Engineering Cytochrome P450 BM3 for Terminal Alkane Hydroxylation

Abstract

Enzymes that catalyze the terminal hydroxylation of alkanes could be used to produce more valuable chemicals from hydrocarbons. Cytochrome P450 BM3 from Bacillus megaterium hydroxylates medium-chain fatty acids at subterminal positions at high rates. To engineer BM3 for terminal alkane hydroxylation, we performed saturation mutagenesis at selected active-site residues of a BM3 variant that hydroxylates alkanes. Recombination of beneficial mutations generated a library of BM3 mutants that hydroxylate linear alkanes with a wide range of regioselectivities. Mutant 77-9H exhibits 52% selectivity for the terminal position of octane. This regioselectivity is octane-specific and does not transfer to other substrates, including shorter and longer hydrocarbons or fatty acids. These results show that BM3 can be readily molded for regioselective oxidation.

Synthesis of 1‐Naphthol by a Natural Peroxygenase Engineered by Directed Evolution

“…Indeed, these enzymes can transform naphthalene into 1-naphthol by either harnessing the peroxide shunt pathway or through their natural NAD(P)H dependent activity. [12][13][14][15] More recently, directed evolution of toluene orthomonooxygenase (TOM) with a whole-cell biocatalytic system was described. [16][17][18] However, the poor enzyme stability and the reliance on expensive redox cofactors and reductase domains have precluded the practical application of these enzymes in specific industrial settings.…”

Use of Chemical Auxiliaries to Control P450 Enzymes for Predictable Oxidations at Unactivated C-H Bonds of Substrates

“…Here again, molecules that are not fatty acids are typically not accepted as substrates because the carboxylate group is required for catalytic activity. Although many mutagenic studies have been performed with P450 BM3 with respect to broadening the substrate scope [34][35][36][37][38][39][40][41][42][43][44], this study demonstrated that perfluorocarboxylic acids (PFCs) can be used as decoy molecules to expand the substrate scope of P450 BM3 without resorting to mutagenesis. PFCs are a great alternative to fatty acids because many of the physical properties are maintained, while the greater bond energy of C-F (116 kcal mol −1 ) compared to that of C-H (95-99 kcal mol −1 ) renders them inert toward oxidation [30].…”

Katalytische, milde und selektive Oxyfunktionalisierung von linearen Alkanen: aktuelle Herausforderungen

“…[102,103] Arnold und Mitarbeiter haben beachtliche Anstrengungen unternommen, um mithilfe der molekularen gerichteten Evolution die Spezifität von P450 BM3 bezüglich Fettsäuren auch auf kurzkettige Alkane zu übertragen. [8,34,[104][105][106][107] Die am besten entwickelten Varianten enthalten Mutationen in beiden Subdomänen und zeigen eine hohe Aktivität für gasfçrmige Alkane und endlich auch für Ethan (Tabelle 1, Nr. 11 und 12).…”