Rhus vernicifera

The participation of laccase in luciferin enzymatic synthesis was assayed using commercial laccase from Rhus vernicifera (Sigma, USA) in the presence of hydroquinone (HQ) or dopamine (DA), and d-cysteine. The reactions were initially performed by mixing 10 μL of laccase (0.5 U) and 25 μL of 40 mM HQ or DA, in a final volume of 100 μL of 0.10 M phosphate buffer at pH 6.5, or 90 mM Tris–HCl buffer pH 7.5 in the Elisa plate wells followed by incubation during 1 h at 22 °C under moderate agitation. After that, 12.5 μL of 80 mM d-cysteine were added to the wells with further incubation during 18 h. The control reactions were performed in the absence of d-cysteine or laccase. Luciferin synthesis in each reaction was luminometrically evaluated by bioluminescence in the presence of Amy luciferase and ATP. This assay was carried out by mixing 25 μL of the reaction product, 5 μL of Amy luciferase (~ 3 µg), 5 μL of solution containing 80 mM MgSO₄/40 mM ATP, and 65 μL of 0.10 M Tris–HCl buffer at pH 8.0. The reactions were performed in triplicate, and the bioluminescent activities were normalized in relation to the activity of the control reaction between hydroquinone and cysteine at pH 7.5. In order to check luciferin formation, we also carried out TLC of these samples, as described in the topic 2.3.

The chemicals CuSO₄, MnSO₄, benzyl butyl phthalate (BBP), di-n-butyl phthalate (DBP), dicyclohexyl phthalate (DCP), diethyl phthalate (DEP), di-(2-ethylhexyl) phthalate (DEHP), acetosyringone (AS), syringaldehyde (SA), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), vanillin (VA), N-hydroxyphthalimide (HPI), (2,2,6,6-tetramethylpiperidin-1-yl) oxyl (TEMPO), and hexane, along with other solvents (analytical grade, 99.0% purity), were purchased from Sigma-Aldrich (Merck/Millipore Sigma, St. Louis, Missouri, USA). The chemical structures of the compounds used in this study are shown in Figure 1A and Supplementary Table S1. Laccases from three fungi (Aspergillus sp., Rhus vernicifera, and Trametes versicolor) were purchased from Sigma-Aldrich (Merck/Millipore Sigma, St. Louis, Missouri, USA).

Nondenaturing gel electrophoresis was used to detect the laccase‐like activity in C. albicans as previously described [30]. C. albicans cells were cultured in YPD overnight at 22 °C. After the cells were harvested, the total proteins were extracted with acid‐washed glass beads (Sigma) using a Fast‐Prep homogenizer (FP120; Thermo Electron). The homogenate was centrifuged at 16 200 g for 30 min. The protein concentration of the cell lysate was measured by the Bradford method, and 300 μg total protein in each 40 μL sample was loaded onto gels. Proteins were also estimated using the Coomassie brilliant blue method (Fig. S2). Commercially, laccase (from Rhus vernicifera; Sigma) was used as a positive control, and an equal protein sample was boiled for 5 min as a negative control. Rhus vernicifera laccase (40 U equivalent) and 300 μg total protein of C. albicans cells were loaded onto gels. After electrophoresis, the gels were immersed in 1 mml‐DOPA in 0.1 m citric acid/0.2 m Na₂HPO₄ (pH 6.0) buffer overnight. Positive laccase activity was revealed by C. albicans protein extracts as shown by dark bands, which confirmed that l‐DOPA had polymerized to form melanin.