Role of conserved and non-conserved residues of Escherichia coli formate dehydrogenase H in the CO2–formate interconversion
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School of Chemical Engineering |
Doctoral thesis (article-based)
| Defence date: 2024-12-13
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Authors
Date
2024
Major/Subject
Mcode
Degree programme
Language
en
Pages
81 + app. 109
Series
Aalto University publication series DOCTORAL THESES, 273/2024
Abstract
Molybdenum-dependent formate dehydrogenases (Mo-FDHs) have come to prominence as promising electrocatalysts for CO2 conversion owing to their ability to operate at low redox potentials. Nevertheless, engineering of these enzymes is required prior to application in industrial processes as these differ from physiological conditions under which Mo-FDHs have naturally evolved to increase host fitness. These engineering efforts are currently hampered by the limited understanding of the catalysis of the CO2–formate interconversion and motivated the research presented in this thesis.In publication I, the conserved active-site selenocysteine (U) residue 140 of Escherichia coli formate dehydrogenase H (EcFDH-H) was replaced with cysteine (C) and serine to study its role in catalysis. Kinetic characterization of U140C variants indicated that U140 stabilizes the reduced Mo(IV)–SH state that is required for CO2 reduction. Publication II focuses on the conserved residue lysine (K) 44 in EcFDH-H that is proximal to the Mo and [4Fe-4S] cofactors. The study investigates the role of K44 in catalysis by substitution with six structurally diverse residues. Subsequent kinetic characterization and molecular dynamics simulation of K44 variants suggested that K44 may stabilize the active forms of these cofactors. In publication III, a growth-based screening strategy was developed to extend the structure–function analysis of EcFDH-H beyond conserved residues. This strategy employs the complementation of an incomplete E. coli formate hydrogenlyase complex with EcFDH-H to establish a positive correlation between EcFDH-H variant activity and cell growth. As a proof of concept, five non-conserved residues were analyzed by the designed strategy and the variant A12G with an 82% increased formate oxidation activity was identified. Additionally, unpublished research is included in this thesis that aims at resolving the electron transfer conduit between cathodes and the catalytic center of EcFDH-H. This research was initiated by mapping seventeen potential electrode attachment sites on the EcFDH-H surface by individual replacement of non-conserved residues with cysteine. The formate oxidation activity of EcFDH-H variants was determined and was found to be preserved for the majority of them, providing a basis for the planned bio-electrochemical trials. Overall, the presented results increase the understanding of the role of conserved and non-conserved residues in EcFDH-H catalysis, providing important insights into the reaction mechanism of CO2–formate interconversion by Mo-FDHs. Additionally, this thesis identified potential targets for future engineering works aiming at improving the catalytic properties of Mo-FDHs, thereby promoting the development of proficient catalysts for CO2 utilization.Description
Supervising professor
Scheller, Silvan, Prof., Aalto University, Department of Bioproducts and Biosystems, FinlandThesis advisor
Lienemann, Michael, Dr., VTT Technical Research Centre of Finland, FinlandJuffer, André, Dr., Oulu University, Finland
Keywords
molybdenum enzyme, formate dehydrogenase, CO2–formate interconversion, enzyme engineering, CO2 utilization
Other note
Parts
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[Publication 1]: Li, Feilong; Scheller, Silvan; Lienemann, Michael. 2023. Comparative analysis of CO2 reduction by soluble Escherichia coli formate dehydrogenase H and its selenocysteine-to-cysteine substitution variant. Journal of CO2 utilization, volume 77, 102608. ISSN 2212-9820.
Full text in Acris/Aaltodoc: https://urn.fi/URN:NBN:fi:aalto-202312117230DOI: 10.1016/j.jcou.2023.102608 View at publisher
- [Publication 2]: Li, Feilong; Lienemann, Michael. Role of a highly conserved lysine residue in the stabilization of the catalytically active structure of a Mo-dependent formate dehydrogenase. Under review in The FEBS Journal
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[Publication 3]: Li, Feilong; Scheller, Silvan; Lienemann, Michael. 2024. A growth-based screening strategy for engineering the catalytic activity of an oxygen-sensitive formate dehydrogenase. Applied and Environmental Microbiology, volume 90, e01472-24, ISSN 1098-5336.
Full text in Acris/Aaltodoc: https://urn.fi/URN:NBN:fi:aalto-202410166760DOI: 10.1128/aem.01472-24 View at publisher