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Site-directed mutations at phenylalanine-190 of manganese peroxidase: effects on stability, function, and
K Kishi1, D P Hildebrand, M Kusters-van Someren
1Department of Chemistry, Biochemistry, and Molecular Biology, Oregon Graduate Institute of Science and Technology, Portland 97291-1000, USA.
Abstract:
A series of site-directed mutants, F190Y, F190L, F190I, and F190A, in the gene encoding manganese peroxidase isozyme 1 (mnp1) from Phanerochaete chrysosporium was generated by overlap extension with the polymerase chain reaction. The mutant genes were expressed in P. chrysosporium during primary metabolic growth under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The manganese peroxidase variants (MnPs) were purified and characterized by kinetic and spectroscopic methods. At pH 4.5, the UV-vis spectra of the ferric and oxidized states of the mutant proteins were very similar to those of the wild-type enzyme. Steady-state kinetic analyses showed that the apparent Km and k(cat) values for MnII and H2O2 also were similar to the corresponding values for the wild-type MnP. The apparent Km and k(cat) values for ferrocyanide oxidation by MnP were not affected by the F190Y, F190L, or F190I mutations; however, the apparent Km value for ferrocyanide oxidation by the F190A mutant MnP was approximately 1/8 of that for the wild-type enzyme. Likewise, the apparent k(cat) value for ferrocyanide oxidation by the MnP F190A mutant was approximately 4-fold greater than the corresponding k(cat) for the wild-type MnP. The stabilities of both the native and oxidized states of MnP were significantly affected by several of the mutations at Phe190. Replacement of Phe190 by either Ile or Ala significantly destabilized the resultant proteins to thermal denaturation. Moreover, the rates of spontaneous reduction of the oxidized intermediates, MnP compounds I and II, were dramatically increased for the F190A mutant relative to the rates observed for the wild-type enzyme. The spectroscopic properties of the wild-type and F190 mutant MnPs were examined as a function of pH. At room temperature, increasing pH from 5.0 to 8.5 induced a FeIII high- to low-spin transition for all of the MnP proteins. This transition may involve direct coordination of the distal His residue to the heme iron to produce bishistidinyl coordination as suggested by magnetic circular dichroism spectroscopy. The pH at which this transition occurred was considerably lower for the F190A and F190I variants and suggests that Phe190 plays a critical role in stabilizing the heme environment of MnP.
Insights
Site-directed mutations in manganese peroxidase (MnP) were created and studied. The F190A mutation significantly altered enzyme activity and stability, highlighting Phe190's role in the heme environment.
Area of Science:
- Biochemistry
- Enzymology
- Protein Engineering
Background:
- Manganese peroxidase (MnP) is a key enzyme in lignin degradation.
- Understanding MnP structure-function relationships is crucial for biotechnological applications.
- The role of specific residues, like Phe190, in MnP activity and stability remains to be fully elucidated.
Purpose of the Study:
- To investigate the impact of site-directed mutations at the Phe190 position of manganese peroxidase isozyme 1 (mnp1) from Phanerochaete chrysosporium.
- To characterize the kinetic, spectroscopic, and stability properties of these MnP variants.
- To determine the role of Phe190 in stabilizing the heme environment and influencing enzyme function.
Main Methods:
- Site-directed mutagenesis (F190Y, F190L, F190I, F190A) using overlap extension PCR.
- Expression of mutant mnp1 genes in P. chrysosporium under the glyceraldehyde-3-phosphate dehydrogenase promoter.
- Purification and characterization of manganese peroxidase variants (MnPs) using kinetic and spectroscopic methods (UV-vis, MCD).
- Assays for Mn(II) and H2O2 oxidation, ferrocyanide oxidation, thermal denaturation, and reduction rates of oxidized intermediates.
Main Results:
- Mutant MnPs exhibited similar UV-vis spectra and kinetic parameters for Mn(II) and H2O2 compared to wild-type.
- The F190A mutation significantly altered ferrocyanide oxidation kinetics (lower Km, higher kcat).
- Mutations F190I and F190A destabilized MnP to thermal denaturation.
- The F190A mutant showed dramatically increased rates of reduction for oxidized intermediates (MnP compounds I and II).
- Spectroscopic analysis revealed Phe190 is critical for stabilizing the heme environment, influencing the pH-dependent spin transition of Fe(III).
Conclusions:
- Phe190 plays a critical role in stabilizing the heme environment of manganese peroxidase.
- Mutations at Phe190 can significantly impact enzyme stability, substrate oxidation kinetics, and redox properties.
- The F190A mutant provides insights into the mechanism of ferrocyanide oxidation and enzyme intermediate reduction.