Plants produce an enormous array of biologically active metabolites, often with stereochemical variations on the same molecular scaffold. These changes in stereochemistry dramatically impact ...biological activity. Notably, the stereoisomers of the heteroyohimbine alkaloids show diverse pharmacological activities. We reported a medium chain dehydrogenase/reductase (MDR) from Catharanthus roseus that catalyses formation of a heteroyohimbine isomer. Here we report the discovery of additional heteroyohimbine synthases (HYSs), one of which produces a mixture of diastereomers. The crystal structures for three HYSs have been solved, providing insight into the mechanism of reactivity and stereoselectivity, with mutation of one loop transforming product specificity. Localization and gene silencing experiments provide a basis for understanding the function of these enzymes in vivo. This work sets the stage to explore how MDRs evolved to generate structural and biological diversity in specialized plant metabolism and opens the possibility for metabolic engineering of new compounds based on this scaffold.
Abstract
Calcium release to the nucleoplasm of root meristem cells was demonstrated to modulate root development. The calcium channel encoded by cyclic nucleotide-gated channel (CNGC) 15 localizes at ...the nuclear envelope in young Arabidopsis seedlings. In contrast, at later stages of root growth, overexpression analysis showed that AtCNGC15 can relocalize to the plasma membrane to mediate primary nitrate-induced gene expression. This raises the question as to whether nuclear localized AtCNGC15 is required for root apical meristem development in young Arabidopsis seedlings, and whether nitrate signalling occurs independently of nuclear localized AtCNGC15 at this developmental stage. In this study, we characterize a novel mutant allele of AtCNGC15 and demonstrate that the mutation of a highly conserved aspartic acid in the C-linker domain is sufficient to impair the gating of AtCNCG15. We demonstrate that AtCNGC15 mediates the nuclear calcium release that modulates root apical meristem development and nitrate-induced LBD39 expression. We also show that, in the presence of nitrate, the relocalization of AtCNGC15 at the plasma membrane occurs specifically in the columella cells. Our results further suggest that the induction of LBD37, LBD38, and LBD39 in the presence of nitrate is modulated by different inputs of cytoplasmic or nuclear calcium release.
Nuclear calcium release, mediated by cyclic nucleotide-gated channel 15, modulates root meristem development and nitrate-induced gene expression in Arabidopsis primary root meristems.
Graphical abstract Schematic describing species-specific natural variation in developmental and dynamic acclimation in response to light environment. Development in the prevailing growth light ...environment (illustrated in yellow) is determined by acclimatory responses that set the plant's overall maximum potential photosynthetic capacity. Plant dynamic responses (illustrated in blue) are modulated in by the natural fluctuating light environment and influence biochemical and cellular processes that determine the plant's actual photosynthetic rate. The time scale for developmental acclimation is in the time scale of days to weeks whilst dynamic responses occur from seconds to tens of minutes. Developmental capacity and dynamic responses ultimately determine overall net capacity. ET — photosynthetic electron transport and CC — Calvin cycle. Highlights ► Light conditions in natural environments are unpredictable and change rapidly ► Plant responses to changes in light availability varies between species ► The genetic basis of this variation can be exploited to improve photosynthesis.
Novel bacterial type II topoisomerase inhibitors (NBTIs) stabilize single-strand DNA cleavage breaks by DNA gyrase but their exact mechanism of action has remained hypothetical until now. We have ...designed a small library of NBTIs with an improved DNA gyrase-binding moiety resulting in low nanomolar inhibition and very potent antibacterial activity. They stabilize single-stranded cleavage complexes and, importantly, we have obtained the crystal structure where an NBTI binds gyrase-DNA in a single conformation lacking apparent static disorder. This directly proves the previously postulated NBTI mechanism of action and shows that they stabilize single-strand cleavage through asymmetric intercalation with a shift of the scissile phosphate. This crystal stucture shows that the chlorine forms a halogen bond with the backbone carbonyls of the two symmetry-related Ala68 residues. To the best of our knowledge, such a so-called symmetrical bifurcated halogen bond has not been identified in a biological system until now.
