Discovery and biocatalytic characterization of opine dehydrogenases by metagenome mining.

Enzymatic processes play an increasing role in synthetic organic chemistry which requires the access to a broad and diverse set of enzymes. Metagenome mining is a valuable and efficient way to discover novel enzymes with unique properties for biotechnological applications. Here, we report the discovery and biocatalytic characterization of six novel metagenomic opine dehydrogenases from a hot spring environment (mODHs) (EC 1.5.1.X). These enzymes catalyze the asymmetric reductive amination between an amino acid and a keto acid resulting in opines which have defined biochemical roles and represent promising building blocks for pharmaceutical applications. The newly identified enzymes exhibit unique substrate specificity and higher thermostability compared to known examples. The feature that they preferably utilize negatively charged polar amino acids is so far unprecedented for opine dehydrogenases. We have identified two spatially correlated positions in their active sites that govern this substrate specificity and demonstrated a switch of substrate preference by site-directed mutagenesis. While they still suffer from a relatively narrow substrate scope, their enhanced thermostability and the orthogonality of their substrate preference make them a valuable addition to the toolbox of enzymes for reductive aminations. Importantly, enzymatic reductive aminations with highly polar amines are very rare in the literature. Thus, the preparative-scale enzymatic production, purification, and characterization of three highly functionalized chiral secondary amines lend a special significance to our work in filling this gap. KEY POINTS: • Six new opine dehydrogenases have been discovered from a hot spring metagenome • The newly identified enzymes display a unique substrate scope • Substrate specificity is governed by two correlated active-site residues.

Gluing GAP to RAS Mutants: A New Approach to an Old Problem in Cancer Drug Development.

Mutated genes may lead to cancer development in numerous tissues. While more than 600 cancer-causing genes are known today, some of the most widespread mutations are connected to the RAS gene; RAS mutations are found in approximately 25% of all human tumors. Specifically, KRAS mutations are involved in the three most lethal cancers in the U.S., namely pancreatic ductal adenocarcinoma, colorectal adenocarcinoma, and lung adenocarcinoma. These cancers are among the most difficult to treat, and they are frequently excluded from chemotherapeutic attacks as hopeless cases. The mutated KRAS proteins have specific three-dimensional conformations, which perturb functional interaction with the GAP protein on the GAP-RAS complex surface, leading to a signaling cascade and uncontrolled cell growth. Here, we describe a gluing docking method for finding small molecules that bind to both the GAP and the mutated KRAS molecules. These small molecules glue together the GAP and the mutated KRAS molecules and may serve as new cancer drugs for the most lethal, most difficult-to-treat, carcinomas. As a proof of concept, we identify two new, drug-like small molecules with the new method; these compounds specifically inhibit the growth of the PANC-1 cell line with KRAS mutation G12D in vitro and in vivo. Importantly, the two new compounds show significantly lower IC50 and higher specificity against the G12D KRAS mutant human pancreatic cancer cell line PANC-1, as compared to the recently described selective G12D KRAS inhibitor MRTX-1133.

Mechanism-Based Redesign of GAP to Activate Oncogenic Ras.

Ras GTPases play a crucial role in cell signaling pathways. Mutations of the Ras gene occur in about one third of cancerous cell lines and are often associated with detrimental clinical prognosis. Hot spot residues Gly12, Gly13, and Gln61 cover 97% of oncogenic mutations, which impair the enzymatic activity in Ras. Using QM/MM free energy calculations, we present a two-step mechanism for the GTP hydrolysis catalyzed by the wild-type Ras.GAP complex. We found that the deprotonation of the catalytic water takes place via the Gln61 as a transient Brønsted base. We also determined the reaction profiles for key oncogenic Ras mutants G12D and G12C using QM/MM minimizations, matching the experimentally observed loss of catalytic activity, thereby validating our reaction mechanism. Using the optimized reaction paths, we devised a fast and accurate procedure to design GAP mutants that activate G12D Ras. We replaced GAP residues near the active site and determined the activation barrier for 190 single mutants. We furthermore built a machine learning for ultrafast screening, by fast prediction of the barrier heights, tested both on the single and double mutations. This work demonstrates that fast and accurate screening can be accomplished via QM/MM reaction path optimizations to design protein sequences with increased catalytic activity. Several GAP mutations are predicted to re-enable catalysis in oncogenic G12D, offering a promising avenue to overcome aberrant Ras-driven signal transduction by activating enzymatic activity instead of inhibition. The outlined computational screening protocol is readily applicable for designing ligands and cofactors analogously.

