Use of directed evolution of mammalian cytochromes P450 for investigating the molecular basis of enzyme function and generating novel biocatalysts

Santosh Kumar, James R. Halpert

Research output: Contribution to journalReview article

34 Citations (Scopus)

Abstract

Directed evolution has been successfully applied to the design of industrial biocatalysts for enhanced catalytic efficiency and stability, and for examining the molecular basis of enzyme function. Xenobiotic-metabolizing mammalian cytochromes P450 with their catalytic versatility and broad substrate specificity offer the possibility of widespread applications in industrial synthesis, medicine, and bioremediation. However, the requirement for NADPH-cytochrome P450 reductase, often cytochrome b5, and an expensive cofactor, NADPH, complicates the design of mammalian P450 enzymes as biocatalysts. Recently, Guengerich and colleagues have successfully performed directed evolution of P450s 1A2 and 2A6 initially by using colony-based colorimetric and genotoxicity screening assays, respectively, followed by in vitro fluorescence-based activity screening assays. More recently, our laboratory has developed a fluorescence-based in vitro activity screening assay system for enhanced catalytic activity of P450s 2B1 and 3A4. The studies indicate an important role of amino acid residues outside of the active site, which would be difficult to target by other methods. The approach can now be expanded to design these as well as new P450s using more targeted substrates of environmental, industrial, and medical importance.

Original languageEnglish (US)
Pages (from-to)456-464
Number of pages9
JournalBiochemical and Biophysical Research Communications
Volume338
Issue number1
DOIs
StatePublished - Dec 9 2005

Fingerprint

Cytochrome P-450 Enzyme System
Assays
Screening
Enzymes
Fluorescence
Cytochromes b5
NADPH-Ferrihemoprotein Reductase
Environmental Biodegradation
Occupational Medicine
Bioremediation
Xenobiotics
Substrates
Substrate Specificity
NADP
Medicine
Catalyst activity
Catalytic Domain
Amino Acids
In Vitro Techniques

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

@article{02aca07d08054d599ec66ec213335d4b,
title = "Use of directed evolution of mammalian cytochromes P450 for investigating the molecular basis of enzyme function and generating novel biocatalysts",
abstract = "Directed evolution has been successfully applied to the design of industrial biocatalysts for enhanced catalytic efficiency and stability, and for examining the molecular basis of enzyme function. Xenobiotic-metabolizing mammalian cytochromes P450 with their catalytic versatility and broad substrate specificity offer the possibility of widespread applications in industrial synthesis, medicine, and bioremediation. However, the requirement for NADPH-cytochrome P450 reductase, often cytochrome b5, and an expensive cofactor, NADPH, complicates the design of mammalian P450 enzymes as biocatalysts. Recently, Guengerich and colleagues have successfully performed directed evolution of P450s 1A2 and 2A6 initially by using colony-based colorimetric and genotoxicity screening assays, respectively, followed by in vitro fluorescence-based activity screening assays. More recently, our laboratory has developed a fluorescence-based in vitro activity screening assay system for enhanced catalytic activity of P450s 2B1 and 3A4. The studies indicate an important role of amino acid residues outside of the active site, which would be difficult to target by other methods. The approach can now be expanded to design these as well as new P450s using more targeted substrates of environmental, industrial, and medical importance.",
author = "Santosh Kumar and Halpert, {James R.}",
year = "2005",
month = "12",
day = "9",
doi = "10.1016/j.bbrc.2005.08.080",
language = "English (US)",
volume = "338",
pages = "456--464",
journal = "Biochemical and Biophysical Research Communications",
issn = "0006-291X",
publisher = "Academic Press Inc.",
number = "1",

}

TY - JOUR

T1 - Use of directed evolution of mammalian cytochromes P450 for investigating the molecular basis of enzyme function and generating novel biocatalysts

AU - Kumar, Santosh

AU - Halpert, James R.

PY - 2005/12/9

Y1 - 2005/12/9

N2 - Directed evolution has been successfully applied to the design of industrial biocatalysts for enhanced catalytic efficiency and stability, and for examining the molecular basis of enzyme function. Xenobiotic-metabolizing mammalian cytochromes P450 with their catalytic versatility and broad substrate specificity offer the possibility of widespread applications in industrial synthesis, medicine, and bioremediation. However, the requirement for NADPH-cytochrome P450 reductase, often cytochrome b5, and an expensive cofactor, NADPH, complicates the design of mammalian P450 enzymes as biocatalysts. Recently, Guengerich and colleagues have successfully performed directed evolution of P450s 1A2 and 2A6 initially by using colony-based colorimetric and genotoxicity screening assays, respectively, followed by in vitro fluorescence-based activity screening assays. More recently, our laboratory has developed a fluorescence-based in vitro activity screening assay system for enhanced catalytic activity of P450s 2B1 and 3A4. The studies indicate an important role of amino acid residues outside of the active site, which would be difficult to target by other methods. The approach can now be expanded to design these as well as new P450s using more targeted substrates of environmental, industrial, and medical importance.

AB - Directed evolution has been successfully applied to the design of industrial biocatalysts for enhanced catalytic efficiency and stability, and for examining the molecular basis of enzyme function. Xenobiotic-metabolizing mammalian cytochromes P450 with their catalytic versatility and broad substrate specificity offer the possibility of widespread applications in industrial synthesis, medicine, and bioremediation. However, the requirement for NADPH-cytochrome P450 reductase, often cytochrome b5, and an expensive cofactor, NADPH, complicates the design of mammalian P450 enzymes as biocatalysts. Recently, Guengerich and colleagues have successfully performed directed evolution of P450s 1A2 and 2A6 initially by using colony-based colorimetric and genotoxicity screening assays, respectively, followed by in vitro fluorescence-based activity screening assays. More recently, our laboratory has developed a fluorescence-based in vitro activity screening assay system for enhanced catalytic activity of P450s 2B1 and 3A4. The studies indicate an important role of amino acid residues outside of the active site, which would be difficult to target by other methods. The approach can now be expanded to design these as well as new P450s using more targeted substrates of environmental, industrial, and medical importance.

UR - http://www.scopus.com/inward/record.url?scp=27544466682&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=27544466682&partnerID=8YFLogxK

U2 - 10.1016/j.bbrc.2005.08.080

DO - 10.1016/j.bbrc.2005.08.080

M3 - Review article

VL - 338

SP - 456

EP - 464

JO - Biochemical and Biophysical Research Communications

JF - Biochemical and Biophysical Research Communications

SN - 0006-291X

IS - 1

ER -