Engineering of methionine-auxotroph Escherichia coli via parallel evolution of two enzymes from Corynebacterium glutamicum's direct-sulfurylation pathway enables its recovery in minimal medium.

Methionine biosynthesis relies on the sequential catalysis of multiple enzymes. Escherichia coli, the main bacteria used in research and industry for protein production and engineering, utilizes the three-step trans-sulfurylation pathway catalyzed by L-homoserine O-succinyl transferase, cystathionine gamma synthase and cystathionine beta lyase to convert L-homoserine to L-homocysteine. However, most bacteria employ the two-step direct-sulfurylation pathway involving L-homoserine O-acetyltransferases and O-acetyl homoserine sulfhydrylase. We previously showed that a methionine-auxotroph Escherichiacoli strain (MG1655) with deletion of metA, encoding for L-homoserine O-succinyl transferase, and metB, encoding for cystathionine gamma synthase, could be complemented by introducing the genes metX, encoding for L-homoserine O-acetyltransferases and metY, encoding for O-acetyl homoserine sulfhydrylase, from various sources, thus altering the Escherichia coli methionine biosynthesis metabolic pathway to direct-sulfurylation. However, introducing metX and metY from Corynebacterium glutamicum failed to complement methionine auxotrophy. Herein, we generated a randomized genetic library based on the metX and metY of Corynebacterium glutamicum and transformed it into a methionine-auxotrophic Escherichia coli strain lacking the metA and metB genes. Through multiple enrichment cycles, we successfully isolated active clones capable of growing in M9 minimal media. The dominant metX mutations in the evolved methionine-autotrophs Escherichia coli were L315P and H46R. Interestingly, we found that a metY gene encoding only the N-terminus 106 out of 438 amino acids of the wild-type MetY enzyme is functional and supports the growth of the methionine auxotroph. Recloning the new genes into the original plasmid and transforming them to methionine auxotroph Escherichia coli validated their functionality. These results show that directed enzyme-evolution enables fast and simultaneous engineering of new active variants within the Escherichia coli methionine direct-sulfurylation pathway, leading to efficient complementation.

A novel computationally engineered collagenase reduces the force required for tooth extraction in an ex-situ porcine jaw model.

The currently employed tooth extraction methods in dentistry involve mechanical disruption of the periodontal ligament fibers, leading to inevitable trauma to the bundle bone comprising the socket walls. In our previous work, we have shown that a recombinantly expressed truncated version of clostridial collagenase G (ColG) purified from Escherichia coli efficiently reduced the force needed for tooth extraction in an ex-situ porcine jaw model, when injected into the periodontal ligament. Considering that enhanced thermostability often leads to higher enzymatic activity and to set the basis for additional rounds of optimization, we used a computational protein design approach to generate an enzyme to be more thermostable while conserving the key catalytic residues. This process generated a novel collagenase (ColG-variant) harboring sixteen mutations compared to ColG, with a nearly 4℃ increase in melting temperature. Herein, we explored the potential of ColG-variant to further decrease the physical effort required for tooth delivery using our established ex-situ porcine jaw model. An average reduction of 11% was recorded in the force applied to extract roots of mandibular split first and second premolar teeth treated with ColG-variant, relative to those treated with ColG. Our results show for the first time the potential of engineering enzyme properties for dental medicine and further contribute to minimally invasive tooth extraction.

Application of In Silico Filtering and Isothermal Titration Calorimetry for the Discovery of Small Molecule Inhibitors of MDM2.

The initial discovery phase of protein modulators, which consists of filtering molecular libraries and in vitro direct binding validation, is central in drug discovery. Thus, virtual screening of large molecular libraries, together with the evaluation of binding affinity by isothermal calorimetry, generates an efficient experimental setup. Herein, we applied virtual screening for discovering small molecule inhibitors of MDM2, a major negative regulator of the tumor suppressor p53, and thus a promising therapeutic target. A library of 20 million small molecules was screened against an averaged model derived from multiple structural conformations of MDM2 based on published structures. Selected molecules originating from the computational filtering were tested in vitro for their direct binding to MDM2 via isothermal titration calorimetry. Three new molecules, representing distinct chemical scaffolds, showed binding to MDM2. These were further evaluated by exploring structure-similar chemical analogues. Two scaffolds were further evaluated by de novo synthesis of molecules derived from the initial molecules that bound MDM2, one with a central oxoazetidine acetamide and one with benzene sulfonamide. Several molecules derived from these scaffolds increased wild-type p53 activity in MCF7 cancer cells. These set a basis for further chemical optimization and the development of new chemical entities as anticancer drugs.