Opine dehydrogenases, an underexplored enzyme family for the enzymatic synthesis of chiral amines.

Opines and opine-type chemicals are valuable natural products with diverse biochemical roles, and potential synthetic building blocks of bioactive compounds. Their synthesis involves reductive amination of ketoacids with amino acids. This transformation has high synthetic potential in producing enantiopure secondary amines. Nature has evolved opine dehydrogenases for this chemistry. To date, only one enzyme has been used as biocatalyst, however, analysis of the available sequence space suggests more enzymes to be exploited in synthetic organic chemistry. This review summarizes the current knowledge of this underexplored enzyme class, highlights key molecular, structural, and catalytic features with the aim to provide a comprehensive general description of opine dehydrogenases, thereby supporting future enzyme discovery and protein engineering studies.

Immobilization of the Aspartate Ammonia-Lyase from Pseudomonas fluorescens R124 on Magnetic Nanoparticles: Characterization and Kinetics.

Aspartate ammonia-lyases (AALs) catalyze the non-oxidative elimination of ammonia from l-aspartate to give fumarate and ammonia. In this work the AAL coding gene from Pseudomonas fluorescens R124 was identified, isolated, and cloned into the pET-15b expression vector and expressed in E. coli. The purified enzyme (PfAAL) showed optimal activity at pH 8.8, Michaelis-Menten kinetics in the ammonia elimination from l-aspartate, and no strong dependence on divalent metal ions for its activity. The purified PfAAL was covalently immobilized on epoxy-functionalized magnetic nanoparticles (MNP), and effective kinetics of the immobilized PfAAL-MNP was compared to the native solution form. Glycerol addition significantly enhanced the storability of PfAAL-MNP. Inhibiting effect of the growing viscosity (modulated by addition of glycerol or glucose) on the enzymatic activity was observed for the native and immobilized form of PfAAL, as previously described for other free enzymes. The storage stability and recyclability of PfAAL-MNP is promising for further biocatalytic applications.

Primary Founder Mutations in the PRKDC Gene Increase Tumor Mutation Load in Colorectal Cancer.

The clonal composition of a malignant tumor strongly depends on cellular dynamics influenced by the asynchronized loss of DNA repair mechanisms. Here, our aim was to identify founder mutations leading to subsequent boosts in mutation load. The overall mutation burden in 591 colorectal cancer tumors was analyzed, including the mutation status of DNA-repair genes. The number of mutations was first determined across all patients and the proportion of genes having mutation in each percentile was ranked. Early mutations in DNA repair genes preceding a mutational expansion were designated as founder mutations. Survival analysis for gene expression was performed using microarray data with available relapse-free survival. Of the 180 genes involved in DNA repair, the top five founder mutations were in PRKDC (n = 31), ATM (n = 26), POLE (n = 18), SRCAP (n = 18), and BRCA2 (n = 15). PRKDC expression was 6.4-fold higher in tumors compared to normal samples, and higher expression led to longer relapse-free survival in 1211 patients (HR = 0.72, p = 4.4 × 10-3). In an experimental setting, the mutational load resulting from UV radiation combined with inhibition of PRKDC was analyzed. Upon treatments, the mutational load exposed a significant two-fold increase. Our results suggest PRKDC as a new key gene driving tumor heterogeneity.

Structure-based inhibitor design of mutant RAS proteins-a paradigm shift.

