MOESM1 of Biochemical characterization of three new α-olefin-producing P450 fatty acid decarboxylases with a halophilic property
Creators: name: Jiang, Yuanyuan
name: Li, Zhong
name: Wang, Cong
name: Zhou, Yongjin
name: Xu, Huifang
name: Li, Shengying
Publisher: name: figshare
Year: 2019
Subjects: subject: Biophysics
subject: Biochemistry
subject: Microbiology
subject: FOS: Biological sciences
subject: Pharmacology
subject: Biotechnology
subject: Chemical Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Inorganic Chemistry
subject: FOS: Chemical sciences
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Plant Biology

description: Additional file 1: Figure S1. Protein sequence alignment of the P450 fatty acid decarboxylases OleTJE from Jeotgalicoccus sp. ATCC 8456 (GenBank accession number: ADW41779) and CYP-Sm46Δ29 (with the N-terminal redundant 29 amino acids deleted) from Staphylococcus massiliensis S46 (GenBank accession number: WP_039990689), with the newly identified FADCs of OleTJH from Jeotgalicoccus halophilus (GenBank accession number: WP_092595307), OleTSQ from Salinicoccus qingdaonensis (GenBank accession number: WP_092983663), and OleTSA from Staphylococcus aureus (GenBank accession number: WP_049319149). The orange stars indicate the residues (79F, 85H, 170I, 245R) that have been reported to be important for decarboxylation by P450 OleTJE. The blue stars indicate the key different amino acid residues. Figure S2. SDS-PAGE analysis of the purified His6-tagged OleTJH (lane A), OleTSQ (lane B), OleTSA (lane C), and protein marker (M). Figure S3. UV-visible spectra of OleTJE (A), OleTJH (B), OleTSQ (C) and OleTSA (D). The purified enzymes were diluted in buffer (pH 7.4) containing 50 mM NaH2PO4, 500 mM NaCl and 10% glycerol. (Black lines show the spectra for the oxidized ferric form of CYPs and red lines show the spectra for the Na2S2O4-reduced ferrous-CO complex of CYPs; Insets exhibit the reduced CO-bound difference spectra of P450 enzyme). Figure S4. Kinetic curves of OleTJE, OleTJH, OleTSQ and OleTSA against their optimal substrate (lauric acid) were fitted to Michaelis-Menten equation respectively. (A) Lauric acid (C12) substrate consumption rates by OleTJE; (B) Lauric acid (C12) substrate consumption rates by OleTJH; (C) Lauric acid (C12) substrate consumption rates by OleTSQ; (D) Lauric acid (C12) substrate consumption rates by OleTSA. The steady state kinetic parameters were calculated using OriginPro 8.5 and are summarized in Table 2. Figure S5. UV-visible spectra of the OleTSA mutants: T47F (A), I177L (B), V319A (C), L405I (D) and T47F/I177L/V319A/L405I (E). The purified enzymes were diluted in buffer (pH 7.4) containing 50 mM NaH2PO4, 500 mM NaCl and 10% glycerol, respectively. (Black lines show the spectra for the oxidized ferric form of CYPs and red lines show the spectra for the Na2S2O4-reduced ferrous-CO complex of CYPs; Insets exhibit the reduced CO-bound difference spectra of P450 enzyme). Figure S6. SDS-PAGE analysis of the purified His6-tagged mutants, including T47F (lane A), I177L (lane B), V319A (lane C), L405I (lane D), T47F/I177L/V319A/L405I (lane E), and protein marker (M). Figure S7. Decarboxylation (DC) versus hydroxylation (HD) activities (ratios) of OleTSA (WT) and its mutants including T47F (M1), I177L (M2), V319A (M3), L405I (M4), and T47F-I177L-V319A-L405I (M5) towards mid-chain fatty acids (C8–C12). Figure S8. Analysis of acidic amino acids (aspartic acid in red and glutamic acid in cyan) in the protein structure of OleTJE (A: PDB ID #: 4L40) and the modeled protein structures by Phyre2 (B: OleTJH; C: OleTSQ; D: OleTSA). Figure S9. The effect of H2O2 concentration on substrate conversion ratios for four FADCs (A: OleTJE; B: OleTJH; C: OleTSQ; D: OleTSA) in our standard reaction system (200 μM lauric acid substrate, 1 μM purified OleT enzyme, 30 °C for 2 h). Table S1. Primers used for cloning and site-directed mutagenesis in this study. Table S2. Major differences in substrate-binding-site residues composition among OleTJE and the three newly identified P450 FADCs.