Collagenase Administration into Periodontal Ligament Reduces the Forces Required for Tooth Extraction in an Ex situ Porcine Jaw Model.

Minimally invasive exodontia is among the long-sought-for development aims of safe dental medicine. In this paper, we aim, for the first time, to examine whether the enzymatic disruption of the periodontal ligament fibers reduces the force required for tooth extraction. To this end, recombinantly expressed clostridial collagenase G variant purified from Escherichia coli was injected into the periodontal ligament of mesial and distal roots of the first and second split porcine mandibular premolars. The vehicle solution was injected into the corresponding roots on the contralateral side. Following sixteen hours, the treated mandibles were mounted on a loading machine to measure the extraction force. In addition, the effect of the enzyme on the viability of different cell types was evaluated. An average reduction of 20% in the applied force (albeit with a large variability of 50 to 370 newton) was observed for the enzymatically treated roots, reaching up to 50% reduction in some cases. Importantly, the enzyme showed only a minor and transient effect on cellular viability, without any signs of toxicity. Using an innovative model enabling the analytical measurement of extraction forces, we show, for the first time, that the enzymatic disruption of periodontal ligament fibers substantially reduces the force required for tooth extraction. This novel technique brings us closer to atraumatic exodontia, potentially reducing intra- and post-operative complications and facilitating subsequent implant placement. The development of novel enzymes with enhanced activity may further simplify the tooth extraction process and present additional clinical relevance for the broad range of implications in the oral cavity.

Screening Collagenase Activity in Bacterial Lysate for Directed Enzyme Applications.

Collagenases are essential enzymes capable of digesting triple-helical collagen under physiological conditions. These enzymes play a key role in diverse physiological and pathophysiological processes. Collagenases are used for diverse biotechnological applications, and it is thus of major interest to identify new enzyme variants with improved characteristics such as expression yield, stability, or activity. The engineering of new enzyme variants often relies on either rational protein design or directed enzyme evolution. The latter includes screening of a large randomized or semirational genetic library, both of which require an assay that enables the identification of improved variants. Moreover, the assay should be tailored for microplates to allow the screening of hundreds or thousands of clones. Herein, we repurposed the previously reported fluorogenic assay using 3,4-dihydroxyphenylacetic acid for the quantitation of collagen, and applied it in the detection of bacterial collagenase activity in bacterial lysates. This enabled the screening of hundreds of E. coli colonies expressing an error-prone library of collagenase G from C. histolyticum, in 96-well deep-well plates, by measuring activity directly in lysates with collagen. As a proof-of-concept, a single variant exhibiting higher activity than the starting-point enzyme was expressed, purified, and characterized biochemically and computationally. This showed the feasibility of this method to support medium-high throughput screening based on direct evaluation of collagenase activity.

EnzyDock: Protein-Ligand Docking of Multiple Reactive States along a Reaction Coordinate in Enzymes.

Enzymes play a pivotal role in all biological systems. These biomachines are the most effective catalysts known, dramatically enhancing the rate of reactions by more than 10 orders of magnitude relative to the uncatalyzed reactions in solution. Predicting the correct, mechanistically appropriate binding modes for substrate and product, as well as all reaction intermediates and transition states, along a reaction pathway is immensely challenging and remains an unsolved problem. In the present work, we developed an effective methodology for identifying probable binding modes of multiple ligand states along a reaction coordinate in an enzyme active site. The program is called EnzyDock and is a CHARMM-based multistate consensus docking program that includes a series of protocols to predict the chemically relevant orientation of substrate, reaction intermediates, transition states, product, and inhibitors. EnzyDock is based on simulated annealing molecular dynamics and Monte Carlo sampling and allows ligand, as well as enzyme side-chain and backbone flexibility. The program can employ many user-defined constraints and restraints and classical force field potentials, as well as a range of hybrid quantum mechanics-molecular mechanics potentials. Herein, we apply EnzyDock to several different kinds of problems. First, we study two terpene synthase reactions, namely bornyl diphosphate synthase and the bacterial diterpene synthase CotB2. Second, we use EnzyDock to predict reaction coordinate states in a pair of Diels-Alder reactions in the enzymes spirotetronate AbyU and LepI. Third, we study a couple of racemases: the cofactor-dependent serine racemase and the cofactor independent proline racemase. Finally, we study several cases of covalent docking involving the Michael addition reaction. For all systems we predict binding modes that are consistent with available experimental observations, as well as with theoretical modeling studies from the literature. EnzyDock provides a platform for generating mechanistic insight into enzyme reactions, useful and reliable starting points for in-depth multiscale modeling projects, and rational design of noncovalent and covalent enzyme inhibitors.

