Stabilization of the Reductase Domain in the Catalytically Self‐Sufficient Cytochrome P450BM3 by Consensus‐Guided Mutagenesis

The multidomain, catalytically self‐sufficient cytochrome P450 BM‐3 from Bacillus megaterium (P450_BM3) constitutes a versatile enzyme for the oxyfunctionalization of organic molecules and natural products. However, the limited stability of the diflavin reductase domain limits the utility of this enzyme for synthetic applications. In this work, a consensus‐guided mutagenesis approach was applied to enhance the thermal stability of the reductase domain of P450_BM3. Upon phylogenetic analysis of a set of distantly related P450s (>38 % identity), a total of 14 amino acid substitutions were identified and evaluated in terms of their stabilizing effects relative to the wild‐type reductase domain. Recombination of the six most stabilizing mutations generated two thermostable variants featuring up to tenfold longer half‐lives at 50 °C and increased catalytic performance at elevated temperatures. Further characterization of the engineered P450_BM3 variants indicated that the introduced mutations increased the thermal stability of the FAD‐binding domain and that the optimal temperature (T_opt) of the enzyme had shifted from 25 to 40 °C. This work demonstrates the effectiveness of consensus mutagenesis for enhancing the stability of the reductase component of a multidomain P450. The stabilized P450_BM3 variants developed here could potentially provide more robust scaffolds for the engineering of oxidation biocatalysts.     

Biocatalytic Synthesis of Nitriles through Dehydration of Aldoximes: The Substrate Scope of Aldoxime Dehydratases

Nitriles, which are mostly needed and produced by the chemical industry, play a major role in various industry segments, ranging from high‐volume, low‐price sectors, such as polymers, to low‐volume, high‐price sectors, such as chiral pharma drugs. A common industrial technology for nitrile production is ammoxidation as a gas‐phase reaction at high temperature. Further popular approaches are substitution or addition reactions with hydrogen cyanide or derivatives thereof. A major drawback, however, is the very high toxicity of cyanide. Recently, as a synthetic alternative, a novel enzymatic approach towards nitriles has been developed with aldoxime dehydratases, which are capable of converting an aldoxime in one step through dehydration into nitriles. Because the aldoxime substrates are easily accessible, this route is of high interest for synthetic purposes. However, whenever a novel method is developed for organic synthesis, it raises the question of substrate scope as one of the key criteria for application as a “synthetic platform technology”. Thus, the scope of this review is to give an overview of the current state of the substrate scope of this enzymatic method for synthesizing nitriles with aldoxime dehydratases. As a recently emerging enzyme class, a range of substrates has already been studied so far, comprising nonchiral and chiral aldoximes. This enzyme class of aldoxime dehydratases shows a broad substrate tolerance and accepts aliphatic and aromatic aldoximes, as well as arylaliphatic aldoximes. Furthermore, aldoximes with a stereogenic center are also recognized and high enantioselectivities are found for 2‐arylpropylaldoximes, in particular. It is further noteworthy that the enantiopreference depends on the E and Z isomers. Thus, opposite enantiomers are accessible from the same racemic aldehyde and the same enzyme.     

Strictly Conserved Residues in Euphorbia tirucalli β‐Amyrin Cyclase: Trp612 Stabilizes Transient Cation through Cation–π Interaction and CH–π Interaction of Tyr736 with Leu734 Confers Robust Local Protein Architecture

