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

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

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

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

The previously laboratory-evolved cytochrome P450 2B1 quadruple mutant V183L/F202L/L209A/S334P (QM), which showed enhanced H(2)O(2)-mediated substrate oxidation, has now been shown to exhibit a >3.0-fold decrease in K(m,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 degrees C) and maintained approximately 1.4-fold higher activity than QM at 45 degrees 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.

Engineering mammalian cytochrome P450 2B1 by directed evolution for enhanced catalytic tolerance to temperature and dimethyl sulfoxide. Publishing Authors By Initials

S Kumar,L Sun,H Liu,BK Muralidhara,JR Halpert,

For similar environment and public health: environment: environment, controlled: temperature research abstracts see: environment and public health: environment: environment, controlled: temperature research

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

PUBLICATION TYPE: Research Support, N.I.H., Extr

Journal: Protein engineering, design & selection : PEDS

VOLUME: 19

Page Numbers: 547-54

Journal Abbreviation: Protein Eng. Des. Sel.

ISSN: 1741-0126

DAY: 18

MONTH: 10

YEAR: 2006

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

Number of References:

LANGUAGE: eng

NlmUniqueID: 101186484

Engineering mammalian cytochrome P450 2B1 by directed evolution for enhanced catalytic tolerance to temperature and dimethyl sulfoxide. Keywords Mesh Terms:

KEYWORDS: Temperature

MESH TERMS: metabolism

Chemical & Substance for Abstract: Engineering mammalian cytochrome P450 2B1 by directed evolution for enhanced catalytic tolerance to temperature and dimethyl sulfoxide. Information

Substance Name: Cytochrome P-450 CYP2B1

Registry Number: EC 1.14.14.1

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

AFFILIATION: Department of Pharmacology and Toxicology, University of Texas Medical Branch 301 University Boulevard, Galveston, TX 77555-1031, USA. sakumar@utmb.edu

Country: England

AGENCY: United States NIEHS

GRANT: ES06676

ACRONYM: ES

MEDLINETA: Protein Eng Des Sel

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