Crystal structure of LepI, a multifunctional SAM-dependent enzyme which catalyzes pericyclic reactions in leporin biosynthesis.

LepI is a novel multifunctional enzyme that catalyzes stereoselective dehydration, Diels-Alder reaction, and retro-Claisen rearrangement. Here we report the crystal structure of LepI in complex with its co-factor S-adenosyl methionine (SAM). LepI forms a tetramer via the N-terminal helical domain and binds to a SAM molecule in the C-terminal catalytic domain. The binding modes of various LepI substrates are investigated by docking simulations, which suggest that the substrates are bound via both hydrophobic and hydrophilic forces, as well as cation-π interactions with the positively charged SAM. The reaction starts with a dehydration step in which H133 possibly deprotonates the pyridone hydroxyl group of the substrate, while D296 might protonate an alkyl-chain hydroxyl group. Subsequent pericyclization may be facilitated by the correct fold of the substrate's alkyl chain and a thermodynamic driving force towards σ-bonds at the expense of π-bonds. These results provide structural insights into LepI catalysis and are important in understanding the mechanism of enzymatic pericyclization.

Slow-Starter Enzymes: Role of Active-Site Architecture in the Catalytic Control of the Biosynthesis of Taxadiene by Taxadiene Synthase.

Taxadiene synthase (TXS) catalyzes the formation of natural product taxa-4(5),11(12)-diene (henceforth taxadiene). Taxadiene is the precursor in the formation of Taxol, which is an important natural anticancer agent. In the current study, we present a detailed mechanistic view of the biosynthesis of taxadiene by TXS using a hybrid quantum mechanics-molecular mechanics potential in conjunction with free energy simulation methods. The obtained free-energy landscape displays initial endergonic steps followed by a stepwise downhill profile, which is an emerging free-energy fingerprint for type I terpene synthases. We identify an active-site Trp residue (W753) as a key feature of the TXS active-site architecture and propose that this residue stabilized intermediate cations via π-cation interactions. To validate our proposed active TXS model, we examine a previously reported W753H mutation, which leads to the exclusive formation of side product cembrene A. The simulations of the W753H mutant show that, in the mutant structure, the His side chain is in the perfect position to deprotonate the cembrenyl cation en route to cembrene formation and that this abortive deprotonation is an energetically facile process. On the basis of the current model, we propose that an analogous mutation of Y841 to His could possibly lead to verticillane. The current simulations stress the importance of the precise positioning of key active-site residues in stabilizing intermediate carbocations. In view of the great pharmaceutical importance of taxadiene, a detailed understanding of the TXS mechanism can provide important clues toward a synthetic strategy for Taxol manufacturing.

Copper-chaperones with dicoordinated Cu(I)--unique protection mechanism.

Cu(I) dicoordination with thiolate ligands is not common. Yet, different from its homologue proteins, human copper chaperone is known to bind Cu(I) using this low coordination number while binding Cu(I) only via the two conserved Cysteine residues, Cys12 and Cys15. Based on structural analysis, this work determines that the protein possesses two distinct conformations referred to as "in" and "out" due to the relative positioning of Cys12 (one of Cu(I) binding residues). The "out" conformation, with Cys12 pointing out, imposes a buried Cu(I) position, whereas the "in" conformation with Cys12 pointing inwards results in a more exposed Cu(I) thus, available for transfer. Using QM/MM methods along with thermodynamic cycles these two conformations are shown to exhibit different coordination preference, suggesting that the protein has evolved to have a unique Cu(I) protection mechanism. It is proposed that the "out" conformation with a preference to dicoordination prevents Cu(I) interaction with external ligands and/or Cu(I) release to the solvent, whereas the "in" conformation with preference to tricoordinated Cu(I), facilitates Cu(I) transfer to target proteins, where additional ligands are involved.

