Purification and properties of reductase of the three-component p-cymene methyl hydroxylase from Pseudomonas chlororaphis subsp. aureofaciens

A novel three-component p-cymene methyl hydroxylase from Pseudomonas chlororaphis subsp. aureofaciens was reported earlier on the basis of genetic characterization and their expression catalyzing methyl group hydroxylation. This enzyme system was inductively synthesized when grown on p-cymene and had an important role in initiating p-cymene metabolism in vivo. In the present study, a NADH-dependent cytochrome c reductase protein has been purified to an electrophoretically homogeneous state and found to be involved in the hydroxylation of methyl group of p-cymene. Molecular mass of the reductase appears to be 38 kDa by SDS/PAGE and 39 kDa by gel filtration apart from one molecule of tightly bound FAD and two atoms each of iron and acid-labile sulfur per molecule of the enzyme. An apparent Km value of the enzyme for NADH is 32 ± 1.2 μM. To the best of our knowledge, this is the first report on the purification of reductase component of p-cymene methyl hydroxylase.

► This is the first report on the purification of reductase component of p-cymene methyl hydroxylase.
► Chromatographic procedures led to a 92-fold purification of the reductase.
► Purified reductase was found to be a monomer of 38 kDa polypeptide.
► Reductase component was characterized as an iron-sulfur flavoprotein.
► The purified reductase contained 1 molecule each of FAD and [2Fe–2S] center.