Induction and catalytic properties of an intracellular NADH-dependent 1,4-benzoquinone reductase from the brown-rot basidiomycete Gloeophyllum trabeum

The expression of intracellular NADH-dependent 1,4-benzoquinone reductase in the brown-rot fungus Gloeophyllum trabeum was increased in the presence of 2,6-dimethoxy-1,4-benzoquinone (2,6-DMBQ) and, to a lesser extent, vanillic acid. Expression was time-dependent and influenced by nitrogen levels. An intracellular NADH-dependent 1,4-benzoquinone reductase was purified from G. trabeum. The subunit molecular mass was 22 kDa. Flavin mononucleotide was the coenzyme. The pI of the enzyme was 4.2. The quinone reductase catalyzed the reduction of multiple 1,4-quinones and exhibited Ping–Pong kinetics. For enzyme-catalyzed 2,6-DMBQ reduction, the apparent KM was 6.8 μM and the kcat1.0×103 s−1; the pH optimum was between 5.5 and 7; the activation energy of the reaction was estimated at 43.7 kJ mol−1. The one-to-one stoichiometric ratio of NADH oxidation versus 2,6-DMBQ reduction suggested a two-electron transfer mechanism for the enzyme. Dicumarol and Cibacron blue were competitive inhibitors with Ki values of 0.5 and 0.2 μM, respectively.