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Photosynthetic organisms, fungi, and animals comprise distinct pathways for vitamin C biosynthesis. Besides this diversity, the final biosynthetic step consistently involves an oxidation reaction carried out by the aldonolactone oxidoreductases. Here, we study the origin and evolution of the diversified activities and substrate preferences featured by these flavoenzymes using molecular phylogeny, kinetics, mutagenesis, and crystallographic experiments. We find clear evidence that they share a common ancestor. A flavin-interacting amino acid modulates the reactivity with the electron acceptors, including oxygen, and determines whether an enzyme functions as an oxidase or a dehydrogenase. We show that a few side chains in the catalytic cavity impart the reaction stereoselectivity. Ancestral sequence reconstruction outlines how these critical positions were affixed to specific amino acids along the evolution of the major eukaryotic clades. During Eukarya evolution, the aldonolactone oxidoreductases adapted to the varying metabolic demands while retaining their overarching vitamin C-generating function. Photosynthetic organisms, fungi, and animals contain distinct pathways for vitamin C biosynthesis, but the final biosynthetic step consistently involves an oxidation reaction catalysed by the aldonolactone oxidoreductases. Here, the authors investigate the origin and evolution of the diversified activities and substrate preferences featured by these enzymes using different methods and find evidence that they share a common ancestor. [ABSTRACT FROM AUTHOR]

The thioredoxin pathway is an antioxidant system present in most organisms. Electrons flow from a thioredoxin reductase to thioredoxin at the expense of a specific electron donor. Most known thioredoxin reductases rely on NADPH as a reducing cofactor. Yet, in 2016, a new type of thioredoxin reductase was discovered in Archaea which utilize instead a reduced deazaflavin cofactor (F420H2). For this reason, the respective enzyme was named deazaflavin‐dependent flavin‐containing thioredoxin reductase (DFTR). To have a broader understanding of the biochemistry of DFTRs, we identified and characterized two other archaeal representatives. A detailed kinetic study, which included pre‐steady state kinetic analyses, revealed that these two DFTRs are highly specific for F420H2 while displaying marginal activity with NADPH. Nevertheless, they share mechanistic features with the canonical thioredoxin reductases that are dependent on NADPH (NTRs). A detailed structural analysis led to the identification of two key residues that tune cofactor specificity of DFTRs. This allowed us to propose a DFTR‐specific sequence motif that enabled for the first time the identification and experimental characterization of a bacterial DFTR. [ABSTRACT FROM AUTHOR]

The thioredoxin pathway is an antioxidant system present in most organisms. Electrons flow from a thioredoxin reductase to thioredoxin at the expense of a specific electron donor. Most known thioredoxin reductases rely on NADPH as a reducing cofactor. Yet, in 2016, a new type of thioredoxin reductase was discovered in Archaea which utilize instead a reduced deazaflavin cofactor (F 420 H 2 ). For this reason, the respective enzyme was named deazaflavin-dependent flavin-containing thioredoxin reductase (DFTR). To have a broader understanding of the biochemistry of DFTRs, we identified and characterized two other archaeal representatives. A detailed kinetic study, which included pre-steady state kinetic analyses, revealed that these two DFTRs are highly specific for F 420 H 2 while displaying marginal activity with NADPH. Nevertheless, they share mechanistic features with the canonical thioredoxin reductases that are dependent on NADPH (NTRs). A detailed structural analysis led to the identification of two key residues that tune cofactor specificity of DFTRs. This allowed us to propose a DFTR-specific sequence motif that enabled for the first time the identification and experimental characterization of a bacterial DFTR., (© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Ancestral Sequence Reconstruction (ASR) allows one to infer the sequences of extinct proteins using the phylogeny of extant proteins. It consists of disclosing the evolutionary history-i.e., the phylogeny-of a protein family of interest and then inferring the sequences of its ancestors-i.e., the nodes in the phylogeny. Assisted by gene synthesis, the selected ancestors can be resurrected in the lab and experimentally characterized. The crucial step to succeed with ASR is starting from a reliable phylogeny. At the same time, it is of the utmost importance to have a clear idea on the evolutionary history of the family under study and the events that influenced it. This allows us to implement ASR with well-defined hypotheses and to apply the appropriate experimental methods. In the last years, ASR has become popular to test hypotheses about the origin of functionalities, changes in activities, understanding physicochemical properties of proteins, among others. In this context, the aim of this chapter is to present the ASR approach applied to the reconstruction of enzymes-i.e., proteins with catalytic roles. The spirit of this contribution is to provide a basic, hands-to-work guide for biochemists and biologists who are unfamiliar with molecular phylogenetics., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)