Inventarisasi Tanaman Peneduh Jalan Penjerap Timbal di Purwokerto

Tanaman peneduh jalan adalah tanaman yang berada di tepi jalan. Tanaman peneduh jalan memiliki dua fungsi yaitu sebagai estetika dan ekologis. Salah satu fungsi ekologis tanaman peneduh jalan adalah mengakumulasi bahan pencemar. Jenis pencemaran yang memerlukan penanganan secara sistematis dan komprehensif adalah pencemaran timbal (Pb). Pb banyak dihasilkan oleh aktivitas pembakaran bahan bakar minyak kendaraan bermotor. Jenis tanaman peneduh jalan yang berpotensi mengakumulasi Pb belum tereksplorasi sehingga dilakukan riset yang dapat menghasilkan database jenis spesies yang mampu mengurangi Pb di lingkungan. Tujuan penelitian adalah menginventarisasi jenis tanaman peneduh jalan penjerap Pb. Manfaat penelitian adalah mendapatkan jenis tanaman peneduh jalan yang berpotensi penjerap Pb. Metode penelitian yang digunakan adalah survai di 8 (delapan) jalan di wilayah Purwokerto. Sampel daun tanaman peneduh jalan diambil secara acak terpilih di sepanjang jalan tersebut. Hasil penelitian menunjukkan jenis-jenis tanaman peneduh jalan yang berpotensi menjerap Pb adalah Glodogan (Polyalthea longifolia), Angsana (Pterocarpus indicus), Filicium (Filicium decipiends), Ketapang (Terminalia catappa), Beringin (Ficus benjamina), Kupu-kupu (Bauhinia tomentosa), Puspa (Schima wallichii), Kenari (Canarium ovatum) dan Genitu (Chrysophyllum cainito)

Characterization of the Artemisinin Binding Site for Translationally Controlled Tumor Protein (TCTP) by Bioorthogonal Click Chemistry

Despite the fact that multiple artemisinin-alkylated proteins in Plasmodium falciparum have been identified in recent studies, the alkylation mechanism and accurate binding site of artemisinin–protein interaction have remained elusive. Here, we report the chemical-probe-based enrichment of the artemisinin-binding peptide and characterization of the artemisinin-binding site of P. falciparum translationally controlled tumor protein (TCTP). A peptide fragment within the N-terminal region of TCTP was enriched and found to be alkylated by an artemisinin-derived probe. MS2 fragments showed that artemisinin could alkylate multiple amino acids from Phe12 to Tyr22 of TCTP, which was supported by labeling experiments upon site-directed mutagenesis and computational modeling studies. Taken together, the “capture-and-release” strategy affords consolidated advantages previously unavailable in artemisinin–protein binding site studies, and our results deepened the understanding of the mechanism of protein alkylation via heme-activated artemisinin

Study of IspH, a Key Enzyme in the Methylerythritol Phosphate Pathway Using Fluoro-Substituted Substrate Analogues

IspH, a [4Fe-4S]-cluster-containing enzyme, catalyzes the reductive dehydroxylation of 4-hydroxy-3-methyl-butenyl diphosphate (HMBPP) to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the methylerythritol phosphate pathway. Studies of IspH using fluoro-substituted substrate analogues to dissect the contributions of several factors to IspH catalysis, including the coordination of the HMBPP C₄–OH group to the iron–sulfur cluster, the H-bonding network in the active site, and the electronic properties of the substrates, are reported

Insights into Pipecolic Acid Biosynthesis in <i>Huperzia serrata</i>

For the biosynthesis of Pip in <i>Huperzia serrata,</i> the mechanistic studies were evaluated. Through a series of biochemical analyses, Pip is biosynthesized through a two-step cascade reaction. Three intermediates possibly exist simultaneously as an equilibrium matter in the first-step reaction catalyzed by <i>Hs</i>Ald1, while <i>Hs</i>Sard4 performs as a ketimine reductase and chemoselectively and stereoselectively takes 1,2-dehydropipecolic acid as the preferred substrate in vitro

Quaternary Ammonium Oxidative Demethylation: X-ray Crystallographic, Resonance Raman, and UV–Visible Spectroscopic Analysis of a Rieske-Type Demethylase