As a member of small GTPase family, KRAS protein is a key physiological modulator of various cellular activities including proliferation. However, mutations of KRAS present in numerous cancer types, most frequently in pancreatic (> 60%), colorectal (> 40%), and lung cancers, drive oncogenic processes through overactivation of proliferation. The G12C mutation of KRAS protein is especially abundant in the case of these types of malignancies. Despite its key importance in human disease, KRAS was assumed to be non-druggable for a long time since the protein seemingly lacks potential drug-binding pockets except the nucleotide-binding site, which is difficult to be targeted due to the high affinity of KRAS for both GDP and GTP. Recently, a new approach broke the ice and provided evidence that upon covalent targeting of the G12C mutant KRAS, a highly dynamic pocket was revealed. This novel targeting is especially important since it serves with an inherent solution for drug selectivity. Based on these results, various structure-based drug design projects have been launched to develop selective KRAS mutant inhibitors. In addition to the covalent modification strategy mostly applicable for G12C mutation, different innovative solutions have been suggested for the other frequently occurring oncogenic G12 mutants. Here we summarize the latest advances of this field, provide perspectives for novel approaches, and highlight the special properties of KRAS, which might issue some new challenges.

Assessment of Tractable Cysteines for Covalent Targeting by Screening Covalent Fragments.

Targeted covalent inhibition and the use of irreversible chemical probes are important strategies in chemical biology and drug discovery. To date, the availability and reactivity of cysteine residues amenable for covalent targeting have been evaluated by proteomic and computational tools. Herein, we present a toolbox of fragments containing a 3,5-bis(trifluoromethyl)phenyl core that was equipped with chemically diverse electrophilic warheads showing a range of reactivities. We characterized the library members for their reactivity, aqueous stability and specificity for nucleophilic amino acids. By screening this library against a set of enzymes amenable for covalent inhibition, we showed that this approach experimentally characterized the accessibility and reactivity of targeted cysteines. Interesting covalent fragment hits were obtained for all investigated cysteine-containing enzymes.

Detection of Genomic Uracil Patterns.

The appearance of uracil in the deoxyuridine moiety of DNA is among the most frequently occurring genomic modifications. Three different routes can result in genomic uracil, two of which do not require specific enzymes: spontaneous cytosine deamination due to the inherent chemical reactivity of living cells, and thymine-replacing incorporation upon nucleotide pool imbalances. There is also an enzymatic pathway of cytosine deamination with multiple DNA (cytosine) deaminases involved in this process. In order to describe potential roles of genomic uracil, it is of key importance to utilize efficient uracil-DNA detection methods. In this review, we provide a comprehensive and critical assessment of currently available uracil detection methods with special focus on genome-wide mapping solutions. Recent developments in PCR-based and in situ detection as well as the quantitation of genomic uracil are also discussed.

What's in a name? From "fluctuation fit" to "conformational selection": rediscovery of a concept.

Rediscoveries are not rare in biology. A recent example is the re-birth of the "fluctuation fit" concept developed by F. B. Straub and G. Szabolcsi in the sixties of the last century, under various names, the most popular of which is the "conformational selection". This theory offers an alternative to the "induced fit" concept by Koshland for the interpretation of the mechanism of protein-ligand interactions. A central question is whether the ligand induces a conformational change (as described by the induced fit model) or rather selects and stabilizes a complementary conformation from a pre-existing equilibrium of various states of the protein (according to the fluctuation fit/conformational selection model). Straub and Szabolcsi's role and the factors hindering the spread of the fluctuation fit theory are discussed in the context of the history of the Hungarian biology in the 1950s and 1960s.

Structural insights into the tyrosine phosphorylation-mediated inhibition of SH3 domain-ligand interactions.

