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

On the Evolution of Catalysis: The changing kinetics of core metabolic enzymes

Enzymes are biological catalysts that are essential to life: they are largely very efficient and exquisitely specialised to their substrate. They are, however, predicted to have evolved from simple promiscuous catalysts (able to catalyse multiple reactions on multiple substrates). The goal of this thesis was to explore the multiple models of evolution with three model enzymes. In a complementary model for enzyme evolution, it is hypothesised that some ancient enzymes may have exhibited higher catalytic rates than their extant descendants. This model was investigated through the reconstruction of core bacterial enzymes AroA (aromatic amino acid biosynthesis) and MurA (peptidoglycan biosynthesis), from the common ancestor of modern Streptoccocci species. The ~300 million year old ancestral enzyme conformed to the model, with 20-fold higher activity than MurA enzymes in modern Streptococci. Several models for enzyme evolution, both primordial and contemporary, require a multifunctional precursor enzyme as a starting point. This was the case in a previous study, in which model enzyme HisA (histidine biosynthesis) from Salmonella enterica was evolved to acquire novel activity towards the TrpF (tryptophan biosynthesis) substrate (Näsvall et al., 2012). In the current study, the variant enzymes representing a mutational trajectory between HisA and TrpF were kinetically characterised and the structure-function link identified for many causative mutations. A three-amino acid duplication was key for establishing the novel TrpF function, altering the induced fit mechanism of the enzyme and repositioning the general acid side chain. Other amino acid substitutions improved novel activity and substrate specificity by excluding the HisA substrate. The S. enterica HisA active site and that of the variant specialised to TrpF activity were characterised through site-directed mutagenesis and kinetic assays. It was found that the new TrpF enzyme employed two different catalytic mechanisms. In addition to an entirely enzyme-based mechanism (as in HisA), the enzyme also employed substrate-assisted catalysis, whereby a functional group of the substrate contributed to catalysis. Such primitive mechanisms might – like multifunctional enzymes – be common intermediates during the evolution of enzymatic function. The requirements for TrpF function in the S. enterica HisA active site were further probed through site-directed mutagenesis to find the relative contribution of different residues to function. A duplicated arginine residue was important to alter the orientation of the active site aspartate to optimise its role as a general acid. A library generated through site-saturation mutagenesis in the active site of a bifunctional HisA variant was selected for improved TrpF function and yielded an enzyme with 13-fold improved activity and absolute substrate specificity. This study has demonstrated the ruggedness of the extended HisA landscape and how highly connected the enzyme is with other functions. The elegant complexity of enzymatic function and its evolution is demonstrated on many different fronts

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

The next generation's Frankenstein films (Letter)

In Mary Shelley's Frankenstein, Victor Frankenstein's well-intentioned research goes awry, creating a monster. The novel was first adapted to film in 1910, and many movie remakes and variations followed. We asked young scientists to craft their own Frankenstein-inspired science fiction by pitching a movie plot answering this question: What modern research could serve as the basis for the next box office hit? According to the responses, the research discoveries most likely to play a part in this year's blockbuster are gene-editing technology, xenotransplantation, and artificial intelligence. Microbes and viruses also played a starring role. Several scripts were set in the future against a backdrop of extreme climate change. Read on for a selection of our scientific Oscar line-up

Structural and functional innovations in the real-time evolution of new (beta alpha)(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 (beta alpha)(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 (beta alpha)(8) barrel as an inherently evolvable protein scaffold.Funding Agencies|Marsden Fund; Rutherford Discovery Fellowship; Swedish Research Council; European Community [283570]</p

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 E. 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