Engineering mammalian cytochrome P450 2B1 by directed evolution for enhanced catalytic tolerance to temperature and dimethyl sulfoxide

Santosh Kumar, Ling Sun, Hong Liu, B. K. Muralidhara, James R. Halpert

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

The previously laboratory-evolved cytochrome P450 2B1 quadruple mutant V183L/F202L/L209A/S334P (QM), which showed enhanced H2O 2-mediated substrate oxidation, has now been shown to exhibit a >3.0-fold decrease in Km,HOOH for 7-ethoxy-4- trifluoromethylcoumarin (7-EFC) O-deethylation compared with the parental enzyme L209A. Subsequently, a streamlined random mutagenesis and a high-throughput screening method were developed using QM to screen and select mutants with enhanced tolerance of catalytic activity to temperature and dimethyl sulfoxide (DMSO). Upon screening >3000 colonies, we identified QM/L295H and QM/K236I/D257N with enhanced catalytic tolerance to temperature and DMSO. QM/L295H exhibited higher activity than QM at a broad range of temperatures (35-55°C) and maintained ∼1.4-fold higher activity than QM at 45°C for 6 h. In addition, QM/L295H showed a significant increase in T m,app compared with L209A. QM/L295H and QM/K236I/D257N exhibited higher activity than QM at a broad range of DMSO concentrations (2.5-15%). Furthermore, QM/K236I/D257N/L295H was constructed by combining QM/K236I/D257N with L295H using site-directed mutagenesis and exhibited a >2-fold higher activity than QM at nearly the entire range of DMSO concentrations. In conclusion, in addition to engineering mammalian cytochromes P450 for enhanced activity, directed evolution can also be used to optimize catalytic tolerance to temperature and organic solvent.

Original languageEnglish (US)
Pages (from-to)547-554
Number of pages8
JournalProtein Engineering, Design and Selection
Volume19
Issue number12
DOIs
StatePublished - Dec 1 2006
Externally publishedYes

Fingerprint

Cytochrome P-450 CYP2B1
Dimethyl sulfoxide
Dimethyl Sulfoxide
Mutagenesis
Temperature
Screening
High-Throughput Screening Assays
Site-Directed Mutagenesis
Application programs
Organic solvents
Cytochrome P-450 Enzyme System
Catalyst activity
Thermodynamic properties
Enzymes
Throughput
Oxidation
Substrates

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Molecular Biology

Cite this

Engineering mammalian cytochrome P450 2B1 by directed evolution for enhanced catalytic tolerance to temperature and dimethyl sulfoxide. / Kumar, Santosh; Sun, Ling; Liu, Hong; Muralidhara, B. K.; Halpert, James R.

In: Protein Engineering, Design and Selection, Vol. 19, No. 12, 01.12.2006, p. 547-554.

Research output: Contribution to journalArticle

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