The functions of Trp612, Leu734, and Tyr736 of Euphorbia tirucalli β‐amyrin synthase were examined. The aliphatic variants (Ala, Val, Met) of Trp612 showed almost no activity, but the aromatic variants exhibited high activities: 12.5 % of the wild‐type activity for the W612H variant, 43 % for W612F, and 63 % for W612Y. That is, the enzymatic activities of the variants increased in proportion to the increase in π‐electron density. Thus, the major function of Trp612 is to stabilize transient cations through a cation–π interaction. The Phe and Tyr variants caused a distorted folding conformation, especially at the E‐ring site, which generated the aberrantly cyclized products germanicol and lupeol. The L734G and L734A variants exhibited significantly decreased activities but yielded taraxerol in a high production ratio. The Val, Ile, and Met variants showed markedly high activities (56–78 % of wild‐type activity); therefore, appropriate steric bulk is required at this position. The aliphatic variants of Tyr736 showed markedly decreased activities, but the Phe mutant exhibited high activity (67 %), which indicates that the π electrons are critical for catalysis. Homology modeling indicated that Tyr736 and Leu734 are perpendicular to the substrate and are situated face to face, which suggests that a CH–π interaction occurs between Tyr736 and Leu734, reinforcing the protein architecture, and that Tyr736 cannot stabilize cationic intermediates through a cation–π interaction.     

Peptide‐Based Fluorescent Probes for Deacetylase and Decrotonylase Activity: Toward a General Platform for Real‐Time Detection of Lysine Deacylation

Histone deacetylases regulate the acetylation levels of numerous proteins and play key roles in physiological processes and disease states. In addition to acetyl groups, deacetylases can remove other acyl modifications on lysines, the roles and regulation of which are far less understood. A peptide‐based fluorescent probe for single‐reagent, real‐time detection of deacetylase activity that can be readily adapted for probing broader lysine deacylation, including decrotonylation, is reported. Following cleavage of the lysine modification, the probe undergoes rapid intramolecular imine formation that results in marked optical changes, thus enabling convenient detection of deacylase activity with good statistical Z′ factors for both absorption and fluorescence modalities. The peptide‐based design offers broader isozyme scope than that of small‐molecule analogues, and is suitable for probing both metal‐ and nicotinamide adenine dinucleotide (NAD⁺)‐dependent deacetylases. With an effective sirtuin activity assay in hand, it is demonstrated that iron chelation by Sirtinol, a commonly employed sirtuin inhibitor, results in an enhancement in the inhibitory activity of the compound that may affect its performance in vivo.     

Discovery of a Short‐Chain Dehydrogenase from Catharanthus roseus that Produces a New Monoterpene Indole Alkaloid

Plant monoterpene indole alkaloids, a large class of natural products, derive from the biosynthetic intermediate strictosidine aglycone. Strictosidine aglycone, which can exist as a variety of isomers, can be reduced to form numerous different structures. We have discovered a short‐chain alcohol dehydrogenase (SDR) from plant producers of monoterpene indole alkaloids (Catharanthus roseus and Rauvolfia serpentina) that reduce strictosidine aglycone and produce an alkaloid that does not correspond to any previously reported compound. Here we report the structural characterization of this product, which we have named vitrosamine, as well as the crystal structure of the SDR. This discovery highlights the structural versatility of the strictosidine aglycone biosynthetic intermediate and expands the range of enzymatic reactions that SDRs can catalyse. This discovery further highlights how a sequence‐based gene mining discovery approach in plants can reveal cryptic chemistry that would not be uncovered by classical natural product chemistry approaches.     

Hedgehog Antagonist Pyrimidine–Indole Hybrid Molecule Inhibits Ciliogenesis through Microtubule Destabilisation

One of the crucial regulators of embryonic patterning and tissue development is the Hedgehog‐glioma (Hh‐Gli) signalling pathway; its uncontrolled activation has been implicated in different types of cancer in adult tissues. Primary cilium is one of the important factors required for the activation of Hh signalling, as it brings the critical components together for key protein–protein interactions required for Hh pathway regulation. Most of the synthetic and natural small molecule modulators of the pathway primarily antagonise Smoothened (Smo) or other effectors like Hh ligand or Gli. Here, we report a previously described Hh antagonist, with a pyrimidine–indole hybrid (PIH) core structure, as an inhibitor of ciliogenesis. The compound is unique in its mode of action, as it shows perturbation of microtubule dynamics in both cell‐based assays and in vivo systems (zebrafish embryos). Further studies revealed that the probable targets are α‐tubulin and its acetylated form, found in the cytoplasm and primary cilia. PIH also showed axonal defasiculation in developing zebrafish embryos. We thus propose that PIH antagonises Hh signalling by repressing cilia biogenesis and disassembling α‐tubulin from its stabilised form.     

