Natural Substrate Research Articles

Characterization and evaluation of photolabile (µ-peroxo)(µ-hydroxo)bis[bis(bipyridyl)cobalt caged oxygen compounds to facilitate time-resolved crystallographic studies of cytochrome c oxidase

Photolabile (µ-peroxo)(µ-hydroxo)bis[bis(bipyridyl)-cobalt-based caged oxygen compounds have been synthesized and characterized by optical absorbance spectroscopy, X-ray crystallography. and the quantum yield and redox stability were investigated. Furthermore, conditions were established where redox incompatibilities encountered between caged oxygen compounds and oxygen-dependant cytochrome c oxidase (CcO) could be circumvented. Herein, we demonstrate that millimolar concentrations of molecular oxygen can be released from a caged oxygen compound with spatio-temporal control upon laser excitation, triggering enzymatic turnover in cytochrome c oxidase. Spectroscopic evidence confirms the attainment of a homogeneous reaction initiation at concentrations and conditions relevant for further crystallography studies. This was demonstrated by the oxidizing microcrystals of reduced CcO by liberation of millimolar concentrations of molecular oxygen from a caged oxygen compound. We believe this will expand the scope of available techniques for the detailed investigation of oxygen-dependant enzymes with its native substrate and facilitate further time-resolved X-ray based studies such as wide/small angle X-ray scattering and serial femtosecond crystallography.Graphical abstract

Influence of habitat features on the colonisation of native and non-indigenous species

Marine artificial structures provide substrates on which organisms can settle and grow. These structures facilitate establishment and spread of non-indigenous species, in part due to their distinct physical features (substrate material, movement, orientation) compared to natural habitat analogues such as rocky shores, and because following construction, they have abundant resources (space) for species to colonise. Despite the perceived importance of these habitat features, few studies have directly compared distributions of native and non-indigenous species or considered how functional identity and associated environmental preferences drive associations. We undertook a meta-analysis to investigate whether colonisation of native and non-indigenous species varies between artificial structures with features most closely resembling natural habitats (natural substrates, fixed structures, surfaces oriented upwards) and those least resembling natural habitats (artificial materials, floating structures, downfacing or vertical surfaces), or whether functional identity is the primary driver of differences. Analyses were done at global and more local (SE Australia) scales to investigate if patterns held regardless of scale. Our results suggest that functional group (i.e., algae, ascidians. barnacles, bryozoans, polychaetes) rather than species classification (i.e., native or non-indigenous) are the main drivers of differences in communities between different types of artificial structures. Specifically, there were differences in the abundance of ascidians, barnacles, and polychaetes between (1) upfacing and downfacing/vertical surfaces, and (2) floating and fixed substrates. When differences were detected, taxa were most abundant on features least resembling natural habitats. Results varied between global and SE Australian analyses, potentially due to reduced variability across studies in the SE Australian dataset. Thus, the functional group and associated preferences of the highest threat NIS in the area should be considered in design strategies (e.g., ecological engineering) to limit their establishment on newly built infrastructure.

Enzymatic one-step synthesis of natural 2-pyrones and new-to-nature derivatives from coenzyme A esters

The 2-pyrone moiety is present in a wide range of structurally diverse natural products with various biological activities. The plant biosynthetic routes towards these compounds mainly depend on the activity of either type III polyketide synthase-like 2-pyrone synthases or hydroxylating 2-oxoglutarate dependent dioxygenases. In the present study, the substrate specificity of these enzymes is investigated by a systematic screening using both natural and artificial substrates with the aims of efficiently forming (new) products and understanding the underlying catalytic mechanisms. In this framework, we focused on the in vitro functional characterization of a 2-pyrone synthase Gh2PS2 from Gerbera x hybrida and two dioxygenases AtF6’H1 and AtF6’H2 from Arabidopsis thaliana using a set of twenty aromatic and aliphatic CoA esters as substrates. UHPLC-ESI-HRMSn based analyses of reaction intermediates and products revealed a broad substrate specificity of the enzymes, enabling the facile "green" synthesis of this important class of natural products and derivatives in a one-step/one-pot reaction in aqueous environment without the need for halogenated or metal reagents and protective groups. Using protein modeling and substrate docking we identified amino acid residues that seem to be important for the observed product scope.

Craspase Orthologs Cleave a Nonconserved Site in Target Protein Csx30.

The Craspase CRISPR-Cas effector consists of the RNA-guided ribonuclease gRAMP and the protease TPR-CHAT, coupling target RNA recognition to protease activation. The natural substrate of Craspase is Csx30, a protein cleaved in two fragments that subsequently activates downstream antiviral pathways. Here, we determined the protease substrate specificity of Craspase from Candidatus "Jettenia caeni" (Jc-Craspase). We find that Jc-Craspase cleaves Jc-Csx30 in a target RNA-dependent fashion in A|S, which is different from the sites found in two other studied Craspases (L|D and M|K for Candidatus "Scalindua brodae" and Desulfonema ishimotonii, respectively). The fact that Craspase cleaves a nonconserved site across orthologs indicates the evolution of specific protein interactions between Craspase and its respective Csx30 target protein. The Craspase family thus represents a panel of proteases with different substrate specificities, which we exploited for the development of a readout for multiplexed RNA detection.

