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Fig. 7 in Mutagenesis of a Lotus japonicus GSK3β/Shaggy-like kinase reveals functionally conserved regulatory residues

Fig. 7. Appearance of whole plants and nodules of L. japonicus hairy root lines transformed with the different LjSK1 variants. A, F) Control (empty vector); B, G) native LjSK1; C, H) LjSK1 K167A; D, I) LjSK1 Y298A; E, J) LjSK190-467. A-E) Representative whole plants with regenerated hairy roots. F-J) Images of nodules on regenerated hairy roots taken in bright-field (top panels) and with an RFP filter to visualize rhizobium colonisation (bottom panels). Scale bars in A-E are 5 cm and in F-G 2 mm.

Fig. 6. Western blot analysis with a p in Mutagenesis of a Lotus japonicus GSK3β/Shaggy-like kinase reveals functionally conserved regulatory residues

Fig. 6. Western blot analysis with a p-GSK-3α/β (6D3) (Santa Cruz Biotechnology, Inc.) monoclonal antibody raised against a phosphopeptide corresponding to amino acids residues surrounding Tyr279 of human GSK3α and Tyr216 of human GSK3β. Lane 1: recombinant LjSK1 and lane 2: LjSK1 Y298A variant. Both proteins have a GST-tag.

Fig. 4 in Mutagenesis of a Lotus japonicus GSK3β/Shaggy-like kinase reveals functionally conserved regulatory residues

Fig. 4. pH (A) and temperature (B) curves for LjSK1 together with standard error bars. Points represent the average value of three measurements. RLU refers to Relative Light Units and ΔRLU is for the difference between a control sample (without enzyme) and the corresponding sample with enzyme. Measurements were performed as described in section 5.3.

Fig. 2. A in Mutagenesis of a Lotus japonicus GSK3β/Shaggy-like kinase reveals functionally conserved regulatory residues

Fig. 2. A homology model of the LjSK1 – ATP complex based on the structure of human GSK3β (PDB:1J1B) (Aoki et al., 2004) after refinement with molecular dynamics simulations and docking of the ATP molecule at the ATP binding site. N- and C-terminal lobes are indicated by different colors and the G-loop and the activation loop are indicated in purple and green, respectively. Mutated residues are shown as stick models. Figure created using Pymol (DeLano, 2002). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 1 in Mutagenesis of a Lotus japonicus GSK3β/Shaggy-like kinase reveals functionally conserved regulatory residues

Fig. 1. Alignment of the primary structure of LjSK1, human GSK3β and AtSK3-2. Identical residues are shown in bold. Residues mutated to alanine are boxed. Residues constituting G-loop and the activation loop are shown in grey boxes and the respective structural feature is indicated. Black arrows indicate residues predicted to be in close proximity to the phosphates of ATP. Gaps, represented by dashes, were introduced into the sequences to optimize the alignment.

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.

Fig. 3 in Site-directed mutagenesis of β sesquiphellandrene synthase enhances enzyme promiscuity

Fig. 3. Analysis of monomeric fraction of PmSTS Wild Type and mutant proteins using (A) 12% SDS PAGE and (B) 10% Native PAGE. The SDS PAGE showed high purity of PmSTS protein were obtained for wild type and for all mutants except for PmSTSΔ24W286A and PmSTSΔ24Y390S. The native PAGE showed several species of PmSTSΔ24WT, PmSTSΔ24V466E, and PmSTSΔ24Y390S/L454G that can be resolved on a 10% native gel. However, only one species of PmSTSΔ24L454G and PmSTSΔ24L454A was resolved on a 10% native gel. Smearing bands were observed for PmSTSΔ24W286A and PmSTSΔ24Y390S on the 10% native gel, probably due to the low purity as judged by 12% SDS PAGE.

Fig. 1 in Site-directed mutagenesis of β sesquiphellandrene synthase enhances enzyme promiscuity

Fig. 1. Superimposition of PmSTS homology model (green) with the crystal structure of Nicotiana tabacum 5-epi-aristolochene synthase (NtEAS, PDB ID:3M01). NtEAS has a FPP ligand analog (2-trans, 6-trans)-2- fluorofarnesyl diphosphate (FPF) bound in the active site (pink) shows an RMSD of 1.4 Å over 518 aligned Cα atoms. The Mg2+ ions in the active site of NtEAS are shown in yellow spheres. PmSTS and NtEAS share ~38% sequence identify. The putative active residues of PmSTS (W286, Y390, L454 and V466) shown heavier sticks were selected for mutagenesis studies. The structure superimposition was carried out using SSM in COOT (Emsley et al., 2010). (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 β sesquiphellandrene synthase enhances enzyme promiscuity

Fig. 2. Size exclusion chromatography (SEC) profiles of affinity chromatography purified wild-type and mutant proteins. The chromatography was performed on a HiLoad Superdex column 200 PG. Detection was at 280 nm. (A) PmSTSΔ24 WT, (B) PmSTSΔ24 V466E, (C) PmSTSΔ24 W286A, (D) PmSTSΔ24 Y390S, (E) PmSTSΔ24L454A, (F) PmSTSΔ24L454G, and (G) PmSTSΔ24Y390S/L454G were run over a size exclusion column to verify the oligomeric status of generated proteins. Elution profiles of β-sesquiphellandrene synthase proteins are represented together with protein standard: bovine thyroglobulin (670 kDa), bovine gamma globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), vitamin B12 (1.35 kDa). The peaks eluted at ~48 mL correspond to protein aggregated during the purification process, and peaks eluted at ~80 mL correspond to the monomeric size of PmSTSΔ24 protein (65 kDa).