Lysine‐241 Has a Role in Coupling 2OG Turnover with Substrate Oxidation During KDM4‐Catalysed Histone Demethylation

The JmjC histone lysyl demethylases (KDMs) play important roles in modulating histone methylation states and have the potential to be regulated by oxygen availability. Lys241 of the KDM4 subfamily is proposed to be important in oxygen binding by KDM4A. We report evidence that, although Lys241 is unlikely to be directly involved in oxygen binding, it has an important role in coupling 2‐oxoglutarate cosubstrate oxidation with lysine demethylase activity. The results suggest that compounds promoting the uncoupling of substrate oxidation are of interest as JmjC‐KDM inhibitors.     

Transition Metal Ion–Mediated Tyrosine‐Based Short‐Peptide Amphiphile Nanostructures Inhibit Bacterial Growth

We report the design and synthesis of a biocompatible small‐peptide‐based compound for the controlled and targeted delivery of encapsulated bioactive metal ions through transformation of the internal nanostructures of its complexes. A tyrosine‐based short‐peptide amphiphile (sPA) was synthesized and observed to self‐assemble into β‐sheet‐like secondary structures. The self‐assembly of the designed sPA was modulated by application of different bioactive transition‐metal ions, as was confirmed by spectroscopic and microscopic techniques. These bioactive metal‐ion‐conjugated sPA hybrid structures were further used to develop antibacterial materials. As a result of the excellent antibacterial activity of zinc ions the growth of clinically relevant bacteria such as Escherichia coli was inhibited in the presence of zinc ? sPA conjugate. Bacterial testing demonstrated that, due to high biocompatibility with bacterial cells, the designed sPA acted as a metal ion delivery agent and might therefore show great potential in locally addressing bacterial infections.     

Bacterial Catabolism of Biphenyls: Synthesis and Evaluation of Analogues

A series of alkylated 2,3‐dihydroxybiphenyls has been prepared on the gram scale by using an effective Directed ortho Metalation–Suzuki–Miyaura cross‐coupling strategy. These compounds have been used to investigate the substrate specificity of the meta‐cleavage dioxygenase BphC, a key enzyme in the microbial catabolism of biphenyl. Isolation and characterization of the meta‐cleavage products will allow further study of related processes, including the catabolism of lignin‐derived biphenyls.     

Coordination‐Assisted Bioorthogonal Chemistry: Orthogonal Tetrazine Ligation with Vinylboronic Acid and a Strained Alkene

Bioorthogonal chemistry can be used for the selective modification of biomolecules without interfering with any other functionality that might be present. Recent developments in the field include orthogonal bioorthogonal reactions to modify multiple biomolecules simultaneously. During our research, we observed that the reaction rates for the bioorthogonal inverse‐electron‐demand Diels–Alder (iEDDA) reactions between nonstrained vinylboronic acids (VBAs) and dipyridyl‐s‐tetrazines were exceptionally higher than those between VBAs and tetrazines bearing a methyl or phenyl substituent. As VBAs are mild Lewis acids, we hypothesised that coordination of the pyridyl nitrogen atom to the boronic acid promoted tetrazine ligation. Herein, we explore the molecular basis and scope of VBA–tetrazine ligation in more detail and benefit from its unique reactivity in the simultaneous orthogonal tetrazine labelling of two proteins modified with VBA and norbornene, a widely used strained alkene. We further show that the two orthogonal iEDDA reactions can be performed in living cells by labelling the proteasome by using a nonselective probe equipped with a VBA and a subunit‐selective VBA bearing a norbornene moiety.