We study the hyperactivation of α‐chymotrypsin (α‐ChT) using the acrylate polymer poly(2‐carboxyethyl) acrylate (PCEA) in comparison to the commonly used poly(acrylic acid) (PAA). The polymers are added during the enzymatic cleavage reaction of the substrate N‐glutaryl‐L‐phenylalanine p‐nitroanilide (GPNA). Enzyme activity assays reveal a pronounced enzyme hyperactivation capacity of PCEA, which reaches up to 950% activity enhancement, and is significantly superior to PAA (revealing an activity enhancement of approx. 450%). In a combined experimental and computational study, we investigate α‐ChT/polymer interactions to elucidate the hyperactivation mechanism of the enzyme. Isothermal titration calorimetry reveals a pronounced complexation between the polymer and the enzyme. Docking simulations reveal that binding of polymers significantly improves the binding affinity of GPNA to α‐ChT. Notably, a higher binding affinity is found for the α‐ChT/PCEA compared to the α‐ChT/PAA complex. Further molecular dynamics (MD) simulations reveal changes in the size of the active site in the enzyme/polymer complexes, with PCEA inducing a more pronounced alteration compared to PAA, facilitating an easier access for the substrate to the active site of α‐ChT.

Introduction Liverworts are a group of non-vascular plants that possess unique metabolism not found in other plants. Many liverwort metabolites have interesting structural and biochemical characteristics, however the fluctuations of these metabolites in response to stressors is largely unknown. Objectives To investigate the metabolic stress-response of the leafy liverwort Radula complanata. Methods Five phytohormones were applied exogenously to in vitro cultured R. complanata and an untargeted metabolomic analysis was conducted. Compound classification and identification was performed with CANOPUS and SIRIUS while statistical analyses including PCA, ANOVA, and variable selection using BORUTA were conducted to identify metabolic shifts.Results It was found that R. complanata was predominantly composed of carboxylic acids and derivatives, followed by benzene and substituted derivatives, fatty acyls, organooxygen compounds, prenol lipids, and flavonoids. The PCA revealed that samples grouped based on the type of hormone applied, and the variable selection using BORUTA (Random Forest) revealed 71 identified and/or classified features that fluctuated with phytohormone application. The stress-response treatments largely reduced the production of the selected primary metabolites while the growth treatments resulted in increased production of these compounds. 4-(3-Methyl-2-butenyl)-5-phenethylbenzene-1,3-diol was identified as a biomarker for the growth treatments while GDP-hexose was identified as a biomarker for the stress-response treatments. Conclusion Exogenous phytohormone application caused clear metabolic shifts in Radula complanata that deviate from the responses of vascular plants. Further identification of the selected metabolite features can reveal metabolic biomarkers unique to liverworts and provide more insight into liverwort stress responses.

Rational re-design of the substrate pocket of phenylpropanoid-flavonoid O-methyltransferase (PFOMT) from Mesembryanthe-mum crystallinum, an enzyme that selectively methylates the 3’-position (= meta-position) in catechol-moieties of flavonoids to guiacol-moieties, provided the basis for the generation of variants with opposite, i. e. 4’- (para-) regioselectivity and enhanced catalytic efficiency. A double variant (Y51R/N202W) identified through a newly developed colorimetric assay efficiently modified the para-position in flavanone and flavano-nol substrates, providing access to the sweetener molecule hesperetin and other rare plant flavonoids having an isovanil-loid motif.

Catalyst discovery and development requires the screening of large reaction sets necessitating analytic methods with the potential for high‐throughput screening. These techniques often suffer from substrate dependency or the requirement of expert knowledge. Chromatographic techniques (GC/LC) can overcome these limitations but are generally hampered by long analysis time or the need for special equipment. The herein developed multiple injections in a single experimental run (MISER) GC‐MS technique allows a substrate independent 96‐well microtiter plate analysis within 60 min. This method can be applied to any laboratory equipped with a standard GC‐MS. With this concept novel, unspecific peroxygenase (UPO) chimeras, could be identified, consisting of subdomains from three different fungal UPO genes. The GC‐technique was additionally applied to evaluate an YfeX library in an E. coli whole‐cell system for the carbene‐transfer reaction on indole, which revealed the thus far unknown axial heme ligand tryptophan.

Bioinspired, synthetic porphyrin complexes are important catalysts in organic synthesis and play a pivotal role in efficient carbene transfer reactions. The advances in this research area stimulated recent, “chemo‐inspired” developments in biocatalysis. Today, both synthetic iron complexes and enzymes play an important role to conduct carbene transfer reactions. The advances and potential developments in both research areas are discussed in this concept article.

