High-order epistasis in catalytic power of dihydrofolate reductase gives rise to a rugged fitness landscape in the presence of trimethoprim selection

Tamer, Yusuf Talha; Gaszek, Ilona K.; Abdizadeh, Haleh; Batur, Tugce; Reynolds, Kimberly A.; Atılgan, Ali Rana; Atılgan, Canan; Toprak, Erdal

doi:10.1101/398065

Cited by 20 publications

(40 citation statements)

References 53 publications

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“…While we did observe filamentation and a drop in growth rates ( Figure S3), cell length showed a nonmonotonic dependence on TMP concentration at both temperatures ( Figure S4 at 37°C, Figure 2E at 42°C). Cell length increased at low concentrations of TMP to reach a maximum at one-half of MIC (0.5μg/ml, MIC of TMP is 1μg/ml (Rodrigues et al, 2016;Tamer et al, 2019;Toprak et al, 2012)), and progressively decreased to 'no drug' levels at higher concentrations. This phenomenon is much more prominent at 42°C than 37°C, presumably because soluble DHFR amounts are lower at 42°C due to aggregation (Bershtein et al, 2012).…”

Section: Filamentation Is Due To Drop In Dhfr Activitymentioning

confidence: 99%

Metabolic response to point mutations reveals principles of modulation of in vivo enzyme activity and phenotype

Bhattacharyya

Bershtein

Adkar

et al. 2019

Preprint

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Understanding genotype-phenotype relationship is a central problem in modern biology. Here we report that several destabilizing mutations in E. coli Dihydrofolate Reductase (DHFR) enzyme result in pronounced yet reversible filamentation of bacterial cells. We find that drop in DHFR activity in the mutants results in massive metabolic shifts leading to significant excess of dUMP and dCTP in the pyrimidine biosynthesis pathway. Both deoxyribonucleotides inhibit downstream essential enzyme Thymidylate Kinase (Tmk) which phosphorylates dTMP, eventually leading to almost complete loss of the DNA building block dTTP. Severe imbalance in deoxyribonucleotide levels ultimately leads to DNA damage, SOS response and filamentation. Supplementation of dTMP in the medium competitively alleviates Tmk inhibition and rescues filamentation, thereby illustrating a classic case of enzyme inhibition by structural mimic of its cognate ligand. Overall, this study highlights the complex interconnected nature of the metabolic network, and the fundamental role of folate metabolism in shaping the cell.

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Section: Filamentation Is Due To Drop In Dhfr Activitymentioning

confidence: 99%

Metabolic response to point mutations reveals principles of modulation of in vivo enzyme activity and phenotype

Bhattacharyya

Bershtein

Adkar

et al. 2019

Preprint

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“…The more highly-enriched allele lies upstream of the folA CDS and likely increases it’s transcription, a well-described route to TMP resistance 31 . The other is known to increase the catalytic rate of FolA, leading to TMP resistance 31 . A third SNP within folA is not enriched, suggesting little or no effect individually at this concentration of TMP.…”

Section: Resultsmentioning

confidence: 99%

High throughput functional variant screens via in-vivo production of single-stranded DNA

