The recombinant CgPst2 protein is non-functional.

A. 6X-Histidine-FLAG-CgPst2 purification from Escherichia coli. The E. coli BL21 (DE3) strain transformant carrying pET28a⁽⁺⁾-6XHIS-FLAG-CgPST2 plasmid was grown in LB medium, induced with IPTG (0.5 mM) at 18°C for 16 h, and cells were collected. After cell lysis, the recombinant CgPst2 protein was purified using TALON metal affinity resin via affinity purification. 30 μl eluates along with flow through were resolved on 12% SDS-PAGE, and stained with Coomassie Brilliant Blue. The black arrow marks CgPst2 band. Immunoblot analysis of these fractions with anti-His antibody (1:5000 dilution) is shown at the bottom. M, Protein Marker. B. NADH:quinone oxidoreductase activity measurement of recombinant CgPst2. 50 μg of purified CgPst2 was incubated for 1 h with FMN (100 μM) or FAD (100 μM) on ice, followed by addition of menadione (500 μM) in buffer containing Tris-HCl (10 mM; pH 7.4) and NaCl (150 mM). The reaction was started with NADH (500 μM) addition, and absorbance was recorded at 340 nm in a 1-cm-path-length quartz cuvette over a period of 60 seconds at 10 seconds interval on Spectramax M5 plate reader. The commercially available NAD(P)H:FMN oxidoreductase (1 Unit, Roche, # 10476480001) was taken as positive control. Blank contained no protein. Absorbance of the substrate NADH was considered as 100 at 0 h time point, and NADH oxidation was calculated by dividing the absorbance at each time point by 0 h absorbance, and multiplying the number by 100. Data represent mean ± SEM. Grouped multiple t-test was performed, with n = 3 to 5. **, p < 0.0021; ****, p < 0.0001. C. NADH:quinone oxidoreductase activity measurement in cell extracts of wt, Cgpst2Δ and Q-KO strains in enzymatic reaction mixtures that contained exogenously added FMN (50 μM) or FAD (50 μM). Data represent mean ± SD (n = 2–3). The Q-KO strain lacks four flavodoxin-like proteins, CgPst2, CgRfs1, CgPst3 and CgYcp4. D. NADH:quinone oxidoreductase activity measurement in extracts of Cgpst2Δ and Q-KO cells expressing CgPST2. CgPST2 expression restored the activity deficit in Cgpst2Δ and Q-KO strains. Data represent mean ± SEM. Black and blue asterisks indicate statistically significant differences in activity of QKO and Cgpst2Δ samples, respectively, expressing CgPST2, compared to corresponding strains carrying vector. Grouped multiple t-test was performed, with n = 3. *, p < 0.0332; **, p < 0.0021.

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