title: Global incorporation of norleucine in place of methionine in cytochrome P450 BM-3 heme domain increases peroxygenase activity.
abstract: In this study we have replaced all 13 methionine residues in the cytochrome P450 BM-3 heme domain (463 amino acids) with the isosteric methionine analog norleucine. This experiment has provided a means of testing the functional limits of globally incorporating into an enzyme an unnatural amino acid in place of its natural analog, and also an efficient way to test whether inactivation during peroxide-driven P450 catalysis involves methionine oxidation. Although there was no increase in the stability of the P450 under standard reaction conditions (in 10 mM hydrogen peroxide), complete substitution with norleucine resulted in nearly two-fold-increased peroxygenase activity. Thermostability was significantly reduced. The fact that the enzyme can tolerate such extensive amino acid replacement suggests that we can engineer enzymes with unique chemical properties via incorporation of unnatural amino acids while retaining or improving catalytic properties. This system also provides a platform for directing enzyme evolution using an extended set of protein building blocks.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.
authors.forename: Patrick C
authors.initials: PC
authors.lastname: Cirino
authors.forename: Yi
authors.initials: Y
authors.lastname: Tang
authors.forename: Katsuyuki
authors.initials: K
authors.lastname: Takahashi
authors.forename: David A
authors.initials: DA
authors.lastname: Tirrell
authors.forename: Frances H
authors.initials: FH
authors.lastname: Arnold
mesh_terms: D003470:Culture Media; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D008715:Methionine / Q000276:immunology*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009646:Norleucine / Q000737:chemistry*; D015202:Protein Engineering / Q000379:methods*; D013696:Temperature
chemical_list: D003470:Culture Media; D009646:Norleucine; D003577:Cytochrome P-450 Enzyme System; D008715:Methionine; D006899:Mixed Function Oxygenases; C022333:peroxygenase