Src homology 3 (SH3) domains bind proline-rich linear motifs in eukaryotes. By mediating inter- and intramolecular interactions, they regulate the functions of many proteins involved in a wide variety of signal transduction pathways. Phosphorylation at different tyrosine residues in SH3 domains has been reported previously. In several cases, the functional consequences have also been investigated. However, a full understanding of the effects of tyrosine phosphorylation on the ligand interactions and cellular functions of SH3 domains requires detailed structural, atomic-resolution studies along with biochemical and biophysical analyses. Here, we present the first crystal structures of tyrosine-phosphorylated human SH3 domains derived from the Abelson-family kinases ABL1 and ABL2 at 1.6 and 1.4 Å resolutions, respectively. The structures revealed that simultaneous phosphorylation of Tyr89 and Tyr134 in ABL1 or the homologous residues Tyr116 and Tyr161 in ABL2 induces only minor structural perturbations. Instead, the phosphate groups sterically blocked the ligand-binding grooves, thereby strongly inhibiting the interaction with proline-rich peptide ligands. Although some crystal contact surfaces involving phosphotyrosines suggested the possibility of tyrosine phosphorylation-induced dimerization, we excluded this possibility by using small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and NMR relaxation analyses. Extensive analysis of relevant databases and literature revealed not only that the residues phosphorylated in our model systems are well-conserved in other human SH3 domains, but that the corresponding tyrosines are known phosphorylation sites in vivo in many cases. We conclude that tyrosine phosphorylation might be a mechanism involved in the regulation of the human SH3 interactome.

p53 controls expression of the DNA deaminase APOBEC3B to limit its potential mutagenic activity in cancer cells.

Cancer genome sequencing has implicated the cytosine deaminase activity of apolipoprotein B mRNA editing enzyme catalytic polypeptide-like (APOBEC) genes as an important source of mutations in diverse cancers, with APOBEC3B (A3B) expression especially correlated with such cancer mutations. To better understand the processes directing A3B over-expression in cancer, and possible therapeutic avenues for targeting A3B, we have investigated the regulation of A3B gene expression. Here, we show that A3B expression is inversely related to p53 status in different cancer types and demonstrate that this is due to a direct and pivotal role for p53 in repressing A3B expression. This occurs through the induction of p21 (CDKN1A) and the recruitment of the repressive DREAM complex to the A3B gene promoter, such that loss of p53 through mutation, or human papilloma virus-mediated inhibition, prevents recruitment of the complex, thereby causing elevated A3B expression and cytosine deaminase activity in cancer cells. As p53 is frequently mutated in cancer, our findings provide a mechanism by which p53 loss can promote cancer mutagenesis.

The Fourth Wave of Biocatalysis Emerges- The 13 th International Symposium on Biocatalysis and Biotransformations.

Ride the wave! Biocatalysis uses nature's catalysts, enzymes and whole cell systems, for synthetic purposes. In a biotransformation, the biocatalyst transforms a well-defined substrate to the desired product, in contrast to the fermentation process, which produces the desired product from a complex mixture of nutrients. Biocatalysis has reached an industrially established level through several waves of technological evolution; participants of the BioTrans 2017 conference in Budapest could witness the newest wave of this technology.

Detection of uracil within DNA using a sensitive labeling method for in vitro and cellular applications.

The role of uracil in genomic DNA has been recently re-evaluated. It is now widely accepted to be a physiologically important DNA element in diverse systems from specific phages to antibody maturation and Drosophila development. Further relevant investigations would largely benefit from a novel reliable and fast method to gain quantitative and qualitative information on uracil levels in DNA both in vitro and in situ, especially since current techniques does not allow in situ cellular detection. Here, starting from a catalytically inactive uracil-DNA glycosylase protein, we have designed several uracil sensor fusion proteins. The designed constructs can be applied as molecular recognition tools that can be detected with conventional antibodies in dot-blot applications and may also serve as in situ uracil-DNA sensors in cellular techniques. Our method is verified on numerous prokaryotic and eukaryotic cellular systems. The method is easy to use and can be applied in a high-throughput manner. It does not require expensive equipment or complex know-how, facilitating its easy implementation in any basic molecular biology laboratory. Elevated genomic uracil levels from cells of diverse genetic backgrounds and/or treated with different drugs can be demonstrated also in situ, within the cell.

