine synthase. The Madagascar periwinkle [Catharanthus roseus (L.) G. Don] is a commercially important horticultural flower species and is a valuable source for several monoterpenoid indole alkaloids (MIAs), such as the powerful antihypertensive drug ajmalicine and the antineoplastic agents, vinblastine and vincristine. While biosynthesis of the common MIA precursor strictosidine and its reactive aglycones has been elucidated, the branch point steps leading to the formation of different classes of MIAs remain poorly characterized. Screening of 3600 ethyl methyl sulfonate mutagenized C. roseus plants using a simple thin-layer chromatography screen yielded a mutant (M2-0754) accumulating high levels of ajmalicine together with significantly lower levels of catharanthine and vindoline. Comparative bioinformatic analyses, virus-induced gene silencing, and biochemical characterization identified geissoschizine synthase, the gateway enzyme that controls flux for the formation of iboga and aspidosperma MIAs. The reduction of geissoschizine synthase transcripts in this high ajmalicine mutant, together with increased transcripts and enzyme activities of strictosidine ß-glucosidase and of heteroyohimbine synthase, explains the preferential formation of ajmalicine in the mutant instead of catharanthine and vindoline that accumulates in the wild-type parent. Reciprocal crosses established that that the high ajmalicine phenotype is inherited as a Mendelian recessive trait.

A tabersonine 3-reductase Catharanthus roseus mutant accumulates vindoline pathway intermediates.

MAIN CONCLUSION: Monoterpenoid indole alkaloids (MIAs) have remarkable biological properties that have led to their medical uses for a variety of human diseases. Mutagenesis has been used to generate plants with new alkaloid profiles and a useful screen for rapid comparison of MIA profiles is described. The MIA mutants identified are useful for investigating MIA biosynthesis and for targeted production of these specialised metabolites. The Madagascar periwinkle (Catharanthus roseus) is the sole source of the dimeric anticancer monoterpenoid indole alkaloids (MIAs), 3',4'-anhydrovinblastine and derivatives, which are formed via the coupling of the MIAs, catharanthine and vindoline. While intense efforts to identify parts of the complex pathways involved in the assembly of these dimers have been successful, our understanding of MIA biochemistry in C. roseus remains limited. A simple thin layer chromatography screen of 4000 ethyl methanesulfonate-metagenized M2 plants is described to identify mutant lines with altered MIA profiles. One mutant (M2-1865) accumulated reduced levels of vindoline inside the leaves in favour of high levels of tabersonine-2,3-epoxide and 16-methoxytabersonine-2,3-epoxide on the leaf surface. This MIA profile suggested that changes in tabersonine 3-reductase (T3R) activity might be responsible for the observed phenotype. Molecular cloning of mutant and wild type T3R revealed two nucleotide substitutions at cytosine residues 565 (CAT to TAT) and 903 (ACC to ACA) in the mutant corresponding to substitution (H189Y) and silent (T305T) amino acid mutations, respectively, in the protein. The single amino acid substitution in the mutant T3R protein diminished the biochemical activity of T3R by 95% that explained the reason for the low vindoline phenotype of the mutant. This phenotype was recessive and exhibited standard Mendelian single-gene inheritance. The stable formation and accumulation of epoxides in the M2-1865 mutant provides a dependable biological source of these two MIAs.

A strictly monofunctional bacterial hydroxymethylpyrimidine phosphate kinase precludes damaging errors in thiamin biosynthesis.

The canonical kinase (ThiD) that converts the thiamin biosynthesis intermediate hydroxymethylpyrimidine (HMP) monophosphate to the diphosphate can also very efficiently convert free HMP to the monophosphate in prokaryotes, plants, and fungi. This HMP kinase activity enables salvage of HMP, but it is not substrate-specific and so allows toxic HMP analogs and damage products to infiltrate the thiamin biosynthesis pathway. Comparative analysis of bacterial genomes uncovered a gene, thiD2 , that is often fused to the thiamin synthesis gene thiE and could potentially encode a replacement for ThiD. Standalone ThiD2 proteins and ThiD2 fusion domains are small (~130-residues) and do not belong to any previously known protein family. Genetic and biochemical analyses showed that representative standalone and fused ThiD2 proteins catalyze phosphorylation of HMP monophosphate, but not of HMP or its toxic analogs and damage products such as bacimethrin and 5-(hydroxymethyl)-2-methylpyrimidin-4-ol. As strictly monofunctional HMP monophosphate kinases, ThiD2 proteins eliminate a potentially fatal vulnerability of canonical ThiD, at the cost of the ability to reclaim HMP formed by thiamin turnover.

