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Fig. 5 in Site directed mutagenesis of Catharanthus roseus (+)-vincadifformine 19-hydroxylase (CYP71BY3) results in two distinct enzymatic functions

Fig. 5. The four residues in the V19H (left) and T3O (right) binding sites that were predicted to affect the opposite enantioselectivity of these highly homologous enzymes using models created on the 3ruk template. Residues shown in the V19H pocket are K106, S312, A376, and F377. Residues shown in the T3O pocket are R105, T311, P375, and L377. The model generated for T19H (3ruk template) looks identical to the T3O pocket, and the correlating residues are R101, T310, P374, and L375.

Fig. 3 in Site directed mutagenesis of Catharanthus roseus (+)-vincadifformine 19-hydroxylase (CYP71BY3) results in two distinct enzymatic functions

Fig. 3. Saturation kinetics of V19H: comparative (þ)-vincadifformine saturation curve in the absence or presence of (¡)-vincadifformine. The rate of (+)-vincadifformine consumption by V19H was reduced in the presence of 3 μM of ()-vincadifformine within the range of 15–20 μM of substrate yet remains unchanged at low and saturated concentrations. Error bars indicate the standard deviation from three technical replicate assays.

Fig. 7. The V19H four-point mutant coupled with T3R in Site directed mutagenesis of Catharanthus roseus (+)-vincadifformine 19-hydroxylase (CYP71BY3) results in two distinct enzymatic functions

Fig. 7. The V19H four-point mutant coupled with T3R converts (¡)-tabersonine to 2,3-dihydro-3-hydroxytabersonine. Traces from in vitro experiments with ()-tabersonine (1) and V19H four-point mutant microsomes or T3O microsomes in the absence or presence of purified recombinant T3R are shown. Yeast microsomes containing the V19H 4-point mutant (red) (V19HL106R–S312T-A376P–F377L) convert ()-tabersonine to tabersonine 2,3-epoxides (5) in the absence of T3R, and to 2,3-dihydro-3-hydroxytabersonine in the presence of T3R. Yeast microsomes containing wild-type T3O (blue) were used as positive controls for tabersonine-2,3-epoxide (5) and 2,3-dihydro-3-hydroxytabersonine biosynthesis in the absence and presence of T3R, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2 in Site directed mutagenesis of Catharanthus roseus (+)-vincadifformine 19-hydroxylase (CYP71BY3) results in two distinct enzymatic functions

Fig. 2. Representation of the 3ruk V19H model superimposed onto the 3ruk template and the location of the catalytic heme. V19H model (purple) is shown in purple, and the 3ruk template is shown in pink with the heme binding site in yellow. The H-bond interaction (green) between PHE443 and CYS450, the first and eighth residues of the heme binding site, which is responsible for the beta bulge (yellow) around the heme cofactor (black). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 1 in Site directed mutagenesis of Catharanthus roseus (+)-vincadifformine 19-hydroxylase (CYP71BY3) results in two distinct enzymatic functions

Fig. 1. The formation of (þ)- and (¡)-aspidosperma MIAs in Catharanthus roseus. In multiple C. roseus tissues, the ()-aspidosperma pathways begin with the biosynthesis of ()-tabersonine (1) from O-acetylstemmadenine (18) through acetylstemmadenine oxygenase (ASO), geissoschizine synthase (GS), and hydrolase 1 (HL1), whereas the (+)-aspidosperma pathway requires hydrolase 3 or 4 (HL3/4) to produce (+)-vincadifformine (15). Leaf-specific enzymes (black) convert ()-tabersonine (1) to vindoline (6), which accumulates in the leaves, while root-specific enzymes (purple) are responsible for the formation of two major root alkaloids, lochnericine (7) and h¨orhammericine (8). A third major root alkaloid, (+)-echitovenine (17) is derived from (+)-vincadifformine (15) via (+)-minovincinine (16) by separate root-specific (blue) enzymes. The remaining ()-aspidosperma alkaloids shown are derived chemically (grey box) from ()-tabersonine (1) to generate ()-vincadifformine (12) or enzymatic conversion by T19H to form 19-hydroxytabersonine (10) or its 19-O acetyltabersonine (11) by the action of TAT. These MIAs [()-vincadifformine (12), 19-hydroxytabersonine (10) and 19-O acetyltabersonine (11)] do not naturally accumulate in C. roseus. T16H: tabersonine 16-hydroxylase [CYP71D12 (T16H1) or CYP71D351 (T16H2); 16OMT: tabersonine 16-O-methyltransferase; T3O: tabersonine 3-oxygenase (CYP71D1V2); T3R: tabersonine 3-reductase; NMT: N-methyltransferase; D4H: desacetoxyvindoline 4-hydroxylase; DAT: deacetylvindoline O-acetyltransferase; TEX: tabersonine epoxidase [CYP71D521 (TEX1) or CYP71D347 (TEX2)]; T19H: tabersonine 19-hydroxylase (CYP71BJ1); TAT: tabersonine 19-acetyltranferase; V19H: (+)-vincadifformine 19-hydroxylase (CYP71BY3); MAT: minovincinine 19-O-acetyltransferase. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

