Functional expression and purification of CYP93C20, a plant membrane-associated cytochrome P450 from Medicago truncatula

Abstract

Plants possess very large numbers of biosynthetic cytochrome P450 enzymes. In spite of the importance of these enzymes for the synthesis of bioactive plant secondary metabolites, only two plant P450 structures has been obtained to date. Isoflavone synthase (IFS) is a membrane-associated cytochrome P450 enzyme catalyzing the entry-point reaction into isoflavonoid biosynthesis. IFS from the model legume Medicago truncatula (CYP93C20) was engineered by deleting the membrane-spanning domain and inserting a hydrophilic polypeptide in the N-terminus and a four histidine tag at the C-terminus. The truncated form exhibited dramatically enhanced expression and solubility. The engineered enzyme was expressed in Escherichia coli XL1-blue cells and was purified by Ni2+-NTA affinity chromatograph and size-exclusion chromatograph. The purified enzyme was characterized by enzyme assay, reduced carbon monoxide difference spectroscopy and peptide mass fingerprinting. The engineered soluble enzyme exhibited the same activity as the full length membrane-associated enzyme expressed in yeast. These studies suggest an approach for engineering plant membrane-associated P450s with enhanced expression and solubility for mechanistic and structural studies.