Cytochrome P450 BM-3 with the mutations A74G, F87V, and L188Q could catalyze indole to produce indigo and indirubin. To further enhance this capability, site-directed and random mutageneses on the monooxygenase domain of P450 BM-3 mutant (A74G/F87V/L188Q; 3X) were performed. The mutant libraries created by error-prone polymerase chain reaction were screened using a colorimetric colony-based method on agar plates followed by a spectroscopic assay involving in absorption of indigo at 670 nm and NADPH at 340 nm in microtiter plate. Three mutants (K434R/3X, E435D/3X, and D168N/ A225V/K440N/3X) exhibited higher hydroxylation activity toward indole in comparison to parent enzyme. Moreover, using saturation site-directed mutagenesis at amino acid positions 168, 225, 434, 435, and 440, two P450 BM-3 variants (D168H/3X, E435T/3X) with an up to sixfold increase in catalytic efficiency (kcat/Km) were identified, and the mutant D168H/3X acquired higher regioselectivity resulting in more indigo (dimerized 3hydroxy-indole) compared to parent mutant (93 vs 72%).
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1007/s12010-007-8002-5 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Kinetic Evidence for an Induced Fit Mechanism in the Binding of the Substrate Camphor by Cytochrome P450.
ACS Catal
January 2021
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, United States.
Bacterial cytochrome P450 (P450) 101A1 (P450) has served as a prototype among the P450 enzymes and has high catalytic activity towards its cognate substrate, camphor. X-ray crystallography and NMR and IR spectroscopy have demonstrated the existence of multiple conformations of many P450s, including P450. Kinetic studies have indicated that substrate binding to several P450s is dominated by a conformational selection process, in which the substrate binds an individual conformer(s) of the unliganded enzyme.
View Article and Find Full Text PDFMechanism of deep eutectic solvents enhancing catalytic function of cytochrome P450 enzymes in biosynthesis and organic synthesis.
J Biotechnol
November 2020
Department of Cardiology, the Second Hospital of Jilin University, No. 218, Ziqiang Road, Nanguan District, Changchun, 130041, Jilin Province, PR China. Electronic address:
Indigo is an insoluble blue dye, which generates serious pollution in its production process. Increasing focus has come to the biosynthesis of indigo that are more environment-preserved and high-efficient. Hence, this study was designed to explore the specific role of various deep eutectic solvents (DESs) on cytochromeP45-BM-3 catalyzing indole to produce indigo.
View Article and Find Full Text PDFMethylene Oxidation of Alkyl Sulfates by Cytochrome P450 and a Role for Conformational Selection in Substrate Recognition.
ACS Catal
May 2020
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 U.S.A.
Cytochrome P450 (P450) is a flavoprotein reductase-heme fusion protein from the bacterium that has been well-characterized in many biophysical aspects. Although the enzyme is known to catalyze the hydroxylation of medium and long-chain fatty acids at high rates, no definitive physiological function has been associated with this process in the organism other than a possible protective role. We found that P450 rapidly hydroxylates alkyl sulfates, particularly those with 12-16 carbons (i.
View Article and Find Full Text PDFArticle Synopsis
- The study highlights the health benefits of ω3 fatty acids, specifically identifying 17,18-epoxyeicosatetraenoic acid (17,18-EpETE) as a valuable lipid with anti-allergic and anti-inflammatory properties.
- Researchers found that the enantiomers of 17,18-EpETE differ in their effectiveness, with only one enantiomer (17(),18()-EpETE) exhibiting significant anti-inflammatory effects in reducing skin hypersensitivity.
- The research also emphasizes the potential of using a bacterial enzyme, cytochrome P450 BM-3, to produce the beneficial enantiomer in a stereoselective way, presenting a promising method for mass-producing useful lipid metabolites.
Interpretation of cytochrome P450 monooxygenase kinetics by modeling of thermodynamic activity.
J Inorg Biochem
June 2018
Institute of Biochemistry and Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany. Electronic address:
The experimentally determined Michaelis constant K results from a combination of two effects: the recognition of the substrate by the enzyme and the molecular interactions between substrate and solvent. By separating substrate recognition from solvent effects, the thermodynamic activity-based Michaelis constant K allows for an unambiguous comparison of how different substrates fit into the substrate binding site. K of a poorly water-soluble substrate is calculated from the experimentally determined concentration-based Michaelis constant K and its activity coefficient at infinite dilution γ.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!