opencc-by-4.0Jul 2022View details →

Direct strain correlations at the single-atom level in three-dimensional core-shell interface structures

The data and source codes for the paper [H.J., D.H.W., T.L., Y.K., C.J., J.L., H.B., A. J. P., W.T., C.O., P.E., Y.-L.L., S.R., S.W.H., and Y.Y., "Direct strain correlations at the single-atom level in three-dimensional core-shell interface structures", Nat. Commun. 13, 5957 (2022)] are posted below.   Nanomaterials with core-shell architectures are prominent examples of strain-engineered materials. The lattice mismatch between the core and shell materials can cause strong interface strain, which affects the surface structures. Therefore, surface functional properties such as catalytic activities can be designed by fine-tuning the misfit strain at the interface. To precisely control the core-shell effect, it is essential to understand how the surface and interface strains are related at the atomic scale. Here, we elucidate the surface-interface strain relations by determining the full 3D atomic structure of Pd@Pt core-shell nanoparticles at the single-atom level via atomic electron tomography. Full 3D displacement fields and strain profiles of core-shell nanoparticles were obtained, which revealed a direct correlation between the surface and interface strain. The strain distributions show a strong shape-dependent anisotropy, whose nature was further corroborated by molecular statics simulations. From the observed surface strains, the surface oxygen reduction reaction activities were predicted. These findings give a deep understanding of structure-property relationships in strain-engineerable core-shell systems, which can lead to direct control over the resulting catalytic properties.   1. Raw experimental tilt series images for Particle 1 and Particle 2 (Raw tilt series images.zip). 2. Denoised and aligned tilt series images for Particle 1 and Particle 2 (Finalized tilt series images.zip). 3. Finalized tilt angles for Particle 1 and Particle 2 (Finalized tilt angles.zip). 4. 3D tomographic reconstruction for Particle 1 and Particle 2 (Final reconstruction.zip). 5. Final atomic structure for Particle 1 and Particle 2 (Final atomic structure.zip). 6. Indices of surface Pd atoms for Particle 1 and Particle 2 (Indices of surface Pd.zip). 7. Source codes for the assignment of atomic coordinates, displacement/strain calculation and ORR activity calculation (Code_PdPt.zip).   If you use any of the above data or source codes in your publications and/or presentations, our paper should be properly cited: H.J., D.H.W., T.L., Y.K., C.J., J.L., H.B., A. J. P., W.T., C.O., P.E., Y.-L.L., S.R., S.W.H., and Y.Y., "Direct strain correlations at the single-atom level in three-dimensional core-shell interface structures", Nat. Commun. 13, 5957, (2022). If you have any questions regarding the above data or source codes, please contact Yongsoo Yang, Department of Physics, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Korea. Email: <a href="mailto:yongsoo.yang@kaist.ac.kr">yongsoo.yang@kaist.ac.kr</a>

opencc-by-4.0Aug 2023View details →

The effects of two different fatigue protocols on movement quality during unanticipated change of direction in female soccer players (data file)

This dataset contains the results of soccer players who participated in our study. In our study, we investigated the effects of two fatigue protocols on movement quality during change of direction in anticipated and unanticipated conditions.

opencc-by-4.0Aug 2023View details →

First records of direct kleptoparasitism in yellowjacket wasps (Vespidae: Vespula)-Supplementary files

Compilation of video recordings and original files: first video (yellowjacket_1) begins after the first yellowjacket decapitates the honeybee and ends after the wasps departure with the honeybee abdomen; second video (yellowjacket_2) begins after the second yellowjackets arrival and ends after it departs with the honeybee thorax.

