Directed reconstruction of a novel ancestral alcohol dehydrogenase featuring shifted pH-profile, enhanced thermostability and expanded substrate spectrum

Correlation Analysis of Key Residue Sites between Computational-Aided Design Thermostability d-Amino Acid Oxidase and Ancestral Enzymes

“…ASR was conducted concurrently using 19 DAAOs from different evolutionary families as starting sequences, resulting in the generation of 18 ancestral sequences within the phylogenetic tree (Figure a). Generally, the ancestral enzyme derived from the root node, designated as ASR20, is considered the best ancestral sequence, and so on in descending order. , Consequently, ancestral enzymes ASR20, ASR21, ASR22, and ASR29 were constructed with sequence identities ranging from 30 to 50%. However, during multiple construction attempts, full-length recombinant ASR20 could not be obtained, which is often unpredictable and elusive.…”

“…16,17 Nevertheless, the practical implementation of ASR still faces some uncertain limitations, such as low solubility during expression and a lack of comprehensive quantitative characterization. 18 Therefore, exploring the relationship between ASR and the original enzyme is crucial as potential beneficial residues identified through ASR could contribute to enhancing the thermostability of this specific enzyme class.…”

“…One of the main rational strategies involves enhancing the folding stability of natural proteins, which includes improving hydrophobic surfaces, substituting electrostatic surfaces, and flexible regions. ,, Computer algorithms have aided in accelerating the exploration of potential stabilizing substitutions within the vast sequence space, increasing the success rate of these strategies . Semirational strategies based on consensus and ancestral sequence reconstruction (ASR) exploiting the greater contribution of consensus amino acids to protein stability without structural requirements are widely used. − It is worth noting that ASR is generated within a related group, taking branch lengths into consideration, and is distinct from consensus proteins. , Nevertheless, the practical implementation of ASR still faces some uncertain limitations, such as low solubility during expression and a lack of comprehensive quantitative characterization . Therefore, exploring the relationship between ASR and the original enzyme is crucial as potential beneficial residues identified through ASR could contribute to enhancing the thermostability of this specific enzyme class.…”

Curse and Blessing of Non‐Proteinogenic Parts in Computational Enzyme Engineering

“…However, although binding motifs and catalytic residues are often conserved, the introduced mutations can affect cofactor binding and specificity. For example, ancestral proteins were reported to exhibit a switched preference between nicotinamide cofactors [277] or flavins [278] …”

“…However, although binding motifs and catalytic residues are often conserved, the introduced mutations can affect cofactor binding and specificity. For example, ancestral proteins were reported to exhibit a switched preference between nicotinamide cofactors [277] or flavins. [278] In contrast, energy-based predictions require a protein structure, and typically calculate the ΔΔG Fold , i. e. the difference in the free energy of (un)folding between a mutant and a wildtype structure.…”