Abstract
Cytochrome P450 2B1 has been subjected to directed evolution to investigate the role of amino acid residues outside of the active site and to engineer novel, more active P450 catalysts. A high throughput screening system was developed to measure H(2)O(2)-supported oxidation of the marker fluorogenic substrate 7-ethoxy-4-trifluoromethylcoumarin (7-EFC). Random mutagenesis by error-prone polymerase chain reaction and activity screening were optimized using the L209A mutant of P450 2B1 in an N-terminally modified construct with a C-terminal His tag (P450 2B1dH). Two rounds of mutagenesis and screening and one subcloning step yielded the P450 2B1 quadruple mutant V183L/F202L/L209A/S334P, which demonstrated a 6-fold higher k(cat) than L209A. Further random or site-directed mutagenesis did not improve the activity. When assayed in an NADPH-supported reconstituted system, V183L/L209A demonstrated lower 7-EFC oxidation than L209A. Therefore, F202L/L209A/S334P was generated, which showed a 2.5-fold higher k(cat)/K(m) for NADPH-dependent 7-EFC oxidation than L209A. F202L/L209A/S334P also showed enhanced catalytic efficiency with 7-benzyloxyresorufin, benzphetamine, and testosterone, and a 10-fold increase in stereoselectivity for testosterone 16alpha-versus 16beta-hydroxylation compared with 2B1dH. Enhanced catalytic efficiency of F202L/L209A/S334P was also retained in the full-length P450 2B1 background with 7-EFC and testosterone as substrates. Finally, the individual mutants were tested for metabolism of the anti-cancer prodrugs cyclophosphamide and ifosfamide. Several of the mutants showed increased metabolism via the therapeutically beneficial 4-hydroxylation pathway, with L209A/S334P showing 2.8-fold enhancement of k(cat)/K(m) with cyclophosphamide and V183L/L209A showing 3.5-fold enhancement with ifosfamide. Directed evolution can thus be used to enhance P450 2B1 catalytic efficiency across a panel of substrates and to identify functionally important residues distant from the active site.
Highlights
Cytochrome P450 2B1 has been subjected to directed evolution to investigate the role of amino acid residues outside of the active site and to engineer novel, more active P450 catalysts
Purified V183L/F202L/L209A/E322K/S334P showed no further enhancement of kcat, suggesting that random mutagenesis was approaching saturation for enhanced 7-EFC O-deethylation
V183L/F202L/ L209A/I290F/S334P showed no increase in kcat beyond V183L/ F202L/L209A/S334P, again suggesting saturation of activity along this particular directed evolution pathway
Summary
Materials—7-EFC and 7-BR were purchased from Molecular Probes, Inc. (Eugene, OR). Polymyxin B sulfate, CPA, and NADPH were bought from Sigma. [4-14C]Testosterone was obtained from Amersham Biosciences. 4-Hydroperoxy-CPA was obtained from ASTA Pharma (Beilefeld, Germany). Error-prone PCR, ligation, and transformation were standardized for P450 2B1dH L209A to obtain Ͼ1000 clones per PCR reaction by selecting suitable forward and reverse primers, restriction sites, ligation conditions, and transformation procedures. Error-prone PCR was standardized to ensure a mutation rate of 1–2 bp per P450 cDNA essentially as described previously [28]. 50 l of the substrate mixture (300 M 7-EFC containing 4% methanol and 20 units/well polymyxin B sulfate) was incubated with 40 l of whole cells for 5 min at room temperature. The reconstitution system for assaying CPA and IFA metabolism included P450 and purified rat liver CPR in a 1:4 molar ratio (5 pmol of P450/0.1 ml of 0.1 M KPi buffer, pH 7.4, containing 0.1 mM EDTA) and was devoid of lipid and b5. The heme group was copied from 2B4 into the 2B1 model
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
