A method for generating user-defined circular single-stranded DNA from plasmid DNA using Golden Gate intramolecular ligation.

Construction of user-defined long circular single stranded DNA (cssDNA) and linear single stranded DNA (lssDNA) is important for various biotechnological applications. Many current methods for synthesis of these ssDNA molecules do not scale to multikilobase constructs. Here we present a robust methodology for generating user-defined cssDNA employing Golden Gate assembly, a nickase, and exonuclease degradation. Our technique is demonstrated for three plasmids with insert sizes ranging from 2.1 to 3.4 kb, requires no specialized equipment, and can be accomplished in 5 h with a yield of 33%-43% of the theoretical. To produce lssDNA, we evaluated different CRISPR-Cas9 cleavage conditions and reported a 52 ± 8% cleavage efficiency of cssDNA. Thus, our current method does not compete with existing protocols for lssDNA generation. Nevertheless, our protocol can make long, user-defined cssDNA readily available to biotechnology researchers.

Cell Survival Enabled by Leakage of a Labile Metabolic Intermediate.

Many metabolites are generated in one step of a biochemical pathway and consumed in a subsequent step. Such metabolic intermediates are often reactive molecules which, if allowed to freely diffuse in the intracellular milieu, could lead to undesirable side reactions and even become toxic to the cell. Therefore, metabolic intermediates are often protected as protein-bound species and directly transferred between enzyme active sites in multi-functional enzymes, multi-enzyme complexes, and metabolons. Sequestration of reactive metabolic intermediates thus contributes to metabolic efficiency. It is not known, however, whether this evolutionary adaptation can be relaxed in response to challenges to organismal survival. Here, we report evolutionary repair experiments on Escherichia coli cells in which an enzyme crucial for the biosynthesis of proline has been deleted. The deletion makes cells unable to grow in a culture medium lacking proline. Remarkably, however, cell growth is efficiently restored by many single mutations (12 at least) in the gene of glutamine synthetase. The mutations cause the leakage to the intracellular milieu of a highly reactive phosphorylated intermediate common to the biosynthetic pathways of glutamine and proline. This intermediate is generally assumed to exist only as a protein-bound species. Nevertheless, its diffusion upon mutation-induced leakage enables a new route to proline biosynthesis. Our results support that leakage of sequestered metabolic intermediates can readily occur and contribute to organismal adaptation in some scenarios. Enhanced availability of reactive molecules may enable the generation of new biochemical pathways and the potential of mutation-induced leakage in metabolic engineering is noted.

How to Recruit a Promiscuous Enzyme to Serve a New Function.

Promiscuous enzymes can be recruited to serve new functions when a genetic or environmental change makes catalysis of a novel reaction important for fitness or even survival. Subsequently, gene duplication and divergence can lead to evolution of an efficient and specialized new enzyme. Every organism likely has thousands of promiscuous enzyme activities that provide a vast reservoir of catalytic potential. However, much of this potential may not be accessible. We compiled kinetic parameters for promiscuous reactions catalyzed by 108 enzymes. The median value of kcat/KM is a very modest 31 M-1 s-1. Based upon the fluxes through metabolic pathways in E. coli, we estimate that many, if not most, promiscuous activities are too inefficient to impact fitness. However, mutations can elevate the level of an insufficient promiscuous activity by increasing enzyme expression, improving kcat/KM, or altering concentrations of the promiscuous and native substrates and allosteric regulators. Particularly in large bacterial populations, stochastic mutations may provide a viable pathway for recruitment of even inefficient promiscuous activities.

Structure and kinetics of indole-3-glycerol phosphate synthase from Pseudomonas aeruginosa: Decarboxylation is not essential for indole formation.