IntroductionThe demand to develop efficient and reliable analytical methods for the quality control of nutraceuticals is on the rise, together with an increase in the legal requirements for safe and consistent levels of its active principles.ObjectiveTo establish a reliable model for the quality control of widely used Senna preparations used as laxatives and assess its phyto-equivalency.MethodsA comparative metabolomics approach via NMR and MS analyses was employed for the comprehensive measurement of metabolites and analyzed using chemometrics.ResultsUnder optimized conditions, 30 metabolites were simultaneously identified and quantified including anthraquinones, bianthrones, acetophenones, flavonoid conjugates, naphthalenes, phenolics, and fatty acids. Principal component analysis (PCA) was used to define relative metabolite differences among Senna preparations. Furthermore, quantitative 1H NMR (qHNMR) was employed to assess absolute metabolites levels in preparations. Results revealed that 6-hydroxy musizin or tinnevellin were correlated with active metabolites levels, suggesting the use of either of these naphthalene glycosides as markers for official Senna drugs authentication.ConclusionThis study provides the first comparative metabolomics approach utilizing NMR and UPLC–MS to reveal for secondary metabolite compositional differences in Senna preparations that could readily be applied as a reliable quality control model for its analysis.

IntroductionThe production of marine drugs in its normal habitats is often low and depends greatly on ecological conditions. Chemical synthesis of marine drugs is not economically feasible owing to their complex structures. Biotechnology application via elicitation represents a promising tool to enhance metabolites yield that has yet to be explored in soft corals.ObjectivesStudy the elicitation impact of salicylic acid (SA) and six analogues in addition to a systemic acquired resistance inducer on secondary metabolites accumulation in the soft coral Sarcophyton ehrenbergi along with the symbiont zooxanthellae and if SA elicitation effect is extended to other coral species S. glaucum and Lobophyton pauciliforum.MethodsPost elicitation in the three corals and zooxanthella, metabolites were extracted and analyzed via UHPLC-MS coupled with chemometric tools.ResultsMultivariate data analysis of the UHPLC-MS data set revealed clear segregation of SA, amino-SA, and acetyl-SA elicited samples. An increased level ca. 6- and 8-fold of the diterpenes viz., sarcophytonolide I, sarcophine and a C28-sterol, was observed in SA and amino-SA groups, respectively. Post elicitation, the level of diepoxy-cembratriene increased 1.5-fold and 2.4-fold in 1 mM SA, and acetyl-SA (aspirin) treatment groups, respectively. S. glaucum and Lobophyton pauciliforum showed a 2-fold increase of diepoxy-cembratriene levels.ConclusionThese results suggest that SA could function as a general and somewhat selective diterpene inducing signaling molecule in soft corals. Structural consideration reveals initial structure–activity relationship (SAR) in SA derivatives that seem important for efficient diterpene and sterol elicitation.

Three different reductases have been fused to CYP153 monooxygenase from Marinobacter aquaeolei. The most promising candidate has been analysed in terms of its linker part, which connects the reductase with the haem domain through sequence alignment of the corresponding reductase family CYP116B. To improve the artificial fusion construct, the linker length has been varied, thereby only altering the non‐conserved middle part of the linker. This way seven artificial fusion constructs have been engineered, which varied in linker length between 11 and 32 amino acids (“natural” is 16). These variations showed a substantial impact on the fusion construct. The best mutant, extended by two amino acids, showed an improved activity (67 %), higher stability (67 % more active haem domain after 2 h) and a coupling efficiency of 94 % (55 % higher than before). Presented in this paper is an approach to find and optimise artificial fusion constructs for P450 monooxygenases.

The structure of a P450 ω‐hydroxylase bound to its fatty acid product was determined, which revealed a narrow substrate tunnel that leads to the heme. The introduction of an arginine side chain in proximity to the carboxyl group of the fatty acid led to a reduced KM value for dodecanoic acid, which suggests the importance of an anchoring point in the active site. An increase in the flexibility of the substrate recognition region was also engineered, which resulted in a threefold improved product formation.

The reduction of activated C=C double bonds is an important reaction in synthetic chemistry owing to the potential formation of up to two new stereogenic centers. Artificial nicotinamide cofactors were recently presented as alternative suppliers of hydride equivalents needed for alkene reduction. To study the effect of cofactors on the reduction of activated alkenes, a set of N‐substituted synthetic nicotinamide cofactors with differing oxidation potentials were synthesized and their electrochemical and kinetic behavior was studied. The effects of the synthetic cofactors on enzyme activity of four ene reductases are outlined in this study, where the cofactor mimic with an N‐substituted 4‐hydroxy‐phenyl residue led to a sixfold higher vmax relative to the natural cofactor NADH.