Engineering enhanced cytochromes P450: Understanding thermostability and mechanism through rational design and directed evolution

The importance of P450s in metabolizing pharmaceuticals and steroid biosynthesis as well as catalyzing synthetically difficult chemical transformations has inspired a significant interest for engineering enhanced cytochromes P450. Some of the properties targeted for improvement in P450s include enhanced catalysis rates, altered substrate specificity, and elevated stability. In the work described herein, engineering of cytochromes P450 for these types of properties is demonstrated using both rational and random approaches.;Enhanced thermostability is a target property to engineer into cytochromes P450. Although many studies have been done toward understanding protein stability, until recently little was understood about the mechanisms utilized by cytochromes P450 for thermostability. The isolation of CYP 119 from Sulfolobus solfataricus has provided a natural means for understanding greatly enhanced thermostability in cytochromes P450.;In this work, several approaches were taken to better understand the molecular interactions responsible for conveying elevated stability. Rational approaches, including generation of a homology model, and later, solution of the crystal structure of CYP 119, provided structural insight into these mechanisms. The structural analysis indicated that aromatic stacking interactions, increased numbers of salt bridges and stronger hydrogen bonding may contribute to the stabilization of CYP 119. However, the structural information alone was not sufficient to understand the stabilizing interactions in detail.;The use of random and combinatorial mutagenesis identified residues E114 and F24 as contributors to protein stability through salt link networks and aromatic stacking. However, the relatively low number of mutants identified through this approach was insufficient to establish a global mechanism for stabilizing P450s. By merging the information gained from structural analysis and random mutagenesis, global mechanisms for stabilization of P450s emerged, such as increased salt link networks and the presence of large aromatic clusters. Further characterization of the mutants revealed the importance of stabilizing the interactions between the protein and prosthetic groups at elevated temperature.;Although random and rational approaches for protein engineering are often used as separate approaches, this work has demonstrated the benefits of combining these approaches. Through the use of both methods a better understanding of thermostability, catalytic mechanism and substrate specificity was gained.