A Hidden Active Site in the Potential Drug Target Mycobacterium tuberculosis dUTPase Is Accessible through Small Amplitude Protein Conformational Changes.

dUTPases catalyze the hydrolysis of dUTP into dUMP and pyrophosphate to maintain the proper nucleotide pool for DNA metabolism. Recent evidence suggests that dUTPases may also represent a selective drug target in mycobacteria because of the crucial role of these enzymes in maintaining DNA integrity. Nucleotide-hydrolyzing enzymes typically harbor a buried ligand-binding pocket at interdomain or intersubunit clefts, facilitating proper solvent shielding for the catalyzed reaction. The mechanism by which substrate binds this hidden pocket and product is released in dUTPases is unresolved because of conflicting crystallographic and spectroscopic data. We sought to resolve this conflict by using a combination of random acceleration molecular dynamics (RAMD) methodology and structural and biochemical methods to study the dUTPase from Mycobacterium tuberculosis In particular, the RAMD approach used in this study provided invaluable insights into the nucleotide dissociation process that reconciles all previous experimental observations. Specifically, our data suggest that nucleotide binding takes place as a small stretch of amino acids transiently slides away and partially uncovers the active site. The in silico data further revealed a new dUTPase conformation on the pathway to a relatively open active site. To probe this model, we developed the Trp21 reporter and collected crystallographic, spectroscopic, and kinetic data that confirmed the interaction of Trp21 with the active site shielding C-terminal arm, suggesting that the RAMD method is effective. In summary, our computational simulations and spectroscopic results support the idea that small loop movements in dUTPase allow the shuttlingof the nucleotides between the binding pocket and the solvent.

Elevated APOBEC3B expression drives a kataegic-like mutation signature and replication stress-related therapeutic vulnerabilities in p53-defective cells.

BACKGROUND: Elevated APOBEC3B expression in tumours correlates with a kataegic pattern of localised hypermutation. We assessed the cellular phenotypes associated with high-level APOBEC3B expression and the influence of p53 status on these phenotypes using an isogenic system. METHODS: We used RNA interference of p53 in cells with inducible APOBEC3B and assessed DNA damage response (DDR) biomarkers. The mutational effects of APOBEC3B were assessed using whole-genome sequencing. In vitro small-molecule inhibitor sensitivity profiling was used to identify candidate therapeutic vulnerabilities. RESULTS: Although APOBEC3B expression increased the incorporation of genomic uracil, invoked DDR biomarkers and caused cell cycle arrest, inactivation of p53 circumvented APOBEC3B-induced cell cycle arrest without reversing the increase in genomic uracil or DDR biomarkers. The continued expression of APOBEC3B in p53-defective cells not only caused a kataegic mutational signature but also caused hypersensitivity to small-molecule DDR inhibitors (ATR, CHEK1, CHEK2, PARP, WEE1 inhibitors) as well as cisplatin/ATR inhibitor and ATR/PARP inhibitor combinations. CONCLUSIONS: Although loss of p53 might allow tumour cells to tolerate elevated APOBEC3B expression, continued expression of this enzyme might impart a number of therapeutic vulnerabilities upon tumour cells.

The novel technique of vapor pressure analysis to monitor the enzymatic degradation of PHB by HPLC chromatography.

