Main conclusion The DNA-binding protein WHIRLY1, sharing structural similarities with ferritin, plays a role in the formation of iron cofactor proteins within chloroplasts. Abstract Previous studies indicated that barley plants with a knockdown of HvWHIRLY1 containing a minimal amount of the protein are compromised in chloroplast development and photosynthesis, and get chlorotic leaves when grown at high irradiance. Thereby, the leaves display signs of iron deficiency. Metal determination revealed, however, that leaves of WHIRLY1-deficient plants had a regular iron content. Nevertheless, WHIRLY1-deficiency affected the functionality of photosystem II less than that of photosystem I, which has a higher demand for iron. Immunological analyses revealed that components of both photosystems had reduced levels. Additionally, the levels of other chloroplast proteins containing different classes of iron cofactors were lower in the WHIRLY1-deficient plants compared to the wild type. In contrast, the level of the iron sequestering protein ferritin increased in WHIRLY1-deficient lines, whereby high irradiance intensified this effect. RNA analyses showed that the upregulation of ferritin coincided with an enhanced expression of the corresponding gene, reflecting an apparent overload of chloroplasts with free iron. Ferritin and WHIRLY proteins are known to share the same oligomeric structure. Therefore, the high abundance of ferritin in WHIRLY1-deficient plants might be a compensation for the reduced abundance of WHIRLY1. Enhanced expression levels of genes encoding photosynthesis proteins and iron cofactor proteins indicate a demand for protein formation or assembly of protein complexes. The results support a general role of WHIRLY1 in assembly and/or stabilization of chloroplast proteins and, moreover, suggest a specific function in sequestering and supply of iron in chloroplasts.

The new farnesyl pyrophosphate (FPP) derivative with a shifted olefinic double bond from C6‐C7 to C7‐C8 is accepted and converted by the sesquiterpene cyclases protoilludene synthase (Omp7) as well as viridiflorene synthase (Tps32). In both cases, a so far unknown germacrene derivative was found to be formed, which we name “germacrene F”. Both cases are examples in which a modification around the central olefinic double bond in FPP leads to a change in the mode of initial cyclization (from 1→11 to 1→10). For Omp7 a rationale for this behaviour was found by carrying out molecular docking studies. Temperature‐dependent NMR experiments, accompanied by NOE studies, show that germacrene F adopts a preferred mirror‐symmetric conformation with both methyl groups oriented in the same directions in the cyclodecane ring.

Macrocyclization of peptides is typically used to fix specific bioactive conformations and improve their pharmacological properties. Recently, macrobicyclic peptides have received special attention owing to their capacity to mimic protein structures or be key components of peptide-drug conjugates. Here, we describe the development of novel synthetic strategies for two distinctive types of peptide macrobicycles. A multicomponent macrocyclo-dimerization approach is introduced for the production of interconnected β-turns, allowing two macrocyclic rings to be formed and dimerized in one pot. Also, an on-resin double stapling strategy is described for the assembly of lactam-bridged macrobicycles with stable tertiary folds.

Silyl ether protecting groups are important tools in organic synthesis, ensuring selective reactions of hydroxyl functional groups. Enantiospecific formation or cleavage could simultaneously enable the resolution of racemic mixtures and thus significantly increase the efficiency of complex synthetic pathways. Based on reports that lipases, which today are already particularly important tools in chemical synthesis, can catalyze the enantiospecific turnover of trimethylsilanol (TMS)-protected alcohols, the goal of this study was to determine the conditions under which such a catalysis occurs. Through detailed experimental and mechanistic investigation, we demonstrated that although lipases mediate the turnover of TMS-protected alcohols, this occurs independently of the known catalytic triad, as this is unable to stabilize a tetrahedral intermediate. The reaction is essentially non-specific and therefore most likely completely independent of the active site. This rules out lipases as catalysts for the resolution of racemic mixtures alcohols through protection or deprotection with silyl groups.

4-Hydroxyphenylacetate 3-hydroxylase (4HPA3H), a flavin-dependent monooxygenase from E. coli that catalyzes the hydroxylation of monophenols to catechols, was modified by rational re-design to convert also more bulky substrates, especially phenolic natural products like phenylpropanoids, flavones or coumarins. Selected amino acid positions in the binding pocket of 4HPA3H were exchanged by residues from the homologous protein from Pseudomonas aeruginosa, yielding variants with improved conversion of spacious substrates such as the flavonoid naringenin or the alkaloid mimetic 2-hydroxycarbazole. Reactions were followed by an adapted Fe(III)-catechol chromogenic assay selective for the products. Especially substitution of the residue Y301 facilitated modulation of substrate specificity: introduction of non-aromatic but hydrophobic (iso)leucine resulted in the preference of the substrate ferulic acid (having a guaiacyl (guajacyl) moiety, part of the vanilloid motif) over unsubstituted monophenols. The in vivo (whole-cell biocatalysts) and in vitro (three-enzyme cascade) transformations of substrates by 4HPA3H and its optimized variants was strictly regiospecific and proceeded without generation of by-products.

