In vitro evolution of horse heart myoglobin to increase peroxidase activity
| Autor: | Mauk Ag, Lianglu Wan, M. B. Twitchett, Michael J. Smith, Lindsay D. Eltis |
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| Rok vydání: | 1998 |
| Předmět: |
Models
Molecular Circular dichroism Protein Conformation medicine.disease_cause Polymerase Chain Reaction chemistry.chemical_compound Protein structure Escherichia coli medicine Animals Point Mutation Horses Cloning Molecular Heme DNA Primers Multidisciplinary biology Myoglobin Mutagenesis Genetic Variation Biological Sciences Molecular biology Kinetics Amino Acid Substitution Peroxidases Metmyoglobin chemistry biology.protein Directed Molecular Evolution Peroxidase |
| Zdroj: | Proceedings of the National Academy of Sciences. 95:12825-12831 |
| ISSN: | 1091-6490 0027-8424 |
| DOI: | 10.1073/pnas.95.22.12825 |
| Popis: | Random mutagenesis and screening for enzymatic activity has been used to engineer horse heart myoglobin to enhance its intrinsic peroxidase activity. A chemically synthesized gene encoding horse heart myoglobin was subjected to successive cycles of PCR random mutagenesis. The mutated myoglobin gene was expressed in Escherichia coli LE392, and the variants were screened for peroxidase activity with a plate assay. Four cycles of mutagenesis and screening produced a series of single, double, triple, and quadruple variants with enhanced peroxidase activity. Steady-state kinetics analysis demonstrated that the quadruple variant T39I/K45D/F46L/I107F exhibits peroxidase activity significantly greater than that of the wild-type protein with k 1 (for H 2 O 2 oxidation of metmyoglobin) of 1.34 × 10 4 M −1 s −1 (≈25-fold that of wild-type myoglobin) and k 3 [for reducing the substrate (2, 2′-azino-di-(3-ethyl)benzthiazoline-6-sulfonic acid] of 1.4 × 10 6 M −1 s −1 (1.6-fold that of wild-type myoglobin). Thermal stability of these variants as measured with circular dichroism spectroscopy demonstrated that the T m of the quadruple variant is decreased only slightly compared with wild-type (74.1°C vs. 76.5°C). The rate constants for binding of dioxygen exhibited by the quadruple variant are identical to the those observed for wild-type myoglobin ( k on , 22.2 × 10 −6 M −1 s −1 vs. 22.3 × 10 −6 M −1 s −1 ; k off , 24.3 s −1 vs. 24.2 s −1 ; K O2 , 0.91 × 10 −6 M −1 vs. 0.92 × 10 −6 M −1 ). The affinity of the quadruple variant for CO is increased slightly ( k on , 0.90 × 10 −6 M −1 s −1 vs. 0.51 × 10 −6 M −1 s −1 ; k off , 5.08 s −1 vs. 3.51 s −1 ; K CO , 1.77 × 10 −7 M −1 vs. 1.45 × 10 −7 M −1 ). All four substitutions are in the heme pocket and within 5 Å of the heme group. |
| Databáze: | OpenAIRE |
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