Herein, the structure resulting from <i>in situ</i> turnover in a chemically challenging quaternary ammonium oxidative demethylation reaction was captured via crystallographic analysis and analyzed via single-crystal spectroscopy. Crystal structures were determined for the Rieske-type monooxygenase, stachydrine demethylase, in the unliganded state (at 1.6 Å resolution) and in the product complex (at 2.2 Å resolution). The ligand complex was obtained from enzyme aerobically cocrystallized with the substrate stachydrine (<i>N</i>,<i>N</i>-dimethylproline). The ligand electron density in the complex was interpreted as proline, generated within the active site at 100 K by the absorption of X-ray photon energy and two consecutive demethylation cycles. The oxidation state of the Rieske iron–sulfur cluster was characterized by UV–visible spectroscopy throughout X-ray data collection in conjunction with resonance Raman spectra collected before and after diffraction data. Shifts in the absorption band wavelength and intensity as a function of absorbed X-ray dose demonstrated that the Rieske center was reduced by solvated electrons generated by X-ray photons; the kinetics of the reduction process differed dramatically for the liganded complex compared to unliganded demethylase, which may correspond to the observed turnover in the crystal

Development of Photoaffinity Probe for the Discovery of Steviol Glycosides Biosynthesis Pathway in <i>Stevia rebuadiana</i> and Rapid Substrate Screening

Functional discovery and characterization of the target enzymes responsible for the biosynthesis pathway coded for the genes is ongoing, and the unknown functional diversity of this class of enzymes has been revealed by genome sequencing. Commonly, it is feasible in annotating of biosynthetic genes of prokaryotes due to the existence of gene clusters of secondary metabolites. However, in eukaryotes, the biosynthetic genes are not compactly clustered in the way of prokaryotes. Hence, it remains challenging to identify the biosynthetic pathways of newly discovered natural products in plants. Steviol glycosides are one class of natural sweeteners found in high abundance in the herb <i>Stevia rebaudiana</i>. Here, we applied the chemoproteomic strategy for the proteomic profiling of the biosynthetic enzymes of steviol glycosides in <i>Stevia rebaudiana</i>. We not only identified a steviol-catalyzing UDP-glycosyltransferase (UGT) UGT73E1 involved in steviol glycoside biosynthesis but also built up a probe-based platform for the screening of potential substrates of functional uncharacterized UGT rapidly. This approach would be a complementary tool in mining novel synthetic parts for assembling of synthetic biological systems for the biosynthesis of other complex natural products

Development of Photoaffinity Probe for the Discovery of Steviol Glycosides Biosynthesis Pathway in <i>Stevia rebuadiana</i> and Rapid Substrate Screening

Functional discovery and characterization of the target enzymes responsible for the biosynthesis pathway coded for the genes is ongoing, and the unknown functional diversity of this class of enzymes has been revealed by genome sequencing. Commonly, it is feasible in annotating of biosynthetic genes of prokaryotes due to the existence of gene clusters of secondary metabolites. However, in eukaryotes, the biosynthetic genes are not compactly clustered in the way of prokaryotes. Hence, it remains challenging to identify the biosynthetic pathways of newly discovered natural products in plants. Steviol glycosides are one class of natural sweeteners found in high abundance in the herb <i>Stevia rebaudiana</i>. Here, we applied the chemoproteomic strategy for the proteomic profiling of the biosynthetic enzymes of steviol glycosides in <i>Stevia rebaudiana</i>. We not only identified a steviol-catalyzing UDP-glycosyltransferase (UGT) UGT73E1 involved in steviol glycoside biosynthesis but also built up a probe-based platform for the screening of potential substrates of functional uncharacterized UGT rapidly. This approach would be a complementary tool in mining novel synthetic parts for assembling of synthetic biological systems for the biosynthesis of other complex natural products

The copper chaperone ATX1 interacts with RAN1.

