In vitro evolution of horse heart myoglobin to increase peroxidase activity.

Abstract

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.

Submission Details

ID: aRQLUBMQ3

Submitter: Shu-Ching Ou

Submission Date: Dec. 12, 2018, 11:27 a.m.

Version: 1

Publication Details
Wan L;Twitchett MB;Eltis LD;Mauk AG;Smith M,Proc Natl Acad Sci U S A (1998) In vitro evolution of horse heart myoglobin to increase peroxidase activity. PMID:9788999
Additional Information

Study Summary

Number of data points 55
Proteins Myoglobin
Unique complexes 20
Assays/Quantities/Protocols Experimental Assay: Tm ; Experimental Assay: Kinetics: k3 ; Experimental Assay: Kinetics: k1 ; Experimental Assay: Binding: koff for O2 ; Experimental Assay: Binding: koff for CO ; Experimental Assay: Binding: kon ; Derived Quantity: Binding: Ka for O2 ; Derived Quantity: Binding: Ka for CO ; Derived Quantity: Kinetics: relative k3 ; Derived Quantity: Kinetics: relative k1
Libraries Variants for Binding (O2 and CO) ; Variants for Mg ; Variants for Kinetics

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Myoglobin P68083 MYG_EQUBU
100.0 Myoglobin P68082 MYG_HORSE
91.5 Myoglobin P02181 MYG_INIGE
92.2 Myoglobin P02169 MYG_LEPMU
90.8 Myoglobin P02166 MYG_PERPO
90.8 Myoglobin Q0KIY1 MYG_BALBO
90.8 Myoglobin Q0KIY2 MYG_BALED
90.8 Myoglobin P02177 MYG_ESCRO
90.8 Myoglobin P02189 MYG_PIG
90.1 Myoglobin P02178 MYG_MEGNO
90.2 Myoglobin P02183 MYG_MESCA
90.2 Myoglobin Q0KIY0 MYG_MESST
90.2 Myoglobin P02167 MYG_NYCCO
90.2 Myoglobin P02165 MYG_TUPGL
90.8 Myoglobin P11343 MYG_LUTLU
90.2 Myoglobin P02163 MYG_ROUAE