'Nothing of chemistry disappears in biology': the Top 30 damage-prone endogenous metabolites.

Many common metabolites are intrinsically unstable and reactive, and hence prone to chemical (i.e. non-enzymatic) damage in vivo Although this fact is widely recognized, the purely chemical side-reactions of metabolic intermediates can be surprisingly hard to track down in the literature and are often treated in an unprioritized case-by-case way. Moreover, spontaneous chemical side-reactions tend to be overshadowed today by side-reactions mediated by promiscuous ('sloppy') enzymes even though chemical damage to metabolites may be even more prevalent than damage from enzyme sloppiness, has similar outcomes, and is held in check by similar biochemical repair or pre-emption mechanisms. To address these limitations and imbalances, here we draw together and systematically integrate information from the (bio)chemical literature, from cheminformatics, and from genome-scale metabolic models to objectively define a 'Top 30' list of damage-prone metabolites. A foundational part of this process was to derive general reaction rules for the damage chemistries involved. The criteria for a 'Top 30' metabolite included predicted chemical reactivity, essentiality, and occurrence in diverse organisms. We also explain how the damage chemistry reaction rules ('operators') are implemented in the Chemical-Damage-MINE (CD-MINE) database (minedatabase.mcs.anl.gov/#/top30) to provide a predictive tool for many additional potential metabolite damage products. Lastly, we illustrate how defining a 'Top 30' list can drive genomics-enabled discovery of the enzymes of previously unrecognized damage-control systems, and how applying chemical damage reaction rules can help identify previously unknown peaks in metabolomics profiles.

Proteins of Unknown Biochemical Function: A Persistent Problem and a Roadmap to Help Overcome It.

The number of sequenced genomes is rapidly increasing, but functional annotation of the genes in these genomes lags far behind. Even in Arabidopsis (Arabidopsis thaliana), only approximately 40% of enzyme- and transporter-encoding genes have credible functional annotations, and this number is even lower in nonmodel plants. Functional characterization of unknown genes is a challenge, but various databases (e.g. for protein localization and coexpression) can be mined to provide clues. If homologous microbial genes exist-and about one-half the genes encoding unknown enzymes and transporters in Arabidopsis have microbial homologs-cross-kingdom comparative genomics can powerfully complement plant-based data. Multiple lines of evidence can strengthen predictions and warrant experimental characterization. In some cases, relatively quick tests in genetically tractable microbes can determine whether a prediction merits biochemical validation, which is costly and demands specialized skills.

A pair of tabersonine 16-hydroxylases initiates the synthesis of vindoline in an organ-dependent manner in Catharanthus roseus.

Hydroxylation of tabersonine at the C-16 position, catalyzed by tabersonine 16-hydroxylase (T16H), initiates the synthesis of vindoline that constitutes the main alkaloid accumulated in leaves of Catharanthus roseus. Over the last decade, this reaction has been associated with CYP71D12 cloned from undifferentiated C. roseus cells. In this study, we isolated a second cytochrome P450 (CYP71D351) displaying T16H activity. Biochemical characterization demonstrated that CYP71D12 and CYP71D351 both exhibit high affinity for tabersonine and narrow substrate specificity, making of T16H, to our knowledge, the first alkaloid biosynthetic enzyme displaying two isoforms encoded by distinct genes characterized to date in C. roseus. However, both genes dramatically diverge in transcript distribution in planta. While CYP71D12 (T16H1) expression is restricted to flowers and undifferentiated cells, the CYP71D351 (T16H2) expression profile is similar to the other vindoline biosynthetic genes reaching a maximum in young leaves. Moreover, transcript localization by carborundum abrasion and RNA in situ hybridization demonstrated that CYP71D351 messenger RNAs are specifically located to leaf epidermis, which also hosts the next step of vindoline biosynthesis. Comparison of high- and low-vindoline-accumulating C. roseus cultivars also highlights the direct correlation between CYP71D351 transcript and vindoline levels. In addition, CYP71D351 down-regulation mediated by virus-induced gene silencing reduces vindoline accumulation in leaves and redirects the biosynthetic flux toward the production of unmodified alkaloids at the C-16 position. All these data demonstrate that tabersonine 16-hydroxylation is orchestrated in an organ-dependent manner by two genes including CYP71D351, which encodes the specific T16H isoform acting in the foliar vindoline biosynthesis.

