Engineering A-type Dye-Decolorizing Peroxidases by Modification of a Conserved Glutamate Residue.

Publisher: Wiley-VCH Verlag Country of Publication: Germany NLM ID: 100937360 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1439-7633 (Electronic) Linking ISSN: 14394227 NLM ISO Abbreviation: Chembiochem Subsets: MEDLINE

Original Publication: Weinheim, Germany : Wiley-VCH Verlag, c2000-

Glutamic Acid*/chemistry ; Glutamic Acid*/metabolism ; Coloring Agents*/chemistry ; Coloring Agents*/metabolism ; Bacillus subtilis*/enzymology ; Peroxidases*/chemistry ; Peroxidases*/metabolism ; Peroxidases*/genetics; Molecular Dynamics Simulation ; Protein Engineering ; Mutagenesis, Site-Directed

Dye-decolorizing peroxidases (DyPs) are recently identified microbial enzymes that have been used in several Biotechnology applications from wastewater treatment to lignin valorization. However, their properties and mechanism of action still have many open questions. Their heme-containing active site is buried by three conserved flexible loops with a putative role in modulating substrate access and enzyme catalysis. Here, we investigated the role of a conserved glutamate residue in stabilizing interactions in loop 2 of A-type DyPs. First, we did site saturation mutagenesis of this residue, replacing it with all possible amino acids in bacterial DyPs from Bacillus subtilis (BsDyP) and from Kitasatospora aureofaciens (KaDyP1), the latter being characterized here for the first time. We screened the resulting libraries of variants for activity towards ABTS and identified variants with increased catalytic efficiency. The selected variants were purified and characterized for activity and stability. We furthermore used Molecular Dynamics simulations to rationalize the increased catalytic efficiency and found that the main reason is the electron channeling becoming easier from surface-exposed tryptophans. Based on our findings, we also propose that this glutamate could work as a pH switch in the wild-type enzyme, preventing intracellular damage.
(© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

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W1224 Biomolecular Technology of Proteins; FCT 2022.02027.PTDC Christian Doppler Research Association, Fundação para a Ciência e Tecnologia (FCT), Portugal; UIDB/04612/2020 MOSTMICRO-ITQB; UIDP/04612/2020 MOSTMICRO-ITQB; LA/P/0087/2020 LS4FUTURE

Keywords: Enzymes; Oxidoreductases; Protein engineering; Protein structures; Strucutre-activity relationship

3KX376GY7L (Glutamic Acid)
0 (Coloring Agents)
EC 1.11.1.- (Peroxidases)

Date Created: 20240220 Date Completed: 20240502 Latest Revision: 20240502

20240502

10.1002/cbic.202300872

38376941