In tryptophan biosynthesis, the reaction catalyzed by the enzyme indole-3-glycerol phosphate synthase (IGPS) starts with a condensation step in which the substrate's carboxylated phenyl group makes a nucleophilic attack to form the pyrrole ring of the indole, followed by a decarboxylation that restores the aromaticity of the phenyl. IGPS from Pseudomonas aeruginosa has the highest turnover number of all characterized IGPS enzymes, providing an excellent model system to test the necessity of the decarboxylation step. Since the 1960s, this step has been considered to be mechanistically essential based on studies of the IGPS-phosphoribosylanthranilate isomerase fusion protein from Escherichia coli Here, we present the crystal structure of P. aeruginosa IGPS in complex with reduced CdRP, a nonreactive substrate analog, and using a sensitive discontinuous assay, we demonstrate weak promiscuous activity on the decarboxylated substrate 1-(phenylamino)-1-deoxyribulose-5-phosphate, with an ∼1000× lower rate of IGP formation than from the native substrate. We also show that E. coli IGPS, at an even lower rate, can produce IGP from decarboxylated substrate. Our structure of P. aeruginosa IGPS has eight molecules in the asymmetric unit, of which seven contain ligand and one displays a previously unobserved conformation closer to the reactive state. One of the few nonconserved active-site residues, Phe201 in P. aeruginosa IGPS, is by mutagenesis demonstrated to be important for the higher turnover of this enzyme on both substrates. Our results demonstrate that despite IGPS's classification as a carboxy-lyase (i.e. decarboxylase), decarboxylation is not a completely essential step in its catalysis.

In Vitro Selection of Peptides and Proteins-Advantages of mRNA Display.

mRNA display is a robust in vitro selection technique that allows the selection of peptides and proteins with desired functions from libraries of trillions of variants. mRNA display relies upon a covalent linkage between a protein and its encoding mRNA molecule; the power of the technique stems from the stability of this link, and the large degree of control over experimental conditions afforded to the researcher. This article describes the major advantages that make mRNA display the method of choice among comparable in vivo and in vitro methods, including cell-surface display, phage display, and ribosomal display. We also describe innovative techniques that harness mRNA display for directed evolution, protein engineering, and drug discovery.

Genetic Code Evolution Investigated through the Synthesis and Characterisation of Proteins from Reduced-Alphabet Libraries.

The universal genetic code of 20 amino acids is the product of evolution. It is believed that earlier versions of the code had fewer residues. Many theories for the order in which amino acids were integrated into the code have been proposed, considering factors ranging from prebiotic chemistry to codon capture. Several meta-analyses combined these theories to yield a feasible consensus chronology of the genetic code's evolution, but there is a dearth of experimental data to test the hypothesised order. We used combinatorial chemistry to synthesise libraries of random polypeptides that were based on different subsets of the 20 standard amino acids, thus representing different stages of a plausible history of the alphabet. Four libraries were comprised of the five, nine, and 16 most ancient amino acids, and all 20 extant residues for a direct side-by-side comparison. We characterised numerous variants from each library for their solubility and propensity to form secondary, tertiary or quaternary structures. Proteins from the two most ancient libraries were more likely to be soluble than those from the extant library. Several individual protein variants exhibited inducible protein folding and other traits typical of intrinsically disordered proteins. From these libraries, we can infer how primordial protein structure and function might have evolved with the genetic code.

Enzyme evolution: innovation is easy, optimization is complicated.

Enzymes have been evolving to catalyze new chemical reactions for billions of years, and will continue to do so for billions more. Here, we review examples in which evolutionary biochemists have used big data and high-throughput experimental tools to shed new light on the enormous functional diversity of extant enzymes, and the evolutionary processes that gave rise to it. We discuss the role that gene loss has played in enzyme evolution, as well as the more familiar processes of gene duplication and divergence. We also review insightful studies that relate not only catalytic activity, but also a host of other biophysical and cellular parameters, to organismal fitness. Finally, we provide an updated perspective on protein engineering, based on our new-found appreciation that most enzymes are sloppy and mediocre.

