RESUMO
Computational models of evolution are valuable for understanding the dynamics of sequence variation, to infer phylogenetic relationships or potential evolutionary pathways and for biomedical and industrial applications. Despite these benefits, few have validated their propensities to generate outputs with in vivo functionality, which would enhance their value as accurate and interpretable evolutionary algorithms. We demonstrate the power of epistasis inferred from natural protein families to evolve sequence variants in an algorithm we developed called sequence evolution with epistatic contributions (SEEC). Utilizing the Hamiltonian of the joint probability of sequences in the family as fitness metric, we sampled and experimentally tested for in vivo [Formula: see text]-lactamase activity in Escherichia coli TEM-1 variants. These evolved proteins can have dozens of mutations dispersed across the structure while preserving sites essential for both catalysis and interactions. Remarkably, these variants retain family-like functionality while being more active than their wild-type predecessor. We found that depending on the inference method used to generate the epistatic constraints, different parameters simulate diverse selection strengths. Under weaker selection, local Hamiltonian fluctuations reliably predict relative changes to variant fitness, recapitulating neutral evolution. SEEC has the potential to explore the dynamics of neofunctionalization, characterize viral fitness landscapes, and facilitate vaccine development.
Assuntos
Epistasia Genética , Proteínas , Filogenia , Proteínas/genética , Mutação , Fenótipo , Evolução Molecular , Aptidão Genética , Modelos GenéticosRESUMO
Inferring the historical and biophysical causes of diversity within protein families is a complex puzzle. A key to unraveling this problem is characterizing the rugged topography of sequence-function adaptive landscapes. Using biochemical data from a 29 = 512 combinatorial library of tobacco 5-epi-aristolochene synthase (TEAS) mutants engineered to make the native major product of Egyptian henbane premnaspirodiene synthase (HPS) and a complementary 512 mutant HPS library, we address the question of how product specificity is controlled. These data sets reveal that HPS is far more robust and resistant to mutations than TEAS, where most mutants are promiscuous. We also combine experimental data with a sequence Potts Hamiltonian model and direct coupling analysis to quantify mutant fitness. Our results demonstrate that the Hamiltonian captures variation in product outputs across both libraries, clusters native family members based on their substrate specificities, and exposes the divergent catalytic roles of couplings between the catalytic and noncatalytic domains of TEAS versus HPS. Specifically, we found that the role of the interdomain connectivities in specifying product output is more important in TEAS than connectivities within the catalytic domain. Despite being 75% identical, this property is not shared by HPS, where connectivities within the catalytic domain are more important for specificity. By solving the X-ray crystal structure of HPS, we assessed structural bases for their interdomain network differences. Last, we calculate the product profile Shannon entropies of the two libraries, which showcases that site-site connectivities also play divergent roles in catalytic accuracy.
Assuntos
Alquil e Aril Transferases , Catálise , Domínio Catalítico , MutaçãoRESUMO
We introduce a model of amino acid sequence evolution that accounts for the statistical behavior of real sequences induced by epistatic interactions. We base the model dynamics on parameters derived from multiple sequence alignments analyzed by using direct coupling analysis methodology. Known statistical properties such as overdispersion, heterotachy, and gamma-distributed rate-across-sites are shown to be emergent properties of this model while being consistent with neutral evolution theory, thereby unifying observations from previously disjointed evolutionary models of sequences. The relationship between site restriction and heterotachy is characterized by tracking the effective alphabet dynamics of sites. We also observe an evolutionary Stokes shift in the fitness of sequences that have undergone evolution under our simulation. By analyzing the structural information of some proteins, we corroborate that the strongest Stokes shifts derive from sites that physically interact in networks near biochemically important regions. Perspectives on the implementation of our model in the context of the molecular clock are discussed.
Assuntos
Sequência de Aminoácidos/fisiologia , Evolução Molecular , Deriva Genética , Proteínas/química , Proteínas/fisiologia , Substituição de Aminoácidos , Proteínas de Bactérias/química , Biologia Computacional/métodos , Simulação por Computador , Modelos Biológicos , Modelos Moleculares , Filogenia , Conformação Proteica , Domínios Proteicos , Alinhamento de SequênciaRESUMO
Computational models of evolution are valuable for understanding the dynamics of sequence variation, to infer phylogenetic relationships or potential evolutionary pathways and for biomedical and industrial applications. Despite these benefits, few have validated their propensities to generate outputs with in vivo functionality, which would enhance their value as accurate and interpretable evolutionary algorithms. We demonstrate the power of epistasis inferred from natural protein families to evolve sequence variants in an algorithm we developed called Sequence Evolution with Epistatic Contributions. Utilizing the Hamiltonian of the joint probability of sequences in the family as fitness metric, we sampled and experimentally tested for in vivo ß-lactamase activity in E. coli TEM-1 variants. These evolved proteins can have dozens of mutations dispersed across the structure while preserving sites essential for both catalysis and interactions. Remarkably, these variants retain family-like functionality while being more active than their WT predecessor. We found that depending on the inference method used to generate the epistatic constraints, different parameters simulate diverse selection strengths. Under weaker selection, local Hamiltonian fluctuations reliably predict relative changes to variant fitness, recapitulating neutral evolution. SEEC has the potential to explore the dynamics of neofunctionalization, characterize viral fitness landscapes and facilitate vaccine development.
RESUMO
The alpha/beta hydrolase (ABH) superfamily is a widespread and functionally malleable protein fold recognized for its diverse biochemical activities across all three domains of life. ABH enzymes possess unexpected catalytic activity in the green plant lineage through selective alterations in active site architecture and chemistry. Furthermore, the ABH fold serves as the core structure for phytohormone and ligand receptors in the gibberellin, strigolactone, and karrikin signaling pathways in plants. Despite recent discoveries, the ABH family is sparsely characterized in plants, a sessile kingdom known to evolve complex and specialized chemical adaptations as survival responses to widely varying biotic and abiotic ecologies. This review calls attention to the ABH superfamily in the plant kingdom to highlight the functional adaptability of the ABH fold.
Assuntos
Hidrolases/metabolismo , Plantas/enzimologia , Biocatálise , Hidrolases/química , Plantas/metabolismo , Dobramento de ProteínaRESUMO
The plant terpene synthase (TPS) family is responsible for the biosynthesis of a variety of terpenoid natural products possessing diverse biological functions. TPSs catalyze the ionization and, most commonly, rearrangement and cyclization of prenyl diphosphate substrates, forming linear and cyclic hydrocarbons. Moreover, a single TPS often produces several minor products in addition to a dominant product. We characterized the catalytic profiles of Hyoscyamus muticus premnaspirodiene synthase (HPS) and compared it with the profile of a closely related TPS, Nicotiana tabacum 5-epi-aristolochene synthase (TEAS). The profiles of two previously studied HPS and TEAS mutants, each containing nine interconverting mutations, dubbed HPS-M9 and TEAS-M9, were also characterized. All four TPSs were compared under varying temperature and pH conditions. In addition, we solved the X-ray crystal structures of TEAS and a TEAS quadruple mutant complexed with substrate and products to gain insight into the enzymatic features modulating product formation. These informative structures, along with product profiles, provide new insight into plant TPS catalytic promiscuity.