Fig. 7 in The CYP74B and CYP74D divinyl ether synthases possess a side hydroperoxide lyase and epoxyalcohol synthase activities that are enhanced by the site-directed mutagenesis

Fig. 7. The unrooted phylogenetic tree of the CYP74 family. Classified CYP74 subfamilies are marked with their letter designations (A, B, C, etc.). Subfamilies consisting of more than one member are outlined with unclosed curves (semi-ellipses). The following CYP74s were used for analysis: As, A. sativum; AsDES (CYP74H1), CAI30435.1; At, Arabidopsis thaliana; AtAOS (CYP74A1), NP199079.1; AtHPL (CYP74B2), C74B2ARATH; Ca, C. annuum; CaHPL (CYP74B1), NP001311810.1; CaDES (CYP74D4), NP001311513.1; Cas, Camellia sinensis; CasHPL (CYP74B24), BAU24783.1; Cs, Cucumis sativus; CsHPL/EAS/AOS (CYP74C31), XP004137005.1; CsHPL/EAS (CYP74C1_Cs), NP001274399.1; Cm, Cucumis melo; CmHPL/EAS (CYP74C2), NP001284390.1; Dc, Daucus carota; DcAOS (CYP74B33), XP_017248700.1; Gm, G. max; GmAOS (CYP74A1), NP001236432.1; GmHPL/EAS (CYP74C13_Gm), KRH29541.1; Hv, Hordeum vulgare, HvAOS2 (CYP74A3), CAB86384.1; Le, L. esculentum; LeAOS1 (CYP74A1), CAB88032.1; LeAOS2 (CYP74A2), AAF67141.1; LeAOS3 (CYP74C3), NP001265949.1; LeHPL (CYP74B3), AAF67142.1; LeDES (CYP74D1), NP001234527.1; Lu, L. usitatissimum; LuAOS (CYP74A1), sp|P48417.1; LuDES (CYP74B16), ADP03054.2; Mp, M. polymorpha, MpAOS1, BAS32647.1; MpAOS2, BAS32648.1; Mt, Medicago truncatula; MtHPL/EAS (CYP74C13_Mt), XP003606860.1; MtHPL3 (CYP74B4), AAY30368.1; Nt, N. tabacum; NtDES (CYP74D3), NP001312606.1; Os, Oryza sativa; OsAOS1 (CYP74A4), XP015631686.1; OsHPL2 (CYP74E1), EAY85033.1; Pa, Parthenium argentatum; PaAOS (CYP74A1), sp| Q40778.2; Pd, Prunus dulcis; PdHPL (CYP74C5), CAE18065.1; Pg, Psidium guajava; PgHPL (CYP74B5), AAK15070.1; Pi, Petunia inflata; PiCYP74C9, ABC75838.1; Pp, P. patens; PpAOS1 (CYP74A1), XP024380613.1; PpAOS2 (CYP74A8), XP024372097.1; PpHPL (CYP74G1), CAC86920.2; Ra, R. acris; RaDES (CYP74Q1), AJU57209.1; Rj, R. japonicus; RjEAS (CYP74A88), QCR70269.1; Sm, S. moellendorffii; SmDES1 (CYP74M1), XP002979266.1; SmDES2 (CYP74M3), XP002964012.2; SmEAS (CYP74M2), EFJ26024.1; St, S. tuberosum; StAOS2 (CYP74A6), ABD15175.1; StAOS3 (CYP74C10), CAI30876.1; StHPL/EAS (CYP74C4), XP006365486.1; StDES (CYP74D2), NP001305517.1; Zm, Zea mays; ZmAOS1 (CYP74A19), AAR33048.1; ZmHPL (CYP74F2), NP_001105255.2. The multiple alignments of selected CYP74 amino acid sequences and phylogenetic tree building were made with MEGA7 software. Multiple alignment was performed using the ClustalW method, phylogenetic tree was build using the maximum likelihood method based on the Poisson correction model (Zuckerkandl and Author- Anonymous, 1965); the bootstrap consensus tree was inferred from 1000 replicates (Felsenstein, 1985). The analysis involved 45 amino acid sequences.

Fig. 6 in The CYP74B and CYP74D divinyl ether synthases possess a side hydroperoxide lyase and epoxyalcohol synthase activities that are enhanced by the site-directed mutagenesis

Fig. 6. The mechanisms of fatty acid hydroperoxide conversions by target DESs. R = HOOC(CH2)7–, R' = n-butyl.