Structural and functional innovations in the real-time evolution of new (ßα)8 barrel enzymes.

New genes can arise by duplication and divergence, but there is a fundamental gap in our understanding of the relationship between these genes, the evolving proteins they encode, and the fitness of the organism. Here we used crystallography, NMR dynamics, kinetics, and mass spectrometry to explain the molecular innovations that arose during a previous real-time evolution experiment. In that experiment, the (ßα)8 barrel enzyme HisA was under selection for two functions (HisA and TrpF), resulting in duplication and divergence of the hisA gene to encode TrpF specialists, HisA specialists, and bifunctional generalists. We found that selection affects enzyme structure and dynamics, and thus substrate preference, simultaneously and sequentially. Bifunctionality is associated with two distinct sets of loop conformations, each essential for one function. We observed two mechanisms for functional specialization: structural stabilization of each loop conformation and substrate-specific adaptation of the active site. Intracellular enzyme performance, calculated as the product of catalytic efficiency and relative expression level, was not linearly related to fitness. Instead, we observed thresholds for each activity above which further improvements in catalytic efficiency had little if any effect on growth rate. Overall, we have shown how beneficial substitutions selected during real-time evolution can lead to manifold changes in enzyme function and bacterial fitness. This work emphasizes the speed at which adaptive evolution can yield enzymes with sufficiently high activities such that they no longer limit the growth of their host organism, and confirms the (ßα)8 barrel as an inherently evolvable protein scaffold.

Two-step Ligand Binding in a (ßα)8 Barrel Enzyme: SUBSTRATE-BOUND STRUCTURES SHED NEW LIGHT ON THE CATALYTIC CYCLE OF HisA.

HisA is a (ßα)8 barrel enzyme that catalyzes the Amadori rearrangement of N'-[(5'-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (ProFAR) to N'-((5'-phosphoribulosyl) formimino)-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) in the histidine biosynthesis pathway, and it is a paradigm for the study of enzyme evolution. Still, its exact catalytic mechanism has remained unclear. Here, we present crystal structures of wild type Salmonella enterica HisA (SeHisA) in its apo-state and of mutants D7N and D7N/D176A in complex with two different conformations of the labile substrate ProFAR, which was structurally visualized for the first time. Site-directed mutagenesis and kinetics demonstrated that Asp-7 acts as the catalytic base, and Asp-176 acts as the catalytic acid. The SeHisA structures with ProFAR display two different states of the long loops on the catalytic face of the structure and demonstrate that initial binding of ProFAR to the active site is independent of loop interactions. When the long loops enclose the substrate, ProFAR adopts an extended conformation where its non-reacting half is in a product-like conformation. This change is associated with shifts in a hydrogen bond network including His-47, Asp-129, Thr-171, and Ser-202, all shown to be functionally important. The closed conformation structure is highly similar to the bifunctional HisA homologue PriA in complex with PRFAR, thus proving that structure and mechanism are conserved between HisA and PriA. This study clarifies the mechanistic cycle of HisA and provides a striking example of how an enzyme and its substrate can undergo coordinated conformational changes before catalysis.

Rapid bursts and slow declines: on the possible evolutionary trajectories of enzymes.

The evolution of enzymes is often viewed as following a smooth and steady trajectory, from barely functional primordial catalysts to the highly active and specific enzymes that we observe today. In this review, we summarize experimental data that suggest a different reality. Modern examples, such as the emergence of enzymes that hydrolyse human-made pesticides, demonstrate that evolution can be extraordinarily rapid. Experiments to infer and resurrect ancient sequences suggest that some of the first organisms present on the Earth are likely to have possessed highly active enzymes. Reconciling these observations, we argue that rapid bursts of strong selection for increased catalytic efficiency are interspersed with much longer periods in which the catalytic power of an enzyme erodes, through neutral drift and selection for other properties such as cellular energy efficiency or regulation. Thus, many enzymes may have already passed their catalytic peaks.