Gene engineering, purification, crystallization and preliminary X-ray diffraction of cytochrome P450 p-coumarate-3-hydroxylase (C3H), the Arabidopsis membrane protein

Abstract

Cytochrome P450s (P450s) are the most versatile biological catalysts in plants; however, because the structure of the P450s has not been fully established, their broad substrate specificity has been limitedly discussed. p-coumarate-3-hydroxylase (C3H) is an essential enzyme for the biosynthesis of phenolic natural products in plants, but all attempts to express and purify C3H, have failed. In this research, we developed a bacterial expression of Arabidopsis C3H by combinational mutagenesis and purified C3H as a catalytically active form. The modified C3H could be purified in the absence of detergent, and crystallized in two forms (orthorhombic and trigonal space group) under different conditions. X-ray diffraction was processed to a 4.0 Å resolution (first type crystal) and a 3.8 Å resolution (second type crystal). Although the diffraction results of C3H mod crystals are not enough to determine crystallographic structure due to low resolution, the simplicity and rapidity of this technology are competitive advantages in comparison with other methods, and may contribute to structural analyses of other membrane proteins including P450s family.