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Identification of amino acid residues involved in 4-chloroindole 3-hydroxylation by cytochrome P450 2A6 using screening of random libraries

Paper ID Volume ID Publish Year Pages File Format Full-Text
24750 43535 2009 7 PDF Available
Title
Identification of amino acid residues involved in 4-chloroindole 3-hydroxylation by cytochrome P450 2A6 using screening of random libraries
Abstract

Cytochrome P450 (P450) 2A6 is able to catalyze indole hydroxylation to form the blue dye indigo. The wild-type P450 2A6 enzyme was randomly mutated throughout the whole open reading frame and screened using 4-chloroindole hydroxylation, a substituted indole selected from 30 indole compounds for enhanced color development. Mutants with up to 5-fold increases of catalytic efficiency (kcat/Km) and 2-fold increases in kcat were selected after two rounds of screening. Important residues located both in (e.g., Thr305) and outside the active site (e.g., Ser224) were identified. The study utilized a better substrate for “indigo assay” to obtain new information on the structure-functional relationship of P450 2A6 that was not revealed by previous mutagenesis studies with this enzyme.

Keywords
P450, cytochrome P450; NPR, NADPH-cytochrome P450 reductase; SRS, substrate recognition sites; PCR, polymerase chain reaction; epPCR, error-prone PCR; IPTG, d-isopropyl-β-thiogalactopyranoside; APCI, atmospheric pressure chemical ionization; WT, wild type
First Page Preview
Identification of amino acid residues involved in 4-chloroindole 3-hydroxylation by cytochrome P450 2A6 using screening of random libraries
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 139, Issue 1, 1 January 2009, Pages 12–18
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering