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 k1 (for H2O2 oxidation of metmyoglobin) of 1. 34 x 10(4) M-1 s-1 ( approximately 25-fold that of wild-type myoglobin) and k3 [for reducing the substrate (2, 2'-azino-di-(3-ethyl)benzthiazoline-6-sulfonic acid] of 1.4 x 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 Tm of the quadruple variant is decreased only slightly compared with wild-type (74.1 degreesC vs. 76.5 degreesC). The rate constants for binding of dioxygen exhibited by the quadruple variant are identical to the those observed for wild-type myoglobin (kon, 22.2 x 10(-6) M-1 s-1 vs. 22.3 x 10(-6) M-1 s-1; koff, 24.3 s-1 vs. 24.2 s-1; KO2, 0.91 x 10(-6) M-1 vs. 0.92 x 10(-6) M-1). The affinity of the quadruple variant for CO is increased slightly (kon, 0.90 x 10(-6) M-1s-1 vs. 0.51 x 10(-6) M-1s-1; koff, 5.08 s-1 vs. 3.51 s-1; KCO, 1.77 x 10(-7) M-1 vs. 1.45 x 10(-7) M-1). All four substitutions are in the heme pocket and within 5 A of the heme group.
Submitter: Shu-Ching Ou
Submission Date: Dec. 12, 2018, 11:27 a.m.
|Number of data points||55|
|Assays/Quantities/Protocols||Experimental Assay: Kinetics: k1 ; Experimental Assay: Kinetics: k3 ; Experimental Assay: Tm ; Experimental Assay: Binding: kon ; Experimental Assay: Binding: koff for CO ; Experimental Assay: Binding: koff for O2 ; Derived Quantity: Kinetics: relative k1 ; Derived Quantity: Kinetics: relative k3 ; Derived Quantity: Binding: Ka for CO ; Derived Quantity: Binding: Ka for O2|
|Libraries||Variants for Kinetics ; Variants for Mg ; Variants for Binding (O2 and CO)|