Engineering Cytochrome P-450s: Chimeric Enzymes

Authors: Sukumaran S.¹; Atkins W.M.²; Shanker R.³

Source: Applied Biochemistry and Biotechnology, Volume 103, Numbers 1-3, October 2002 , pp. 291-302(12)

Publisher: Humana Press

Abstract:

Cytochrome P-450 isozymes represent a critical component of nature?s spectrum of detoxification catalysts that could be exploited for bioremediation. The ethanol-inducible human cytochrome P-450 2E1 serves as a model eukaryotic P-450 that complements the bacterial P-450 cam in dehalogenation and detoxification of environmental pollutants. We explored the construction of novel chimeric P-450s using cytochrome P-450 camC and 2E1 genes. For construction of chimera 1 (478 amino acids, 55.14 kDa), 145 amino acids from the N-terminus of P-450 2E1 protein (493 amino acids, 56.84 kDa) were replaced with 130 amino acids from the N-terminus of P-450 camC protein (415 amino acids, 46.66 kDa). In chimera 2 (525 amino acids, 60.24 kDa) the strategy involves replacement of 28 amino acids in the C-terminus of chimera 1 with 75 amino acids from the C-terminus of P-450 camC gene. Homology models of both the chimeric proteins were developed using SWISS-MODEL based on the known crystal structure of cytochrome P-450 camC, BM-3, 1DT6A, and 2C17A. The models indicated that the proposed heme-binding site was intact, which is inevitable for catalytic activity of cytochrome P-450s. The expression of chimera 1 and 2 genes in Escherichia coli DH5 was evident from light-pink cell pellets, protein band in sodium dodecyl sulfate polyacrylamide gel electrophoresis, and diagnostic carbon monoxide-difference spectra. Our studies show that strategies can be developed to exploit the natural diversity of the P-450 superfamily to generate chimeric biocatalysts that would provide new templates amenable to directed evolution.

Keywords: Designer proteins; cytochrome P-450; P-450 101; chimeric P-450 2E1; homology modeling; camphor; detoxification; bacteria

Language: English

Document Type: Research article

Affiliations: 1: National Environmental Engineering Research Institute, Nehru Marg, Nagpur 440020, India 2: Department of Medicinal Chemistry, University of Washington, Seattle, WA 98195, USA 3: National Environmental Engineering Research Institute, Nehru Marg, Nagpur 440020, India

Publication date: 2002-10-01

• In this: publication
• By this: publisher
• In this Subject: Anatomy & Physiology ,  Biotechnology ,  Organic Chemistry ,  Biochemistry
• By this author: Sukumaran S. ;  Atkins W.M. ;  Shanker R.

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