Cytochrome P450 enzymes in Thymus vulgaris

By using RACE-PCR techniques we were able to isolate a new gene of Thymus vulgaris which encodes for a protein belonging to the CYP450 familiy and showing limonene-6-hydroxylase activity. This protein, termed TvL6 H exhibits 76% sequence identity to CYP71D18 a known limonene-6-hydroxylase of Mentha x gracilis. Both T. vulgaris and M. gracilis belong to the familiy of Lamiaceae. M. gracilis contains high amounts of carvone formed from limonene in two enzymatic steps, namely 6-hydroxylation and subsequent dehydrogenation. T. vulgaris posseses only trace amounts of limonene, carveol or carvone in its essential oil. Therefore, we propose that TvL6 H may play a role in carvacrol formation. TvL6 H was expressed in yeast and the recombinant enzyme did not accept p-cymene or γ-terpinene as substrates indicating that a carvacrol pathway via carveol may be operative. It also did not produce the 3-hydroxylated isopiperitenone although TvL6 H shares a high degree of sequence identity with mint limonene 3-hydroxylases. Further genes encoding for cytochrome P450 s are about to be identified using primers binding within the highly conserved heme-binding motif sequence. These candidate genes will also be completed and isolated by RACE techniques and then expressed in S. cerevisiae and their enzymatic functions and properties unraveled.

Keywords: Cytochrome P450 enzymes, Thymus vulgaris, hydroxylases.