Functional characterization of amyrin synthase involved in ursolic acid biosynthesis in Catharanthus roseus leaf epidermis.

Catharanthus roseus accumulates high levels of the pentacyclic triterpene, ursolic acid, as a component of its wax exudate on the leaf surface. Bioinformatic analyses of transcripts derived from the leaf epidermis provide evidence for the specialized role of this tissue in the biosynthesis of ursolic acid. Cloning and functional expression in yeast of a triterpene synthase derived from this tissue showed it to be predominantly an α-amyrin synthase (CrAS), since the α-amyrin to ß-amyrin reaction products accumulated in a 5:1 ratio. Expression analysis of CrAS showed that triterpene biosynthesis occurs predominantly in the youngest leaf tissues and in the earliest stages of seedling development. Further studies using laser capture microdissection to harvest RNA from epidermis, mesophyll, idioblasts, laticifers and vasculature of leaves showed the leaf epidermis to be the preferred sites of CrAS expression and provide conclusive evidence for the involvement of this tissue in the biosynthesis of ursolic acid in C. roseus.

Evidence for the localization of the Arabidopsis cytokinin receptors AHK3 and AHK4 in the endoplasmic reticulum.

Cytokinins are hormones that are involved in various processes of plant growth and development. The model of cytokinin signalling starts with hormone perception through membrane-localized histidine kinase receptors. Although the biochemical properties and functions of these receptors have been extensively studied, there is no solid proof of their subcellular localization. Here, cell biological and biochemical evidence for the localization of functional fluorophor-tagged fusions of Arabidopsis histidine kinase 3 (AHK3) and 4 (AHK4), members of the cytokinin receptor family, in the endoplasmic reticulum (ER) is provided. Furthermore, membrane-bound AHK3 interacts with AHK4 in vivo. The ER localization and putative function of cytokinin receptors from the ER have major impacts on the concept of cytokinin perception and signalling, and hormonal cross-talk in plants.

Vinca drug components accumulate exclusively in leaf exudates of Madagascar periwinkle.

The monoterpenoid indole alkaloids (MIAs) of Madagascar periwinkle (Catharanthus roseus) continue to be the most important source of natural drugs in chemotherapy treatments for a range of human cancers. These anticancer drugs are derived from the coupling of catharanthine and vindoline to yield powerful dimeric MIAs that prevent cell division. However the precise mechanisms for their assembly within plants remain obscure. Here we report that the complex development-, environment-, organ-, and cell-specific controls involved in expression of MIA pathways are coupled to secretory mechanisms that keep catharanthine and vindoline separated from each other in living plants. Although the entire production of catharanthine and vindoline occurs in young developing leaves, catharanthine accumulates in leaf wax exudates of leaves, whereas vindoline is found within leaf cells. The spatial separation of these two MIAs provides a biological explanation for the low levels of dimeric anticancer drugs found in the plant that result in their high cost of commercial production. The ability of catharanthine to inhibit the growth of fungal zoospores at physiological concentrations found on the leaf surface of Catharanthus leaves, as well as its insect toxicity, provide an additional biological role for its secretion. We anticipate that this discovery will trigger a broad search for plants that secrete alkaloids, the biological mechanisms involved in their secretion to the plant surface, and the ecological roles played by them.