Terpene synthase-mediated biotransformation of eleven synthetic sulfur- or oxygen-containing non-natural prenyl diphosphates resulted in the formation of five novel terpenoids and analogues. Uniquely, they trap intermediate steps and form heterocycles or compounds with alkyne side chains. Computational modelling differentiates convertible from inconvertible substrates and thereby provides an understanding of the detailed molecular mechanism of terpene cyclases. Two terpene cyclases were used as biocatalytic tool, namely, limonene synthase from Cannabis sativa (CLS) and 5-epi-aristolochene synthase (TEAS) from Nicotiana tabacum. They showed significant substrate flexibility towards non-natural prenyl diphosphates to form novel terpenoids, including core oxa- and thia-heterocycles and alkyne-modified terpenoids. We elucidated the structures of five novel monoterpene-analogues and a known sesquiterpene-analogue. These results reflected the terpene synthases′ ability and promiscuity to broaden the pool of terpenoids with structurally complex analogues. Docking studies highlight an on-off conversion of the unnatural substrates.

Type 1 secretion systems (T1SS) have a relatively simple architecture compared to other classes of secretion systems and therefore, are attractive to be optimized by protein engineering. Here, we report a KnowVolution campaign for the hemolysin (Hly) enhancer fragment, an untranslated region upstream of the hlyA gene, of the hemolysin T1SS of Escherichia coli to enhance its secretion efficiency. The best performing variant of the Hly enhancer fragment contained five nucleotide mutations at  five positions (A30U, A36U, A54G, A81U, and A116U) resulted in a 2-fold increase in the secretion level of a model lipase fused to the secretion carrier HlyA1. Computational analysis suggested that altered affinity to the generated enhancer fragment towards the S1 ribosomal protein contributes to the enhanced secretion levels. Furthermore, we demonstrate that involving a native terminator region along with the generated Hly enhancer fragment increased the secretion levels of the Hly system up to 5-fold.

The tree species Eucalyptus camaldulensis shows exceptionally high tolerance against aluminum, a widespread toxic metal in acidic soils. In the roots of E. camaldulensis, aluminum is detoxified via the complexation with oenothein B, a hydrolyzable tannin. In our approach to elucidate the biosynthesis of oenothein B, we here report on the identification of E. camaldulensis enzymes that catalyze the formation of gallate, which is the phenolic constituent of hydrolyzable tannins. By systematical screening of E. camaldulensis dehydroquinate dehydratase/shikimate dehydrogenases (EcDQD/SDHs), we found two enzymes, EcDQD/SDH2 and 3, catalyzing the NADP+-dependent oxidation of 3-dehydroshikimate to produce gallate. Based on extensive in vitro assays using recombinant EcDQD/SDH2 and 3 enzymes, we present for the first time a detailed characterization of the enzymatic gallate formation activity, including the cofactor preferences, pH optima, and kinetic constants. Sequence analyses and structure modeling suggest the gallate formation activity of EcDQD/SDHs is based on the reorientation of 3-dehydroshikimate in the catalytic center, which facilitates the proton abstraction from the C5 position. Additionally, EcDQD/SDH2 and 3 maintain DQD and SDH activities, resulting in a 3-dehydroshikimate supply for gallate formation. In E. camaldulensis, EcDQD/SDH2 and 3 are co-expressed with UGT84A25a/b and UGT84A26a/b involved in hydrolyzable tannin biosynthesis. We further identified EcDQD/SDH1 as a “classical” bifunctional plant shikimate pathway enzyme and EcDQD/SDH4a/b as functional quinate dehydrogenases of the NAD+/NADH-dependent clade. Our data indicate that in E. camaldulensis the enzymes EcDQD/SDH2 and 3 provide the essential gallate for the biosynthesis of the aluminum-detoxifying metabolite oenothein B.

The recently described flavin‐dependent halogenase BrvH is able to catalyze both bromination and chlorination of indole, but shows significantly higher bromination activity. BrvH was annotated as a tryptophan halogenase, but does not accept tryptophan as a substrate. Its native substrate remains unknown. A predictive model with the data available for BrvH was analysed. A training set of compounds tested in vitro was docked into the active site of a complete protein model based on the X‐ray structure of BrvH. The atoms not resolved experimentally have been modelled using molecular mechanics force fields to obtain this protein model. Furthermore, docking poses for the substrates and known non‐substrates have been calculated. Parameters like distance, partial charge, and hybridization state have been analysed to derive rules for prediction of activity. With this model for activity of the BrvH, a virtual screening suggested several structures for potential substrates. Some of the thus preselected compounds were tested in vitro and several could be verified as convertible substrates. Based on information on halogenated natural products, a new dataset was created to specifically search for natural products as substrates/products, and virtual screening in this database yielded further hits.

Human drug‐metabolizing cytochrome P450 monooxygenases (CYPs) have enormous substrate promiscuity; this makes them promising tools for the expansion of natural product diversity. Here, we used CYP3A4 for the targeted diversification of a plant biosynthetic route leading to monoterpenoid indole alkaloids. In silico, in vitro and in planta studies proved that CYP3A4 was able to convert the indole alkaloid vinorine into vomilenine, the former being one of the central intermediates in the ajmaline pathway in the medicinal plant Rauvolfia serpentina (L.) Benth. ex Kurz. However, to a much larger extent, the investigated conversion yielded vinorine (19R ,20R)‐epoxide, a new metabolite with an epoxide functional group that is rare for indole alkaloids. The described work represents a successful example of combinatorial biosynthesis towards an increase in biodiversity of natural metabolites. Moreover, characterisation of the products of the in vitro and in planta transformation of potential pharmaceuticals with human CYPs might be indicative of the route of their conversion in the human organism.