Research Article: Structural analysis of Cytochrome P450 BM3 mutant M11 in complex with dithiothreitol

Date Published: May 24, 2019

Publisher: Public Library of Science

Author(s): Karla Frydenvang, Marlies C. A. Verkade-Vreeker, Floor Dohmen, Jan N. M. Commandeur, Maria Rafiq, Osman Mirza, Flemming Steen Jørgensen, Daan P. Geerke, Claudio M. Soares.


The bacterial Cytochrome P450 (CYP) BM3 (CYP102A1) is one of the most active CYP isoforms. BM3 mutants can serve as a model for human drug-metabolizing CYPs and/or as biocatalyst for selective formation of drug metabolites. Hence, molecular and computational biologists have in the last two decades shown strong interest in the discovery and design of novel BM3 variants with optimized activity and selectivity for substrate conversion. This led e.g. to the discovery of mutant M11 that is able to metabolize a variety of drugs and drug-like compounds with relatively high activity. In order to further improve our understanding of CYP binding and reactions, we performed a co-crystallization study of mutant M11 and report here the three-dimensional structure M11 in complex with dithiothreitol (DTT) at a resolution of 2.16 Å. The structure shows that DTT can coordinate to the Fe atom in the heme group. UV/Vis spectroscopy and molecular dynamics simulation studies underline this finding and as first structure of the CYP BM3 mutant M11 in complex with a ligand, it offers a basis for structure-based design of novel mutants.

Partial Text

The CYP enzyme family comprises 57 human isoforms serving various purposes. The human drug-metabolizing CYPs are promiscuous enzymes with broad substrate specificity transforming a variety of compounds to more soluble compounds and, thereby, facilitating their excretion from the human organism [1]. The human CYPs also comprise highly selective enzymes involved in e.g. steroidogenesis [2]. Recently, these CYPs have been shown to be potential targets for treatment of various forms of cancer [3, 4]. The plant kingdom contains 127 CYP families typically with more than 250 CYPs in each, and each CYP usually being responsible for the stereoselective synthesis of a single compound [5]. The bacterial CYPs are interesting as targets for certain diseases (e.g. Mycobacterium tuberculosis) [6]. Furthermore, they can be tailored to mimic human CYPs and often be expressed in higher yield than their human analogues [7].

We presented a crystal structure of CYP BM3 mutant M11 in complex with a ligand. Over the last 10 years, this “humanized” CYP BM3 variant has shown potential as biocatalyst and as template to engineer mutants that selectively metabolize drugs and drug-like compounds. Our crystal structure represents, to our knowledge, the first structure of the CYP BM3 mutant M11 with a ligand, DTT, bound directly to the heme iron. Our crystal structure indicates that binding of a DTT ligand to M11 does not significantly alter the protein conformation, which underlines the use of the structure of this promiscuous enzyme for modeling purposes. Our work also shows that DTT coordinates with the heme iron, with a Fe-S distance of 2.3 Å. Absorbance difference spectra obtained in UV/Vis spectroscopic titration of M11 with DTT demonstrate coordination by the ligand in its anionic form. Docking and molecular dynamics simulations also suggest that the monovalent DTT anion can adopt a heme iron-coordinating binding pose, in which no specific interactions between DTT and the active-site residues are observed. This finding is in line with our interpretation from the obtained electron density map that DTT remains relatively flexible in its observed active-site bound conformation.