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Fig. S1. ClustalW multiple alignment of primary sequences of FlxA from C. pinensis with those of histidine ammonia-lyases (HALs), phenylalanine ammonia-lyases (PALs) and tyrosine ammonia-lyases (TALs) with known substrate specificity. Sequences used for the alignment were abbreviated as follows (with GenBank accession numbers in parentheses): HAL P. putida, HAL Pseudomonas putida (P21310); HAL S. griseus, HAL Streptomyces griseus (AAA26769); PAL P. crispum, PAL Petroselinum crispum (P24481); PAL P. luminescens, PAL Photorhabdus luminescens sp. laumondii TT01 (NP_929491); PAL S. maritimus, PAL Streptomyces maritimus (AAF81735); TAL C. pinensis, TAL Chitinophaga pinensis (YP_003121550); TAL R. sphaeroides, TAL Rhodobacter sphaeroides 2.4.1 (YP_355075); TAL S. espanaensis, TAL Saccharothrix espanaensis (ABC88669); TAL S. sp. Tü 4128, TAL Streptomyces sp. Tü 4128 (AEV23249). The selectivity switch reported by Watts et al. (2006) is boxed in motive 1. The residue at the first position (here residue 140) varied between Phe (F) and His (H) in PAL sequences, whereas TAL or PAL with TAL activity have a His (H) at the first position. Furthermore, a second conserved residue was reported (Berner et al., 2006), which is boxed in motive 2 (here residue 507) and was always Glu (E) for HAL and Gln (Q) for TAL or PAL. The alignment was performed using Geneious 6.1.7 ClustalW default settings. Coloured residues indicate a similarity ≥ 75%.

Fig. S2. SDS-PAGE analysis of purified proteins. Left side: Purified FlxA (expected size 58 kDa). Right side: Purified FlxY (expected size 49 kDa). Marker: PageRuler™ Unstained protein ladder (Fermentas).

Fig. S3. GC-MS analysis of FlxA enzyme assays. A. Mass spectrum of the product observed after incubation of FlxA with L-tyrosine (I) and 4-coumaric acid standard (II). B. Chromatogram of an assay containing FlxA and l-phenylalanine (I), the mass spectrum of the product at 6.1 min from the above chromatogram (II) and the mass spectrum of E-cinnamic acid standard (III).

Fig. S4. Photometric FlxA enzyme assays. A. Endpoint assay detecting the enzymatic conversion of l-tyrosine (blue), l-phenylalanine (red) and l-histidine (green) by measuring the absorption increase of 4-coumaric acid (310 nm), E-cinnamic acid (275 nm) or urocanic acid (277 nm) respectively. B. pH dependence of FlxA activity with l-tyrosine (I) and l-phenylalanine (II). Temperature dependence of FlxA activity with K-tyrosine (III) and l-phenylalanine (IV). TAL: tyrosine ammonia-lyase; PAL: phenylalanine ammonia-lyase; HAL: histidine ammonia-lyase.

Fig. S5. Michaelis–Menten kinetics of FlxA. Substrate saturation plots of FlxA with l-tyrosine (A) and l-phenylalanine (B) and their respective linearization as Lineweaver–Burk (C, D) or Hanes–Woolf plots(E, D). TAL: tyrosine ammonia-lyase; PAL: phenylalanine ammonia-lyase.

Fig. S6. ClustalW multiple alignment of primary sequences of FlxY from Chitinophaga pinensis with three 4-coumarate-CoA ligases (4CL) from Arabidopsis thaliana and the E-cinnamate-CoA ligase (CCL) ScCCL from Streptomyces coelicolor A3(2). Sequences used for the alignment were abbreviated as follows (with GenBank accession numbers in parentheses): At4CL1, A. thaliana 4CL1 (AAA82888.1); At4CL2, A. thaliana 4CL2 (AF106086_1); At4CL3, A. thaliana 4CL3 (AF106088_1); ScCCL, S. coelicolor A3 (2) CCL (CAB95894); Cp4CL, C. pinensis 4CL (YP_003121574.1). Two conserved peptide boxes from 4CL are boxed in red and nine residues that are suggested to form the At4CL substrate binding pocket are marked by red triangles (Kaneko et al., 2003). The alignment was performed using Geneious 6.1.7 ClustalW default settings. Coloured residues indicate a similarity ≥ 75%.

Fig. S7. HPLC-MS analysis of enzyme assays with FlxY. A. Mass spectra of CoA (I), 4-coumaroyl-CoA standard (II) and products detected in enzyme assays containing FlxY and 4-coumaric acid (III) or E-cinnamic acid (IV) as substrates. B. Theoretical fragmentation tree explaining the above mass spectra with randomly positioned charges.

Fig. S8. HPLC-MS analysis of the substrate specificity of FlxY. The structures of the substrates, the EIC from CoA (green) and the EIC of expected substrate-CoA thioesters (blue) are shown.

Fig. S9. Results of enzyme assays with FlxY. pH (A) and temperature optimum (B) of the coupled enzyme assay. Substrate saturation plots of FlxY with 4-coumaric acid (C) and E-cinnamic acid (D) and their respective linearization as Lineweaver–Burk (E, F) or Hanes–Woolf plots (G, H).

Fig. S10. KOH test, mass spectra and HPLC traces from C. pinensis flexirubin 1. A. Crude acetone extract of C. pinensis culture (left) showing the reversible colour shift after addition of base (middle) or base and acid (right). B. HPLC-MS-chromatograms of C. pinensis extract before (upper trace) and after fractionation by column chromatography (lower trace). UV at 420 nm (blue line) and an EIC m/z 633.5 [M − H+] are shown. The occurrence of an additional signal after purification may be explained by isomerization. Chromatograms are drawn to the same scale. C. HPLC-ESI-MS2 of 1 m/z 633.5 [M − H+]. MALDI-iontrap-MS3 spectra of m/z 315.2 (D) and m/z 265.3 (E) with m/z 634.4 [M]+ as precursor. F. On the left side, MALDI-iontrap-MS2 of wild-type 1m/z 634.4 [M]+ (I) and m/z 637.4 [M]+ from feeding experiments with d3-methionine (II) and d4-tyrosine (III). The resulting fragments m/z 343.2 for wild type and m/z 346.2 or m/z 346.3 for the feeding experiments were further fragmented and the corresponding MS3 mass spectra are depicted on the right side.

Table S1. Strains and plasmids used in this work.

Table S2. Primers and PCR products used in this study.

Table S3. High-resolution MALDI-orbitrap-MS of CoA-thioesters from FlxY substrate assays.

Table S4. Predicted gene clusters for flexirubin biosynthesis in C. pinensis. Domain-guided annotation is based on conserved domains detected by BLAST-P of C. pinensis DSM 2588 primary sequences against the genome of F. johnsoniae UW101.

Table S5. Gene cluster for flexirubin biosynthesis in F. johnsoniae UW101. Domain-guided annotation is based on conserved domains detected by BLAST-P.

Table S6. Comparison of the enzymatic properties of bacterial 4CLs (4-coumarate-CoA ligase) and CCLs (cinnamate-CoA ligase). Values were obtained from assays with 4-coumarate (4CA) and E-cinnamate (CA) as substrates.

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