Fig. 4 in The CYP74B and CYP74D divinyl ether synthases possess a side hydroperoxide lyase and epoxyalcohol synthase activities that are enhanced by the site-directed mutagenesis

Fig. 4. GC-MS analyses of products (Me/TMS) of recombinant WT LuDES incubations with 9(S)-HPOD (A), 9(S)-HPOT (B), 13(S)-HPOD (C), and 13(S)- HPOT (D). 2, 9,10-epoxy-11-hydroxy-12-octadecenoic acid (Me/TMS); 3, 9- hydroxynonanoic acid (Me/TMS); 5, 9,10-epoxy-11-hydroxy-12,15-octadecadienoic acid (Me/TMS); 6, 11-hydroxy-12,13-epoxy-9-octadecenoic acid (Me/ TMS); 7, (ω5Z)-etherolenic acid (Me); 8, (9Z)-12-hydroxy-9-dodecenoic acid (Me/TMS); 9, (10E)-12-hydroxy-10-dodecenoic acid (Me/TMS); 10, 11-hydroxy-12,13-epoxy-9,15-octadecadienoic acid (Me/TMS); 12, 9-hydroxy- 12,13-epoxy-10-octadecenoic acid (Me/TMS); 13, 9,10-epoxy-13-hydroxy- 11,15-octadecadienoic acid (Me/TMS); 14, 9,10-epoxy-13-hydroxy-11-octadecenoic acid (Me/TMS). The structural formulae of products are presented in Fig. 2. 9-HOD/T and 13-HOD/T are decrypted in Fig. 3.

Fig. 3 in The CYP74B and CYP74D divinyl ether synthases possess a side hydroperoxide lyase and epoxyalcohol synthase activities that are enhanced by the site-directed mutagenesis

Fig. 3. GC-MS analyses of products (Me/TMS) of recombinant NtDES incubations with 9(S)-HPOD (A), 9(S)-HPOT (B), 13(S)-HPOD (C), and 13(S)- HPOT (D). 1, colneleic acid (Me); 2, 9,10-epoxy-11-hydroxy-12-octadecenoic acid (Me/TMS); 3, 9-hydroxynonanoic acid (Me/TMS); 4, colnelenic acid (Me); 4a, (3′E)-colnelenic acid (Me); 6, 11-hydroxy-12,13-epoxy-9-octadecenoic acid (Me/TMS). The structural formulae of products are presented in Fig. 2. 9-HOD, (9S,10E,12Z)-9-hydroxy-10,12-octadecadienoic acid; 9-HOT, (9S,10E,12Z,15Z)-9-hydroxy-10,12,15-octadecatrienoic acid; 13-HOD, (9Z,11E, 13S)-13-hydroxy-9,11-octadecadienoic acid; 13-HOT, (9Z,11E,13S,15Z)-13- hydroxy-9,11,15-octadecatrienoic acid.

Fig. 2 in The CYP74B and CYP74D divinyl ether synthases possess a side hydroperoxide lyase and epoxyalcohol synthase activities that are enhanced by the site-directed mutagenesis

Fig. 2. Structural formulae of reaction products of target WT enzymes and their mutant forms. 1, colneleic acid; 2, 9,10-epoxy-11-hydroxy-12-octadecenoic acid; 3, 9-hydroxynonanoic acid; 4, colnelenic acid; 4a, (3′E)-colnelenic acid; 5, 9,10-epoxy-11-hydroxy-12,15-octadecadienoic acid; 6, 11-hydroxy-12,13-epoxy-9-octadecenoic acid; 7, (ω5Z)-etherolenic acid; 8, (9Z)-12-hydroxy-9-dodecenoic acid; 9, (10E)-12-hydroxy-10-dodecenoic acid; 10, 11-hydroxy-12,13-epoxy-9,15-octadecadienoic acid; 11, (ω5Z)-etheroleic acid; 12, 9-hydroxy-12,13-epoxy-10-octadecenoic acid; 13, 9,10-epoxy-13-hydroxy-11,15-octadecadienoic acid; 14, 9,10-epoxy- 13-hydroxy-11-octadecenoic acid. (3′E)-Colnelenic acid is a product of thermal isomerization of the ordinary (8E,1′E,3′Z,6′Z)-colnelenic acid occurring during the GC analyses.