Terpene synthases typically form complex molecular scaffolds by concerted activation and cyclization of linear starting materials in a single enzyme active site. Here we show that iridoid synthase, ...an atypical reductive terpene synthase, catalyzes the activation of its substrate 8-oxogeranial into a reactive enol intermediate, but does not catalyze the subsequent cyclization into nepetalactol. This discovery led us to identify a class of nepetalactol-related short-chain dehydrogenase enzymes (NEPS) from catmint (Nepeta mussinii) that capture this reactive intermediate and catalyze the stereoselective cyclisation into distinct nepetalactol stereoisomers. Subsequent oxidation of nepetalactols by NEPS1 provides nepetalactones, metabolites that are well known for both insect-repellent activity and euphoric effects in cats. Structural characterization of the NEPS3 cyclase reveals that it binds to NAD
yet does not utilize it chemically for a non-oxidoreductive formal 4 + 2 cyclization. These discoveries will complement metabolic reconstructions of iridoid and monoterpene indole alkaloid biosynthesis.
The widespread second messenger molecule cyclic di-GMP (cdG) regulates the transition from motile and virulent lifestyles to sessile, biofilm-forming ones in a wide range of bacteria. Many pathogenic ...and commensal bacterial-host interactions are known to be controlled by cdG signaling. Although the biochemistry of cyclic dinucleotide metabolism is well understood, much remains to be discovered about the downstream signaling pathways that induce bacterial responses upon cdG binding. As part of our ongoing research into the role of cdG signaling in plant-associated Pseudomonas species, we carried out an affinity capture screen for cdG binding proteins in the model organism Pseudomonas fluorescens SBW25. The flagella export AAA+ ATPase FliI was identified as a result of this screen and subsequently shown to bind specifically to the cdG molecule, with a KD in the low micromolar range. The interaction between FliI and cdG appears to be very widespread. In addition to FliI homologs from diverse bacterial species, high affinity binding was also observed for the type III secretion system homolog HrcN and the type VI ATPase ClpB2. The addition of cdG was shown to inhibit FliI and HrcN ATPase activity in vitro. Finally, a combination of site-specific mutagenesis, mass spectrometry, and in silico analysis was used to predict that cdG binds to FliI in a pocket of highly conserved residues at the interface between two FliI subunits. Our results suggest a novel, fundamental role for cdG in controlling the function of multiple important bacterial export pathways, through direct allosteric control of export ATPase proteins.
Background: AAA+ ATPase proteins play integral roles in the export apparatus of many bacterial organelles.
Results: The second messenger cyclic di-GMP binds specifically to multiple export ATPases at a highly conserved binding site.
Conclusion: Cyclic di-GMP binding is central to the function of many different bacterial export complexes.
Significance: This profoundly affects our understanding of numerous important bacterial organelles, including flagella, type III, and type VI secretion systems.
The PRMT5•MTA complex has recently emerged as a new synthetically lethal drug target for the treatment of MTAP-deleted cancers. Here, we report the discovery of development candidate MRTX1719. ...MRTX1719 is a potent and selective binder to the PRMT5•MTA complex and selectively inhibits PRMT5 activity in MTAP-deleted cells compared to MTAP-wild-type cells. Daily oral administration of MRTX1719 to tumor xenograft-bearing mice demonstrated dose-dependent inhibition of PRMT5-dependent symmetric dimethylarginine protein modification in MTAP-deleted tumors that correlated with antitumor activity. A 4-(aminomethyl)phthalazin-1(2H)-one hit was identified through a fragment-based screen, followed by X-ray crystallography, to confirm binding to the PRMT5•MTA complex. Fragment growth supported by structural insights from X-ray crystallography coupled with optimization of pharmacokinetic properties aided the discovery of development candidate MRTX1719.
The ability of fungi to establish mycorrhizal associations with plants and enhance the acquisition of mineral nutrients stands out as a key feature of terrestrial life. Evidence indicates that ...arbuscular mycorrhizal (AM) association is a trait present in the common ancestor of land plants,1,2,3,4 suggesting that AM symbiosis was an important adaptation for plants in terrestrial environments.5 The activation of nuclear calcium signaling in roots is essential for AM within flowering plants.6 Given that the earliest land plants lacked roots, whether nuclear calcium signals are required for AM in non-flowering plants is unknown. To address this question, we explored the functional conservation of symbiont-induced nuclear calcium signals between the liverwort Marchantia paleacea and the legume Medicago truncatula. In M. paleacea, AM fungi penetrate the rhizoids and form arbuscules in the thalli.7 Here, we demonstrate that AM germinating spore exudate (GSE) activates nuclear calcium signals in the rhizoids of M. paleacea and that this activation is dependent on the nuclear-localized ion channel DOES NOT MAKE INFECTIONS 1 (MpaDMI1). However, unlike flowering plants, MpaDMI1-mediated calcium signaling is only required for the thalli colonization but not for the AM penetration within rhizoids. We further demonstrate that the mechanism of regulation of DMI1 has diverged between M. paleacea and M. truncatula, including a key amino acid residue essential to sustain DMI1 in an inactive state. Our study reveals functional evolution of nuclear calcium signaling between liverworts and flowering plants and opens new avenues of research into the mechanism of endosymbiosis signaling.
