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Engineering Cytochrome P450 BM-3 for Selective Hydroxylation of Alkanes


Meinhold, Peter (2005) Engineering Cytochrome P450 BM-3 for Selective Hydroxylation of Alkanes. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/aats-ca30.


Cytochromes P450 are of potential synthetic value because they hydroxylate a large array of substrates, often with high regio- and enantioselectivity. In contrast to most P450s, the BM-3 variant from Bacillus megaterium is soluble, easily expressed in E. coli, and does not require additional electron transfer proteins. A highly efficient enzyme for its preferred reaction, hydroxylation of medium-chain (C12 to C18) fatty acids, BM-3 is a good candidate for engineering for applications requiring activity on other substrates.

Using iterations of random mutagenesis, recombination, and high throughput screening, we engineered P450 BM-3 mutants to hydroxylate linear alkanes as short as propane. Activity towards linear alkanes was further increased by changing two key active site residues. The resulting mutants hydroxylate linear alkanes with high regioselectivity and, notably, enantioselectivity.

We further engineered these enzymes with guidance from the crystal structure of substrate-bound P450 BM-3. Eleven active-site residues were chosen for saturation mutagenesis, and the resulting mutants were screened for improved activity towards alkanes, as measured by total product formation. Substitutions at these positions generally did not affect correct folding of the enzyme, and a large fraction of folded proteins retained similar levels of activity as their predecessor. Moreover, several of the 11 selected amino acid substitutions yielded mutants that were both more active and produced various combinations of product regioisomers.

Recombination of these beneficial active-site mutations generated BM-3 variants that catalyze: (a) regio- and enantioselective hydroxylation of linear alkanes; (b) terminal hydroxylation of linear alkanes; (c) regio- and enantioselective hydroxylation of heterocyclic compounds; and (d) ethane hydroxylation.

The selective conversion of ethane to ethanol, not previously reported for any P450, is catalyzed by the most active mutant from this library. In nature, this reaction is solely observed for methane monooxygenases (MMOs) and related enzymes in alkane-assimilating bacteria.

Additionally, we have found that the reductase domain can be engineered to increase the efficiency of these reactions. Our progress in converting BM-3 from a fatty-acid hydroxylase into an enzyme able to selectively hydroxylate smaller alkanes, including ethane, is an important step towards our ultimate goal, achieving selective BM-3 catalyzed conversion of methane to methanol.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Alkane Hydroxylation; Alkane Oxidation; Biocatalysis; Cytochrome P450 BM-3; Directed Evolution; Methane; Protein Engineering
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Biochemistry and Molecular Biophysics
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Arnold, Frances Hamilton
Thesis Committee:
  • Rees, Douglas C. (chair)
  • Newman, Dianne K.
  • Arnold, Frances Hamilton
  • Labinger, Jay A.
Defense Date:31 May 2005
Record Number:CaltechETD:etd-06032005-113204
Persistent URL:
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:2422
Deposited By: Imported from ETD-db
Deposited On:03 Jun 2005
Last Modified:08 Nov 2023 00:11

Thesis Files

PDF (Meinhold_Thesis.pdf) - Final Version
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