Fig. 1 in The CYP74B and CYP74D divinyl ether synthases possess a side hydroperoxide lyase and epoxyalcohol synthase activities that are enhanced by the site-directed mutagenesis

Fig. 1. Multiple alignments of I-helix sequences of following CYP74s: As, Allium sativum; AsDES, CAI30435; Ca, Capsicum annuum; CaDES, ABH03632; CaHPL, AAK27266; Cs, Citrus sinensis; CsAOS, NP_001275835; Gm, Glycine max; GmAOS, NP_001236445; Le, Solanum lycopersicum; LeHPL, CAB43022; LeDES, AAG42261; Lu, Linum usitatissimum; LuDES, ADP03054; Na, Nicotiana attenuata; NaAOS, CAC82911; Nt, Nicotiana tabacum; NtDES, AAL40900; NtHPL, AAZ39884; Ra, Ranunculus acris; RaDES, CYP74Q1, AJU57209; Sm, Selaginella moellendorffii; SmDES1, CYP74M1, EFJ19674; SmDES2, CYP74M3, EFJ34345; St, Solanum tuberosum; StDES, CAC28152. Hydroperoxide-binding domain is circled. Sites with substitutions are marked with arrows.

Fig. 5 in The CYP74B and CYP74D divinyl ether synthases possess a side hydroperoxide lyase and epoxyalcohol synthase activities that are enhanced by the site-directed mutagenesis

Fig. 5. GC-MS analyses of products (Me/TMS) of LuDES F291V mutant form incubations with 9(S)-HPOD (A), 9(S)-HPOT (B), 13(S)-HPOD (C), and 13(S)- HPOT (D). 2, 9,10-epoxy-11-hydroxy-12-octadecenoic acid (Me/TMS); 3, 9- hydroxynonanoic acid (Me/TMS); 5, 9,10-epoxy-11-hydroxy-12,15-octadecadienoic acid (Me/TMS); 6, 11-hydroxy-12,13-epoxy-9-octadecenoic acid (Me/ TMS); 8, (9Z)-12-hydroxy-9-dodecenoic acid (Me/TMS); 9, (10E)-12-hydroxy- 10-dodecenoic acid (Me/TMS); 10, 11-hydroxy-12,13-epoxy-9,15-octadecadienoic acid (Me/TMS). The structural formulae of products are presented in Fig. 2. 9-HOD/T and 13-HOD/T are decrypted in Fig. 3.

Fig. 8 in Site-directed mutagenesis of β sesquiphellandrene synthase enhances enzyme promiscuity

Fig. 8. Proposed mechanisms for the conversion of FPP into sesquiterpene products catalysed by PmSTS WT and its mutants (L454G and L454A). The scheme is derived from the proposed mechanism of monoterpenes synthases (Gatto et al., 2015; Piechulla et al., 2016). The products generated by the PmSTSΔ24WT are shown in black. New products generated by mutant L454G and L454A in this study are shown in red. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 9 in Site-directed mutagenesis of β sesquiphellandrene synthase enhances enzyme promiscuity

Fig. 9. The homology modelling illustration of the active site of PmSTSΔ24WT and PmSTSΔ24L454G. The residue L454 and Y418 are shown as stick and coloured in purple and orange, respectively. (A) The side chain of the L454 provides steric hindrance, preventing the rotation of the Y418 toward the interior of the active site. (B) The mutation of L454G provide sufficient space to allow the Y418 to undergo rotation toward the interior of the active site and (C) thus allowing interaction with other amino acid in the active site. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5 in Site-directed mutagenesis of β sesquiphellandrene synthase enhances enzyme promiscuity

Fig. 5. Relative activity of PmSTSΔ24 WT and mutants (0.1 μM) incubated with the substrate FPP (50 μM). The relative activity of PmSTS were measured using Malachite Green Assay. The relative activity of PmSTS were calculated using the wild type as 100% with error bars representing SE (n = 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7 in Site-directed mutagenesis of β sesquiphellandrene synthase enhances enzyme promiscuity

Fig. 7. Mass spectra and retention index of the hydroxylated sesquiterpene generated by PmSTS mutants L454G and L454A. NIST library mass spectra of (A) sesquicineole, (B) cis-sesquisabinene hydrate, (C) trans-sesquisabinene hydrate, and (D) α-bisabolol are shown at bottom of each respective sample mass spectra.

Fig. 4 in Site-directed mutagenesis of β sesquiphellandrene synthase enhances enzyme promiscuity

Fig. 4. Structural analysis by circular dichroism (CD) of PmSTSΔ24WT and L454 mutants. Far-UV CD spectra (190–260 nm) of PmSTSΔ24 WT, PmSTSΔ24Y390S/L454G, PmSTSΔ24L454G and PmSTSΔ24L454A are shown. Buffer used was 20 mM Tris pH 8.0, 100 mM NaCl 2 mM βME. Protein concentration was 10 μM. Further secondary structure variations in percentages for each L454 mutant and WT were determined using the JASCO SSE program are presented in Table S2.