<p>(A) <i>atx1-1</i> and <i>atx1-2</i> are hypersensitive to triplin. The phenotypes of 3-day-old, dark-grown Col-0, <i>atx1-1</i> and <i>atx1-2</i> seedlings treated with 20 μM triplin are shown. Scale bar represents 1 mm. (B) Hypocotyl lengths of 3-day-old, dark-grown seedlings of Col-0, <i>atx1-1</i>, <i>atx1-2</i>, <i>ran1-2</i>, <i>atx1-1 ran1-2</i> and two <i>35S</i>:<i>ATX1-GFP (atx1-1)</i> transgenic lines treated with different doses of triplin are shown. Each experiment was repeated three times, more than 30 seedlings were used every time. Error bars represent SEM. (C) Subcellular co-localization of ATX1 and RAN1. ATX1-RFP and RAN1-GFP were transiently expressed in N. benthamiana leaves and observed and imaged under a confocal microscope. Scale bars represent 20 μM. (D) ATX1 interacted with RAN1 in yeast two-hybrid assay. ATX1 was fused to a GAL4 DNA-binding domain (BD) and RAN1-N (289 amino-terminal amino acids of RAN1) was ligated to a GAL4 activation domain (AD). The protein interactions were examined on cells grown on synthetic dropout (-Leu/-Trp/-His/-Ade) medium plus X-α-Gal (50mg/L) plates for 3 days. (E) Bimolecular fluorescence complementation assays showed interaction between ATX1 and RAN1 using the split luciferase system. Nicotiana benthamiana leaves were infiltrated with agrobacteria containing different construct combinations harboring both the C- and N-terminal of the luciferase fused to either ATX1 and RAN1-N or just one of them (controls). (F) Co-Immunoprecipitation assays showed interaction between ATX1 and RAN1. Nicotiana benthamiana leaves were infiltrated with agrobacteria containing ATX1-GFP/FLAG and RAN1-N-FLAG/GFP or just one of them (controls). The protein extracts were immunoblotted with anti-FLAG antibody or anti-GFP antibody.</p

<i>ran1-1</i> and <i>ran1-2</i> are hypersensitive to triplin and copper can partially reverse the effects of triplin.

<p>(A) Phenotypes of 3-day-old, dark-grown seedlings of Col-0, <i>ran1-1</i> and <i>ran1-2</i> treated with 10 μM triplin. (B) Triplin dose responses of Col-0, <i>ran1-1</i>, <i>ran1-2</i> and two <i>35S</i>:<i>RAN1-GFP (ran1-2)</i> transgenic lines. Data is the average hypocotyl length under each condition. Each experiment was repeated three times, more than 30 seedlings were used every time. Error bars represent SEM. (C) Phenotypes of 3-day-old, dark-grown seedlings of <i>ran1-2</i>, <i>ran1-2 etr1-1</i> and <i>ran1-2 ein2-5</i> treated with 100 μM triplin. (D) The phenotypes of 3-day-old, dark-grown seedlings of Col-0 treated with 100 μM triplin in the presence of 20 μM ZnSO₄, 20 μM CuSO₄ or H₂O as a control. (E) The hypocotyl length of seedlings as described in (D). Each experiment was repeated three times, more than 30 seedlings were used every time. Error bars represent SEM. **p < 0.01 indicated the difference of the hypocotyl length between the seedlings treated with CuSO₄ compared with H₂O and ZnSO₄ in the presence of 100 μM triplin. Scale bars represent 1 mm.</p

Triplin acts through ethylene signaling pathway to cause a triple response phenotype in <i>Arabidopsis</i> seedlings.

<p>(A) Phenotypes of 3-day-old, dark-grown Col-0 seedlings treated with 100 μM triplin, 50 μM ACC, or 1% (v/v) DMSO as a control. Scale bar represents 1 mm. (B) The chemical structure of triplin. (C) The hypocotyl length of 3-day-old, dark-grown Col-0, <i>etr1-1</i>, <i>etr1-2</i>, <i>ein2-5 and ein3 eil1</i> seedlings treated with 100 μM triplin, 50 μM ACC, or 1% (v/v) DMSO as a control. Each experiment was repeated three times, more than 30 seedlings were used every time. Error bars represent SEM. ***P < 0.0001 (two-tailed Student’s t-test) indicated a significant difference of the hypocotyl length of the mutants compared to Col-0 treated with 100 μM triplin. (D) qRT-PCR analysis the expression of the ethylene response gene <i>ERF1</i> treated by 1% (v/v) DMSO, 100 μM triplin, or 50 μM ACC. Each experiment was repeated three times, and error bars represent SEM.</p