Predicting the coordination number within copper chaperones: Atox1 as case study.

The concentration of copper ions in biological systems is tightly regulated by metallochaperone proteins which are responsible for Cu(I) delivery to designated locations in the cell. These proteins contain a unique motif (MXCXXC) that binds Cu(I) very tightly and specifically but at the same time allows efficient metal transfer to target proteins that often contain a similar copper binding motif. It was found that binding to Cu(I) is achieved through the two cysteine residues in a low coordination number of 2-3 due to possible binding of a third external ligand. Understanding copper transport requires better understanding of copper coordination. Here we therefore focused on establishing a computational method that can predict the coordination number of copper in copper chaperones. The method is shown to be successful in predicting the coordination of Cu(I) within the human copper chaperone (Atox1). Based on the results, a possible rationale for this unique Cu(I) dicoordination in Atox1 is suggested.

Calculation of transition dipole moment in fluorescent proteins--towards efficient energy transfer.

Förster Resonance Energy Transfer (FRET) between fluorescent proteins (FPs) is widely used to construct fluorescent sensor proteins, to study intracellular protein-protein interactions and to monitor conformational changes in multidomain proteins. Although FRET depends strongly on the orientation of the transition dipole moments (TDMs) of the donor and acceptor fluorophores, this orientation dependence is currently not taken into account in FRET sensor design. Similarly, studies that use FRET to derive structural constrains typically assume a κ(2) of 2/3 or use the TDM of green fluorescent protein, as this is the only FP for which the TDM has been determined experimentally. Here we used time-dependent density functional theory (TD-DFT) methods to calculate the TDM for a comprehensive list of commonly used fluorescent proteins. The method was validated against higher levels of calculation. Validation with model compounds and the experimentally determined TDM of GFP shows that the TDM is mostly determined by the structure of the π-conjugated fluorophore and is insensitive to non-conjugated side chains or the protein surrounding. Our calculations not only provide TDM for most of the currently used FPs, but also suggest an empirical rule that can be used to obtain the TDMs for newly developed fluorescent proteins in the future.

Can DFT methods correctly and efficiently predict the coordination number of copper(I) complexes? A case study.

The coordination number of various experimentally known Cu(I) compounds is studied using density functional theory. Various basis sets are tested, aiming to establish a reliable level for prediction of the coordination number of these and other Cu(I) complexes. It is found that most levels exhibit correct trends, namely, the bulkier ligands demonstrate larger preference for coordination of two ligands. Proper absolute values are obtained when dispersion corrections are also included in the calculations. It is concluded that the fairly small modified 6-31+G* basis set due to Pulay represents a good compromise between accuracy and efficiency, followed by Balabanov and Peterson's all-electron aug-cc-pVDZ basis set. The overall energy is decomposed into various components whose relative contribution to the overall tendency of forming a complex with a particular coordination is examined. It is shown that two opposing contributions play a major role: the interaction energy of the ligand being added and the deformation energy of the copper's coordination sphere prior to the ligand addition. The former being a stabilizing contribution, leads to higher coordination numbers while the later, a destabilizing contribution, is shown to favor lower coordination numbers.

New organic noble gas molecules: energetics, stability and potential energy surfaces of HCCXeCCH and HCCKrCCH.

The diacetylides of Xe and Kr, HCCXeCCH and HCCKrCCH, are predicted to exist as metastable, chemically-bound compounds. The electronic structure, properties of the potential energy surfaces and decomposition paths are computed and analyzed. MP2, MCSCF and CASPT2 ab initio methods are used, as appropriate for each of the properties studied. Using transition state theory and the computed barrier for decomposition, the lifetime of HCCXeCCH is calculated as a function of temperature. The molecule is predicted to be stable well above the cryogenic range, with a lifetime of 24 h at 200 K. The implications for organic chemistry of the noble gases are discussed.