A novel method was introduced for the quantitative determination of substances in aqueous solutions by using the evaporative light scattering (ELS) detector of a high performance liquid chromatograph (HPLC). The principle of the measurement is the different equilibrium vapor pressure of the solvent and the analyte resulting in decreasing evaporation rate, larger droplets and stronger signal with increasing concentration. The new technique based on vapor pressure analysis was validated with traditional UV-Vis detection carried out with a diode array detector (DAD). The new technique was used for monitoring the concentration of solutions obtained during the enzymatic degradation of poly(3-hydroxybutyrate) yielding the 3-hydroxybutyrate monomer as the product. The accuracy of the measurement allowed the determination of degradation kinetics as well. The results obtained with the two techniques showed excellent agreement at small concentrations. Deviations at larger concentrations were explained with the non-linear correlation between analyte concentration and detector signal and the linear regression used for calibration. Mathematical analysis of the method made possible the determination of the evaporation enthalpy of the analyte as well. The new approach is especially suitable for the quantitative analysis of compounds, which do not absorb in the detection range of the DAD detector or if their characteristic absorbance is close to the lower end of its wavelength range.

A Methylidene Group in the Phosphonic Acid Analogue of Phenylalanine Reverses the Enantiopreference of Binding to Phenylalanine Ammonia-Lyases.

Aromatic amino acid ammonia-lyases and aromatic amino acid 2,3-aminomutases contain the post-translationally formed prosthetic 3,5-dihydro-4-methylidene-5H-imidazol-5-one (MIO) group. MIO enzymes catalyze the stereoselective synthesis of α- or ß-amino acid enantiomers, making these chemical processes environmentally friendly and affordable. Characterization of novel inhibitors enables structural understanding of enzyme mechanism and recognizes promising herbicide candidates as well. The present study found that both enantiomers of the aminophosphonic acid analogue of the natural substrate phenylalanine and a novel derivative bearing a methylidene at the ß-position inhibited phenylalanine ammonia-lyases (PAL), representing MIO enzymes. X-ray methods unambiguously determined the absolute configuration of all tested enantiomers during their synthesis. Enzyme kinetic measurements revealed the enantiomer of the methylidene-substituted substrate analogue as being a mirror image relation to the natural l-phenylalanine as the strongest inhibitor. Isothermal titration calorimetry (ITC) confirmed the binding constants and provided a detailed analysis of the thermodynamic driving forces of ligand binding. Molecular docking suggested that binding of the (R)- and (S)-enantiomers is possible by a mirror image packing.

Perturbation of genome integrity to fight pathogenic microorganisms.

BACKGROUND: Resistance against antibiotics is unfortunately still a major biomedical challenge for a wide range of pathogens responsible for potentially fatal diseases. SCOPE OF REVIEW: In this study, we aim at providing a critical assessment of the recent advances in design and use of drugs targeting genome integrity by perturbation of thymidylate biosynthesis. MAJOR CONCLUSION: We find that research efforts from several independent laboratories resulted in chemically highly distinct classes of inhibitors of key enzymes within the routes of thymidylate biosynthesis. The present article covers numerous studies describing perturbation of this metabolic pathway in some of the most challenging pathogens like Mycobacterium tuberculosis, Plasmodium falciparum, and Staphylococcus aureus. GENERAL SIGNIFICANCE: Our comparative analysis allows a thorough summary of the current approaches to target thymidylate biosynthesis enzymes and also include an outlook suggesting novel ways of inhibitory strategies. This article is part of a Special Issue entitled "Science for Life" Guest Editor: Dr. Austen Angell, Dr. Salvatore Magazù and Dr. Federica Migliardo.

The First Enantioselective Total Synthesis of (-)-trans-Dihydronarciclasine.

A feasible and enantioselective total synthesis of (-)-trans-dihydronarciclasine [(-)-1], a highly biologically active alkaloid, was devised starting from vanillin (8). The key step of this new synthesis was an asymmetric, organocatalytic Michael addition, in which an optically active nitropentanone [(-)-13] was obtained from a butenone derivative (12). Excellent enantioselectivity (>99% ee) was achieved using the (8S,9S)-9-amino(9-deoxy)epiquinine (16) organocatalyst. The target molecule can be prepared in 13 steps from compound (-)-13. The total synthesis has provided a facile and first access to the ent-form of naturally occurring (+)-trans-dihydronarciclasine, a highly potent cytostatic alkaloid.