Schubert

Goodman

Wannier

et al. 2020

Preprint

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5Tremendous genetic variation exists in nature, but our ability to create and characterize 6 individual genetic variants remains far more limited in scale. Likewise, engineering proteins and 7 phenotypes requires the introduction of synthetic variants, but design of variants outpaces 8 experimental measurement of variant effect. Here, we optimize efficient and continuous 9 generation of precise genomic edits in Escherichia coli, via in-vivo production of single-stranded 10 DNA by the targeted reverse-transcription activity of retrons. Greater than 90% editing 11 efficiency can be obtained using this method, enabling multiplexed applications. We introduce 12 Retron Library Recombineering (RLR), a system for high-throughput screens of variants, 13 wherein the association of introduced edits with their retron elements enables a targeted deep 14 sequencing phenotypic output. We use RLR for pooled, quantitative phenotyping of synthesized 15 variants, characterizing antibiotic resistance alleles. We also perform RLR using sheared 16 genomic DNA of an evolved bacterium, experimentally querying millions of sequences for 17 antibiotic resistance variants. In doing so, we demonstrate that RLR is uniquely suited to utilize 18 non-designed sources of variation. Pooled experiments using ssDNA produced in vivo thus 19 present new avenues for exploring variation, both designed and not, across the entire genome. 20 21 Introduction: 22Constructing genotypes of interest and observing their effect on phenotype critically aids 23 our understanding of genetics and genome function. As methods for editing genomes have 24 progressed, this "reverse genetics" approach has expanded in breadth and scale, from knockout 25 libraries 1 to refactored genomes 2,3 . These experiments can now be performed within multiplexed 26 pools, which allow an ever greater number of mutations to be explored across varied conditions. 27Critically, both creating genotypes and observing phenotype within pools has necessitated 28 2 development of new techniques. Transposon insertions 4 , marked integrations 5 and CRISPR-29 inhibition 6,7 can create thousands of variants simultaneously within pooled experiments, and 30 targeted sequencing of these elements enables pooled measurement of variant phenotypes. These 31 advancements in experimental scale have fundamentally transformed our understanding of 32 genome function 8 . 33However, these current high-throughput genetic techniques remain limited, in that they 34 typically introduce, ablate, or regulate kilobases of DNA to create variation. This contrasts with 35 point mutations, which are ubiquitous in natural variation 9 , and are indispensable for engineering 36 proteins 10 and metabolic pathways 11 . While modifying kilobases of DNA can add and subtract 37 functional elements such as genes and regulatory sequences from the genome, point mutations 38 can alter the function of these elements, accessing a larger phenotypic landscape. 39Point mutations and other precision edits can be performed by oligonucleo...

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“…Quantitative understanding of the molecular mechanisms of protein evolution is a major challenge in evolutionary biology and protein engineering. The availability and the diversity of evolutionary paths leading to proteins with novel biochemical functions are ultimately determined by a complex pattern of energetic interactions between amino acid residues within the protein, as well as protein-ligand interactions [4][5][6][7]. The emergence of novel catalytic functions is a paramount example of evolutionary expansion with profound biological implications.…”

Section: Introductionmentioning

confidence: 99%

Sparse epistatic patterns in the evolution of terpene synthases

Ballal

Laurendon

Salmon

et al. 2019

Preprint

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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 the emergence of terpene cyclization, we have carried out in-depth examination of mutational space around (E)-b-farnesene synthase, an Artemisia annua enzyme which catalyzes production of a linear hydrocarbon. Each mutant enzyme in our synthetic libraries was characterized biochemically, resulting in reaction rate measurements for 7 cyclic and 4 linear products. We used this reaction rate data as input to the Michaelis-Menten model of enzyme kinetics, in which the 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 input of incorrect products, which may be toxic or costly to remove and degrade. This approach results in a fitness landscape which is a non-linear function of Michaelis-Menten free energies and is therefore considerably more epistatic. Overall, our experimental and computational framework provides focused characterization of sequence determinants and molecular mechanisms underlying evolutionary emergence of novel enzymatic functions in the context of micro-evolutionary exploration of sequence space around wild-type enzymes.

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High-order epistasis in catalytic power of dihydrofolate reductase gives rise to a rugged fitness landscape in the presence of trimethoprim selection

Abstract: 15Evolutionary fitness landscapes of certain antibiotic target enzymes have been comprehensively

Cited by 20 publications

References 53 publications

Metabolic response to point mutations reveals principles of modulation of in vivo enzyme activity and phenotype

Metabolic response to point mutations reveals principles of modulation of in vivo enzyme activity and phenotype

High throughput functional variant screens via in-vivo production of single-stranded DNA

Sparse epistatic patterns in the evolution of terpene synthases

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