Engineering the Substrate Specificity of Gallate Dioxygenase for 3-O-Methylgallate
Riihinen, Tommi (2024)
2024
Tekniikan ja luonnontieteiden kandidaattiohjelma - Bachelor's Programme in Engineering and Natural Sciences
Tekniikan ja luonnontieteiden tiedekunta - Faculty of Engineering and Natural Sciences
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Hyväksymispäivämäärä
2024-09-04
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-202409028500
https://urn.fi/URN:NBN:fi:tuni-202409028500
Tiivistelmä
Lignin side streams from biomass fractioning could be used for high value products, but remain underutilized. One extensively studied possibility for microbial lignin valorization is the application of synthetic biology for lignin conversion into 2-pyrone-4,6-dicarboxylic acid (PDC), a promising building block for the manufacture of biodegradable polyesters. There is reason to believe that microbial production of PDC from lignin could benefit from a specific 3-O-methylgallate 3,4-dioxygenase enzyme.
In this study, the extradiol dioxygenases GalA, DesB, DesZ and LigAB were compared in structure and sequence. This study represents the first structural characterization of GalA and DesZ. Both characterizations were based on AlphaFold2 predictions. The gallate 3,4-dioxygenase GalA was investigated in a rational engineering paradigm for potential modifications to shift its substrate specificity towards 3-O-methylgallate. The effects of the resulting set of potential mutations to GalA were computationally assessed with a novel computational method presented in this study. Multiple promising modifications were identified.
The computational method was developed to predict the effect of amino acid mutations to the enzymatic efficiency of enzymes which catalyze a moderately slow and irreversible reaction. The method is based on dividing an active site substrate ensemble into active and inactive categories and assessing their relative probability with regard to the Boltzmann distribution.
This study adds to the understanding of extradiol dioxynases, provides targets for potential creating a 3-O-methylgallate specific 3,4-dioxygenase from GalA and presents a novel method for in silico assessment of the effect of enzyme modifications. Further work should test the candidate mutations on GalA and validate the computational modelling approach on a larger dataset.
In this study, the extradiol dioxygenases GalA, DesB, DesZ and LigAB were compared in structure and sequence. This study represents the first structural characterization of GalA and DesZ. Both characterizations were based on AlphaFold2 predictions. The gallate 3,4-dioxygenase GalA was investigated in a rational engineering paradigm for potential modifications to shift its substrate specificity towards 3-O-methylgallate. The effects of the resulting set of potential mutations to GalA were computationally assessed with a novel computational method presented in this study. Multiple promising modifications were identified.
The computational method was developed to predict the effect of amino acid mutations to the enzymatic efficiency of enzymes which catalyze a moderately slow and irreversible reaction. The method is based on dividing an active site substrate ensemble into active and inactive categories and assessing their relative probability with regard to the Boltzmann distribution.
This study adds to the understanding of extradiol dioxynases, provides targets for potential creating a 3-O-methylgallate specific 3,4-dioxygenase from GalA and presents a novel method for in silico assessment of the effect of enzyme modifications. Further work should test the candidate mutations on GalA and validate the computational modelling approach on a larger dataset.