Catalytically
Lupo Hepperly
Oscillatory systems regulate many biological processes, including key cellular functions such as metabolism and cell division, as well as larger-scale processes such as circadian rhythm and heartbeat1,2,3,4. Abiotic chemical oscillations, discovered originally in inorganic systems5,6, inspired the development of various synthetic oscillators for application as autonomous time-keeping systems in analytical chemistry, materials chemistry and the biomedical field7,8,9,10,11,12,13,14,15,16,17. Expanding their role beyond that of a pacemaker by having synthetic chemical oscillators periodically drive a secondary function would turn them into significantly more powerful tools. However, this is not trivial because the participation of components of the oscillator in the secondary function might jeopardize its time-keeping ability. We now report a small molecule oscillator that can catalyse an independent chemical reaction in situ without impairing its oscillating properties. In a flow system, the concentration of the catalytically active product of the oscillator shows sustained oscillations and the catalysed reaction is accelerated only during concentration peaks. Augmentation of synthetic oscillators with periodic catalytic action allows the construction of complex systems that, in the future, may benefit applications in automated synthesis, systems and polymerization chemistry and periodic drug delivery.
Our catalytically active oscillator is made from small organic molecules and is based on the principles of aminocatalysis. It makes use of autocatalytic 9-fluorenylmethoxycarbonyl (Fmoc) group deprotection29,30 and acetylation of amines. Using an open flow system, sustained oscillations are realized over a range of different conditions. The key component of the oscillator is an organocatalyst that promotes chemical reactions via enamine and/or base catalysis. The reactants that form the oscillator and those involved in the catalysed reaction are mixed in the same reactor and form a system in which each component serves a unique purpose with minimal interference between oscillation and catalysis.
A heterogeneous and dilute suspension of catalytically active colloids is studied as a nonequilibrium analogue of ionic systems, which has the remarkable feature of action-reaction symmetry breaking. Symmetrically coated colloids are found to join up to form self-assembled molecules that could be inert or have spontaneous activity in the form of net translational velocity and spin depending on their symmetry properties and their constituents. The type of activity can be adjusted by changing the surface chemistry and ambient variables that control the surface reactions and the phoretic drift.
Hydroxynitrile lyases (HNL's) belonging to the α/β-hydrolase-fold superfamily evolved from esterases approximately 100 million years ago. Reconstruction of an ancestral hydroxynitrile lyase in the α/β-hydrolase fold superfamily yielded a catalytically active hydroxynitrile lyase, HNL1. Several properties of HNL1 differ from the modern HNL from rubber tree (HbHNL). HNL1 favors larger substrates as compared to HbHNL, is two-fold more catalytically promiscuous for ester hydrolysis (p-nitrophenyl acetate) as compared to mandelonitrile cleavage, and resists irreversible heat inactivation to 35 C higher than for HbHNL. We hypothesized that the x-ray crystal structure of HNL1 may reveal the molecular basis for the differences in these properties. The x-ray crystal structure solved to 1.96-Å resolution shows the expected α/β-hydrolase fold, but a 60% larger active site as compared to HbHNL. This larger active site echoes its evolution from esterases since related esterase SABP2 from tobacco also has a 38% larger active site than HbHNL. The larger active site in HNL1 likely accounts for its ability to accept larger hydroxynitrile substrates. Site-directed mutagenesis of HbHNL to expand the active site increased its promiscuous esterase activity 50-fold, consistent with the larger active site in HNL1 being the primary cause of its promiscuous esterase activity. Urea-induced unfolding of HNL1 indicates that it unfolds less completely than HbHNL (m-value = 0.63 for HNL1 vs 0.93 kcal/molM for HbHNL), which may account for the ability of HNL1 to better resist irreversible inactivation upon heating. The structure of HNL1 shows changes in hydrogen bond networks that may stabilize regions of the folded structure.
Citation: Jones BJ, Evans RL III, Mylrea NJ, Chaudhury D, Luo C, Guan B, et al. (2020) Larger active site in an ancestral hydroxynitrile lyase increases catalytically promiscuous esterase activity. PLoS ONE 15(6): e0235341.
