Coevolutionary Information Captures Catalytic Functions and Reveals Divergent Roles of Terpene Synthase Interdomain Connections.

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.

Sparse Epistatic Patterns in the Evolution of Terpene Synthases.

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)-ß-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.

Quantum Blue Reduces the Severity of Woody Breast Myopathy via Modulation of Oxygen Homeostasis-Related Genes in Broiler Chickens.

The incidence of woody breast (WB) is increasing on a global scale representing a significant welfare problem and economic burden to the poultry industry and for which there is no effective treatment due to its unknown etiology. In this study, using diffuse reflectance spectroscopy (DRS) coupled with iSTAT portable clinical analyzer, we provide evidence that the circulatory- and breast muscle-oxygen homeostasis is dysregulated [low oxygen and hemoglobin (HB) levels] in chickens with WB myopathy compared to healthy counterparts. Molecular analysis showed that blood HB subunit Mu (HBM), Zeta (HBZ), and hephaestin (HEPH) expression were significantly down regulated; however, the expression of the subunit rho of HB beta (HBBR) was upregulated in chicken with WB compared to healthy counterparts. The breast muscle HBBR, HBE, HBZ, and hypoxia-inducible factor prolyl hydroxylase 2 (PHD2) mRNA abundances were significantly down regulated in WB-affected compared to normal birds. The expression of HIF-1α at mRNA and protein levels was significantly induced in breasts of WB-affected compared to unaffected birds confirming a local hypoxic status. The phosphorylated levels of the upstream mediators AKT at Ser473 site, mTOR at Ser2481 site, and PI3K P85 at Tyr458 site, as well as their mRNA levels were significantly increased in breasts of WB-affected birds. In attempt to identify a nutritional strategy to reduce WB incidence, male broiler chicks (Cobb 500, n = 576) were randomly distributed into 48 floor pens and subjected to six treatments (12 birds/pen; 8 pens/treatment): a nutrient adequate control group (PC), the PC supplemented with 0.3% myo-inositol (PC + MI), a negative control (NC) deficient in available P and Ca by 0.15 and 0.16%, respectively, the NC fed with quantum blue (QB) at 500 (NC + 500 FTU), 1,000 (NC + 1,000 FTU), or 2,000 FTU/kg of feed (NC + 2,000 FTU). Although QB-enriched diets did not affect growth performances (FCR and FE), it did reduce the severity of WB by 5% compared to the PC diet. This effect is mediated by reversing the expression profile of oxygen homeostasis-related genes; i.e., significant down regulation of HBBR and upregulation of HBM, HBZ, and HEPH in blood, as well as a significant upregulation of HBA1, HBBR, HBE, HBZ, and PHD2 in breast muscle compared to the positive control.

Emergence of terpene cyclization in Artemisia annua.

The emergence of terpene cyclization was critical to the evolutionary expansion of chemical diversity yet remains unexplored. Here we report the first discovery of an epistatic network of residues that controls the onset of terpene cyclization in Artemisia annua. We begin with amorpha-4,11-diene synthase (ADS) and (E)-ß-farnesene synthase (BFS), a pair of terpene synthases that produce cyclic or linear terpenes, respectively. A library of ~27,000 enzymes is generated by breeding combinations of natural amino-acid substitutions from the cyclic into the linear producer. We discover one dominant mutation is sufficient to activate cyclization, and together with two additional residues comprise a network of strongly epistatic interactions that activate, suppress or reactivate cyclization. Remarkably, this epistatic network of equivalent residues also controls cyclization in a BFS homologue from Citrus junos. Fitness landscape analysis of mutational trajectories provides quantitative insights into a major epoch in specialized metabolism.

Automating gene library synthesis by structure-based combinatorial protein engineering: examples from plant sesquiterpene synthases.

Structure-based combinatorial protein engineering (SCOPE) is a homology-independent recombination method to create multiple crossover gene libraries by assembling defined combinations of structural elements ranging from single mutations to domains of protein structure. SCOPE was originally inspired by DNA shuffling, which mimics recombination during meiosis, where mutations from parental genes are "shuffled" to create novel combinations in the resulting progeny. DNA shuffling utilizes sequence identity between parental genes to mediate template-switching events (the annealing and extension of one parental gene fragment on another) in PCR reassembly reactions to generate crossovers and hence recombination between parental genes. In light of the conservation of protein structure and degeneracy of sequence, SCOPE was developed to enable the "shuffling" of distantly related genes with no requirement for sequence identity. The central principle involves the use of oligonucleotides to encode for crossover regions to choreograph template-switching events during PCR assembly of gene fragments to create chimeric genes. This approach was initially developed to create libraries of hybrid DNA polymerases from distantly related parents, and later developed to create a combinatorial mutant library of sesquiterpene synthases to explore the catalytic landscapes underlying the functional divergence of related enzymes. This chapter presents a simplified protocol of SCOPE that can be integrated with different mutagenesis techniques and is suitable for automation by liquid-handling robots. Two examples are presented to illustrate the application of SCOPE to create gene libraries using plant sesquiterpene synthases as the model system. In the first example, we outline how to create an active-site library as a series of complex mixtures of diverse mutants. In the second example, we outline how to create a focused library as an array of individual clones to distil minimal combinations of functionally important mutations. Through these examples, the principles of the technique are illustrated and the suitability of automating various aspects of the procedure for given applications are discussed.