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Abstract Enzymes provide optimal three-dimensional structures for substrate binding and the subsequent accelerated reaction. Such folding-dependent catalytic behaviors, however, are seldom mechanistically explored with reduced structural complexity. Here, we demonstrate that the α-helix, a much simpler structural motif of enzyme, can facilitate its own growth through the self-catalyzed polymerization ofN-carboxyanhydride (NCA) in dichloromethane. The reversible binding between the N terminus of α-helical polypeptides and NCAs promotes rate acceleration of the subsequent ring-opening reaction. A two-stage, Michaelis–Menten-type kinetic model is proposed by considering the binding and reaction between the propagating helical chains and the monomers, and is successfully utilized to predict the molecular weights and molecular-weight distributions of the resulting polymers. This work elucidates the mechanism of helix-induced, enzyme-mimetic catalysis, emphasizes the importance of solvent choice in the discovery of new reaction type, and provides a route for rapid production of well-defined synthetic polypeptides by taking advantage of self-accelerated ring-opening polymerizations.
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Sparse Epistatic Patterns in the Evolution of Terpene Synthases
Abstract We explore sequence determinants of enzyme activity and specificity in a major enzyme family of terpene synthases. Most enzymes in this family catalyze reactions that produce cyclic terpenes—complex hydrocarbons widely used by plants and insects in diverse biological processes such as defense, communication, and symbiosis. To analyze the molecular mechanisms of emergence of terpene cyclization, we have carried out in-depth examination of mutational space around (E)-β-farnesene synthase, an Artemisia annua enzyme which catalyzes production of a linear hydrocarbon chain. Each mutant enzyme in our synthetic libraries was characterized biochemically, and the resulting reaction rate data were used as input to the Michaelis–Menten model of enzyme kinetics, in which free energies were represented as sums of one-amino-acid contributions and two-amino-acid couplings. Our model predicts measured reaction rates with high accuracy and yields free energy landscapes characterized by relatively few coupling terms. As a result, the Michaelis–Menten free energy landscapes have simple, interpretable structure and exhibit little epistasis. We have also developed biophysical fitness models based on the assumption that highly fit enzymes have evolved to maximize the output of correct products, such as cyclic products or a specific product of interest, while minimizing the output of byproducts. This approach results in nonlinear fitness landscapes that are considerably more epistatic. Overall, our experimental and computational framework provides focused characterization of evolutionary emergence of novel enzymatic functions in the context of microevolutionary exploration of sequence space around naturally occurring enzymes.
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- Award ID(s):
- 1920914
- PAR ID:
- 10163562
- Date Published:
- Journal Name:
- Molecular Biology and Evolution
- ISSN:
- 0737-4038
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract The absence of orthogonal aminoacyl-transfer RNA (tRNA) synthetases that accept non-l-α-amino acids is a primary bottleneck hindering the in vivo translation of sequence-defined hetero-oligomers and biomaterials. Here we report that pyrrolysyl-tRNA synthetase (PylRS) and certain PylRS variants accept α-hydroxy, α-thio andN-formyl-l-α-amino acids, as well as α-carboxy acid monomers that are precursors to polyketide natural products. These monomers are accommodated and accepted by the translation apparatus in vitro; those with reactive nucleophiles are incorporated into proteins in vivo. High-resolution structural analysis of the complex formed between one PylRS enzyme and am-substituted 2-benzylmalonic acid derivative revealed an active site that discriminates prochiral carboxylates and accommodates the large size and distinct electrostatics of an α-carboxy substituent. This work emphasizes the potential of PylRS-derived enzymes for acylating tRNA with monomers whose α-substituent diverges substantially from the α-amine of proteinogenic amino acids. These enzymes or derivatives thereof could synergize with natural or evolved ribosomes and/or translation factors to generate diverse sequence-defined non-protein heteropolymers.more » « less
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Abstract MotivationThousands of genomes are publicly available, however, most genes in those genomes have poorly defined functions. This is partly due to a gap between previously published, experimentally characterized protein activities and activities deposited in databases. This activity deposition is bottlenecked by the time-consuming biocuration process. The emergence of large language models presents an opportunity to speed up the text-mining of protein activities for biocuration. ResultsWe developed FuncFetch—a workflow that integrates NCBI E-Utilities, OpenAI’s GPT-4, and Zotero—to screen thousands of manuscripts and extract enzyme activities. Extensive validation revealed high precision and recall of GPT-4 in determining whether the abstract of a given paper indicates the presence of a characterized enzyme activity in that paper. Provided the manuscript, FuncFetch extracted data such as species information, enzyme names, sequence identifiers, substrates, and products, which were subjected to extensive quality analyses. Comparison of this workflow against a manually curated dataset of BAHD acyltransferase activities demonstrated a precision/recall of 0.86/0.64 in extracting substrates. We further deployed FuncFetch on nine large plant enzyme families. Screening 26 543 papers, FuncFetch retrieved 32 605 entries from 5459 selected papers. We also identified multiple extraction errors including incorrect associations, nontarget enzymes, and hallucinations, which highlight the need for further manual curation. The BAHD activities were verified, resulting in a comprehensive functional fingerprint of this family and revealing that ∼70% of the experimentally characterized enzymes are uncurated in the public domain. FuncFetch represents an advance in biocuration and lays the groundwork for predicting the functions of uncharacterized enzymes. Availability and implementationCode and minimally curated activities are available at: https://github.com/moghelab/funcfetch and https://tools.moghelab.org/funczymedb.more » « less
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null (Ed.)Abstract Background Continuous enzyme kinetic assays are often used in high-throughput applications, as they allow rapid acquisition of large amounts of kinetic data and increased confidence compared to discontinuous assays. However, data analysis is often rate-limiting in high-throughput enzyme assays, as manual inspection and selection of a linear range from individual kinetic traces is cumbersome and prone to user error and bias. Currently available software programs are specialized and designed for the analysis of complex enzymatic models. Despite the widespread use of initial rate determination for processing kinetic data sets, no simple and automated program existed for rapid analysis of initial rates from continuous enzyme kinetic traces. Results An Interactive Continuous Enzyme Kinetics Analysis Tool (ICEKAT) was developed for semi-automated calculation of initial rates from continuous enzyme kinetic traces with particular application to the evaluation of Michaelis-Menten and EC 50 /IC 50 kinetic parameters, as well as the results of high-throughput screening assays. ICEKAT allows users to interactively fit kinetic traces using convenient browser-based selection tools, ameliorating tedious steps involved in defining ranges to fit in general purpose programs like Microsoft Excel and Graphpad Prism, while still maintaining simplicity in determining initial rates. As a test case, we quickly analyzed over 500 continuous enzyme kinetic traces resulting from experimental data on the response of the protein lysine deacetylase SIRT1 to small-molecule activators. Conclusions ICEKAT allows simultaneous visualization of individual initial rate fits and the resulting Michaelis-Menten or EC 50 /IC 50 kinetic model fits, as well as hits from high-throughput screening assays. In addition to serving as a convenient program for practicing enzymologists, ICEKAT is also a useful teaching aid to visually demonstrate in real-time how incorrect initial rate fits can affect calculated Michaelis-Menten or EC 50 /IC 50 kinetic parameters. For the convenience of the research community, we have made ICEKAT freely available online at https://icekat.herokuapp.com/icekat .more » « less
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1093/molbev/msaa052