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•The symbiosis-induced nuclear calcium signal is ancestral in land plants•DMI1 is required for arbuscular mycorrhiza colonization of M. paleacea thalli•AM colonization of M. paleacea rhizoid is independent of nuclear calcium signaling•The activation mechanism of DMI1 has diverged between M. paleacea and flowering plants
Lam et al. show that arbuscular-mycorrhiza-induced nuclear calcium signaling is ancestral in land plants. It is necessary for the colonization of liverwort thalli by arbuscular mycorrhiza, but not for the rhizoid infection. It is dependent on DMI1, whose gating mechanism has diverged between liverwort and flowering plants.
Medium‐chain alcohol dehydrogenases (ADHs) comprise a highly conserved enzyme family that catalyse the reversible reduction of aldehydes. However, recent discoveries in plant natural product ...biosynthesis suggest that the catalytic repertoire of ADHs has been expanded. Here we report the crystal structure of dihydroprecondylocarpine acetate synthase (DPAS), an ADH that catalyses the non‐canonical 1,4‐reduction of an α,β‐unsaturated iminium moiety. Comparison with structures of plant‐derived ADHs suggest the 1,4‐iminium reduction does not require a proton relay or the presence of a catalytic zinc ion in contrast to canonical 1,2‐aldehyde reducing ADHs that require the catalytic zinc and a proton relay. Furthermore, ADHs that catalysed 1,2‐iminium reduction required the presence of the catalytic zinc and the loss of the proton relay. This suggests how the ADH active site can be modified to perform atypical carbonyl reductions, providing insight into how chemical reactions are diversified in plant metabolism.
Medium chain alcohol dehydrogenases usually perform a reversible 1,2‐reduction of aldehydes to form the corresponding alcohols. Here we show how the active site of these dehydrogenases can be modified to perform 1,2‐reduction of iminium moieties, as well 1,4‐reduction of α,β‐unsaturated iminium groups and 1,4‐reduction of α,β‐unsaturated aldehydes.
The heterogeneous behavior of patients with melanoma makes prognostication challenging. To address this, a gene expression profile (GEP) test to predict metastatic risk was previously developed. This ...study evaluates the GEP's prognostic accuracy in an independent cohort of cutaneous melanoma patients.
This multi-center study analyzed primary melanoma tumors from 523 patients, using the GEP to classify patients as Class 1 (low risk) and Class 2 (high risk). Molecular classification was correlated to clinical outcome and assessed along with AJCC v7 staging criteria. Primary endpoints were recurrence-free (RFS) and distant metastasis-free (DMFS) survival.
The 5-year RFS rates for Class 1 and Class 2 were 88% and 52%, respectively, and DMFS rates were 93% versus 60%, respectively (P < 0.001). The GEP was a significant predictor of RFS and DMFS in univariate analysis (hazard ratio HR = 5.4 and 6.6, respectively, P < 0.001 for each), along with Breslow thickness, ulceration, mitotic rate, and sentinel lymph node (SLN) status (P < 0.001 for each). GEP, tumor thickness and SLN status were significant predictors of RFS and DMFS in a multivariate model that also included ulceration and mitotic rate (RFS HR = 2.1, 1.2, and 2.5, respectively, P < 0.001 for each; and DMFS HR = 2.7, 1.3 and 3.0, respectively, P < 0.01 for each).
The GEP test is an objective predictor of metastatic risk and provides additional independent prognostic information to traditional staging to help estimate an individual's risk for recurrence. The assay identified 70% of stage I and II patients who ultimately developed distant metastasis. Its role in consideration of patients for adjuvant therapy should be examined prospectively.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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