Divergent evolution creates superfamilies of enzymes, which share the same protein fold, but differ in substrate specificity or in the type of catalytic activities. The focus of this paper is understanding how evolution creates new catalytic activity during divergent evolution. This question is of interest to evolutionary biologists and also to protein engineers seeking to introduce and optimize new catalytic activities in proteins. Divergent evolution of enzymes to create new catalytic activity is thought to involve intermediate catalytically promiscuous enzymes [1, 2]. Catalytic promiscuity is the ability of enzymes to catalyze additional, chemically distinct reactions besides their primary reaction [3]. Duplication of the genes for these promiscuous enzymes followed by optimization of the promiscuous catalytic activity driven by increased organismal fitness is believed to give rise to enzymes with new primary activities. Support for this notion includes the observation that differing catalytic activities within a superfamily share mechanistic features or transition states and that enzymes within a superfamily often show promiscuous activities that correspond to the primary activities of other enzymes in the superfamily [4, 5]. Characterization of resurrected likely ancestral enzymes supports the hypothesis that new functions evolved from ancestors with multiple functions. Reconstructed ancestral enzymes have shown substrate promiscuity [6] and also catalytic promiscuity [7, 8].
Previous work identified three molecular mechanisms that promote catalytic promiscuity in enzymes. First, catalytic promiscuity may have less to do with the enzyme and more with the two reactions being compared. The two reactions may involve similar transition states so the interactions that stabilize the primary reaction also stabilize the promiscuous reaction. In such cases, almost all enzymes catalyzing these reaction types will show promiscuity. For example, the primary function of proteases is amide hydrolysis, but almost all proteases similarly catalyze ester hydrolysis because both reactions involve similar transition states. Second, a catalytically promiscuous enzyme may change its conformation thereby temporarily creating a different enzyme active site structure with different catalytic abilities. For example, a lactonase with promiscuous phosphotriesterase activity catalyzes lactone hydrolysis via a closed conformation, but phosphate triester hydrolysis via an open conformation [9, 10]. The third mechanism for promiscuity is a larger active site with multiple possibilities for interactions between enzyme and transition state. The enzyme active site remains the same, but substrates adopt different orientations within it. For example, phosphonate monoester hydrolase catalyzes promiscuous hydrolysis of sulfate monoesters and other analogs but contains a rigid active site. The varied substrates presumably adopt different orientations within the active site. Catalytic promiscuity correlates with larger active sites and larger polar solvent-accessible surface area [11, 12]. A large active site accommodates a broader range of substrates and allows them to bind in multiple conformations, while a large polar surface allows multiple alternative electrostatic interactions that can stabilize the transition state.
A catalytically promiscuous enzyme may simultaneously use all three of these mechanisms, for example, the lactonase mentioned above also known as paraoxonase I [9]. The enzyme catalyzed hydrolysis of both lactones and phosphate triesters. The first mechanism applies since both hydrolyses have similar negatively charged intermediates and lactonase/phosphotriesterase catalytic promiscuity is common among these enzymes. The lactone hydrolysis intermediates are tetrahedral and the phosphate triester hydrolysis intermediates are pentavalent, but they are nevertheless similar. The second mechanism occurs when different active site conformations enable the two distinct reactions. Finally, the rich catalytic network within the active site enables the third mechanism by promoting multiple reaction pathways by using subsets of active site residues or using them for different roles. In the paraoxonase I case, three amino acid residues (E53, H115, D269) near the active site calcium stabilize the attack of water on the lactone, while only two of these residues (E53, D269) stabilize the attack of water on the phosphotriester.
A reconstructed ancestral hydroxynitrile lyase, HNL1, from the α/β-hydrolase-fold superfamily is the focus of this paper [8]. Most enzymes in the α/β-hydrolase-fold superfamily are esterases, which catalyze the hydrolysis of esters, but this superfamily also includes lyases, which catalyze the cleavage of hydroxynitriles and the corresponding reverse addition reaction [13]. HNL1 primarily catalyzes the cleavage of hydroxynitriles, but can also catalyze promiscuous ester hydrolysis. Both reactions involve nucleophilic attack on carbonyl compounds with tetrahedral transition states with partial negative charge on the oxygens. However, the differences in the transition states (acyl enzyme formation versus no acyl enzyme formation, hydrophobic versus polar leaving group) leads to a strong trade-off between esterase and hydroxynitrile lyase catalytic activities. Indeed, it is rare that an esterase catalyzes hydroxynitrile cleavage and vice versa. Thus, the mechanistic basis for the catalytically promiscuous esterase activity of HNL1 remains unclear.