Using the Evolutionary History of Proteins to Engineer Insertion-Deletion Mutants from Robust, Ancestral Templates Using Graphical Representation of Ancestral Sequence Predictions (GRASP)

Ross, Connie M.; Foley, Gabriel; Bodén, Mikael; Gillam, Elizabeth M. J.

doi:10.1007/978-1-0716-1826-4_6

Cited by 14 publications

(16 citation statements)

References 43 publications

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“…Thus, we identified the ancestral nodes where the protein acquired/lost remote loops and inferred functional ancestral proteins with different loop combinations (InDels). As previous studies [27][28][29] demonstrated that InDels in loop regions are highly tolerated, our ancestral proteins showed robustness to add/remove loops. Thus, we demonstrate that ancestral sequence reconstruction approach in enzyme family with structural variations in loop regions is useful for designing enzyme with different loop combinations and exploring the potential remote site for loop engineering.…”

Section: Discussionsupporting

confidence: 66%

Beyond the Active Site: The addition of a remote loop reveals a new complex biological function for chitinase enzymes

Kozome

Sljoka

Laurino

2023

Preprint

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Loops are small secondary structural elements that play a crucial role in the emergence of new enzyme functions. However, our understanding of loop functions is mainly limited to the catalytic loops. To understand the function of remote loops in enzymes, we studied Glycoside hydrolase family 19 (GH19) chitinase - an essential enzyme family for pathogen degradation in plants. By revealing the evolutionary history and loops appearance of GH19 chitinase, we discovered that one loop which is remote from the catalytic site, is necessary to acquire the new antifungal activity. We demonstrated that this remote loop directly accesses the fungal cell wall, and surprisingly, it needs to adopt a defined structure supported by long-range intramolecular interactions to perform its function. Our findings prove that Nature applies this new strategy at the molecular level to achieve a complex biological function while maintaining the original activity one in the catalytic pocket, suggesting an alternative way to design new enzyme function.

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Section: Discussionsupporting

confidence: 66%

Beyond the Active Site: The addition of a remote loop reveals a new complex biological function for chitinase enzymes

Kozome

Sljoka

Laurino

2023

Preprint

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“…The tree and alignment were submitted to Graphical Representation of Ancestral Sequence Predictions (GRASP; Foley 2022 ) to infer a series of ancestors using a maximum likelihood, joint reconstruction approach. GRASP models insertion and deletion events via the inference of a partial order graph at each ancestor, representing the inclusion/exclusion of sequence by “jumps” (edges) between positions determined by the original input alignment ( Foley 2022 ; Ross et al 2022 ). Although content is determined by maximum likelihood, inference of edges for an ancestor is based on maximum parsimony.…”

Section: Resultsmentioning

confidence: 99%

Ancestral Sequence Reconstruction of a Cytochrome P450 Family Involved in Chemical Defense Reveals the Functional Evolution of a Promiscuous, Xenobiotic-Metabolizing Enzyme in Vertebrates

Harris

Thomson

Gumulya

et al. 2022

Molecular Biology and Evolution

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The cytochrome P450 family 1 enzymes (CYP1s) are a diverse family of hemoprotein monooxygenases which metabolize many xenobiotics including numerous environmental carcinogens. However, their historical function and evolution remain largely unstudied. Here we investigate CYP1 evolution via the reconstruction and characterization of the vertebrate CYP1 ancestors. Younger ancestors and extant forms generally demonstrated higher activity towards typical CYP1 xenobiotic and steroid substrates than older ancestors, suggesting significant diversification away from the original CYP1 function. Caffeine metabolism appears to be a recently evolved trait of the CYP1A subfamily, observed in the mammalian CYP1A lineage, and may parallel the recent evolution of caffeine synthesis in multiple separate plant species. Likewise, the aryl hydrocarbon receptor agonist, 6-formylindolo[3,2-b]carbazole (FICZ) was metabolised to a greater extent by certain younger ancestors and extant forms, suggesting that activity towards FICZ increased in specific CYP1 evolutionary branches, a process that may have occurred in parallel to the exploitation of land where UV-exposure was higher than in aquatic environments. As observed with previous reconstructions of P450 enzymes, thermostability correlated with evolutionary age; the oldest ancestor was up to 35 °C more thermostable than the extant forms, with a 10T50 (temperature at which 50% of the haemoprotein remains intact after 10 min) of 71 °C. This robustness may have facilitated evolutionary diversification of the CYP1s by buffering the destabilizing effects of mutations that conferred novel functions, a phenomenon which may also be useful in exploiting the catalytic versatility of these ancestral enzymes for commercial application as biocatalysts.

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“…The archaeal branch was used as an outgroup to root the ML tree. The joint reconstructions of all ancestral nodes were calculated with the GRASP (Graphical representation of ancestral sequence predictions) web server (62) using the Jones-Taylor-Thornton (JTT) evolutionary model.…”

Section: Methodsmentioning

confidence: 99%

Translation in a Box: Orthogonal Evolution in theSaccharomyces cerevisiaeMitochondrion

Rothschild-Mancinelli

Álvarez-Carreño

Guo

et al. 2023

Preprint

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The synthesis capabilities of the translation system (TS) provide engineering and directed evolution potential as well as a system for generating novel polymers. Orthogonal translation systems offer a parallel platform for translation engineering, enabling primary and secondary translation systems to operate independently without interfering with each other. However, current translation platforms for engineering lack complete orthogonality between the primary and secondary systems. Using a combination of indirect modifications through antibiotics, truncations of mito-rProteins mL50 and uL23, and replacement of mito-rProtein uL2, we develop and test the mitochondrial translation system in Saccharomyces cerevisiae as a fully orthogonal platform for in vivo directed evolution of translation. Our results show that continuous, comprehensive, creative, directed-evolution of a fully orthogonal TS in vivo appears to be a useful approach for technical control of the TS. This study emphasizes the power of system-wide evolutionary approaches over rational design. Evolution is creative and non-linear and can find unexpected solutions to problems.

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Using the Evolutionary History of Proteins to Engineer Insertion-Deletion Mutants from Robust, Ancestral Templates Using Graphical Representation of Ancestral Sequence Predictions (GRASP)

Cited by 14 publications

References 43 publications

Beyond the Active Site: The addition of a remote loop reveals a new complex biological function for chitinase enzymes

Beyond the Active Site: The addition of a remote loop reveals a new complex biological function for chitinase enzymes

Ancestral Sequence Reconstruction of a Cytochrome P450 Family Involved in Chemical Defense Reveals the Functional Evolution of a Promiscuous, Xenobiotic-Metabolizing Enzyme in Vertebrates

Translation in a Box: Orthogonal Evolution in theSaccharomyces cerevisiaeMitochondrion

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