Urban food waste for soil amendment? Analysis and characterisation of waste-based compost for soil fertility management in agroecological horticultural production systems in the city of Rosario, Argentina

Urban and peri-urban lands can be an important source of food production for localised and sustainable food systems, however, their soils can be of poor quality, degraded or damaged by anthropic activities, and little is known about their suitability or safety. This paper aims to contribute to this knowledge gap by assessing the soil remediation capacity and qualities of different types of compost made from urban and peri-urban organic wastes for agroecological food production. Prepared over the course of 2021, and used in 2022 for food growing, five different composts were observed and analysed, in two different farms in the city of Rosario, Argentina. Four raw materials generated largely by local industries were used to make the composts: chicken manure, rumen (cow’s stomachs), brewer’s bagasse (byproducts of the beer industry) and urban leaves collected from the municipality waste collection. These were mixed in different proportions (all reaching the 20–30 C/N ratio, typical of quality compost) to produce viable growing substrates where radishes and lettuces were grown. The aim of the study was to assess the possibility, quality and limitations to use locally available organic inputs for soil fertility management in agroecological farming, in the context of urbanisation and to assess pathways to develop closed-cycle agroecological agriculture at metropolitan level. Natural manure substrates (raw and composted) were analysed, as well as crops grown and fertilised with each of the substrates. The attributes and limiting factors of each substrate and their response to local soil conditions were compared and physicochemical, biochemical, and microbiological analyses were performed, including among others, the study of microbial biomass, biological activity, biophytotoxicity, pH, aerobic heterotrophs, nitrogen fixation, and the presence of antibiotics, agrochemicals and heavy metals. The results of the analyses show that all the composted materials improved the physical, chemical and biological properties. However, in some cases, pollutants were present even after composting. Analysis carried out on the vegetables generally indicate undetectable levels or levels below the admissible limits, demonstrating the filtering capacity of the different composts and the soil.

The DmsABC S-oxide reductase is an essential component of a novel, hypochlorite-inducible system of extracellular stress defense in Haemophilus influenzae.

Defenses against oxidative damage to cell components are essential for survival of bacterial pathogens during infection, and here we have uncovered that the DmsABC S-/N-oxide reductase is essential for virulence and in-host survival of the human-adapted pathogen, Haemophilus influenzae. In several different infection models, H. influenzae ΔdmsA strains showed reduced immunogenicity as well as lower levels of survival in contact with host cells. Expression of DmsABC was induced in the presence of hypochlorite and paraquat, closely linking this enzyme to defense against host-produced antimicrobials. In addition to methionine sulfoxide, DmsABC converted nicotinamide- and pyrimidine-N-oxide, precursors of NAD and pyrimidine for which H. influenzae is an auxotroph, at physiologically relevant concentrations, suggesting that these compounds could be natural substrates for DmsABC. Our data show that DmsABC forms part of a novel, periplasmic system for defense against host-induced S- and N-oxide stress that also comprises the functionally related MtsZ S-oxide reductase and the MsrAB peptide methionine sulfoxide reductase. All three enzymes are induced following exposure of the bacteria to hypochlorite. MsrAB is required for physical resistance to HOCl and protein repair. In contrast, DmsABC was required for intracellular colonization of host cells and, together with MtsZ, contributed to resistance to N-Chlorotaurine. Our work expands and redefines the physiological role of DmsABC and highlights the importance of different types of S-oxide reductases for bacterial virulence.

Chemoenzymatic synthesis of macrocyclic peptides and polyketides via thioesterase-catalyzed macrocyclization.

Chemoenzymatic strategies that combine synthetic and enzymatic transformations offer efficient approaches to yield target molecules, which have been increasingly employed in the synthesis of bioactive natural products. In the biosynthesis of macrocyclic nonribosomal peptides, polyketides, and their hybrids, thioesterase (TE) domains play a significant role in late-stage macrocyclization. These domains can accept mimics of native substrates in vitro and exhibit potential for use in total synthesis. This review summarizes the recent advances of TE domains in the chemoenzymatic synthesis for these natural products that aim to address the common issues in classical synthetic approaches and increase synthetic efficiencies, which have the potential to facilitate further pharmaceutical research.

Reconstructing dynamics correlation network to simultaneously improve activity and stability of 2,3-butanediol dehydrogenase by design of distal interchain disulfide bonds

The complete enzyme catalytic cycle includes substrate binding, chemical reaction and product release, in which different dynamic conformations are adopted. Due to the complex relationship among enzyme activity, stability and dynamics, the directed evolution of enzymes for improved activity or stability commonly leads to a trade-off in stability or activity. It hence remains a challenge to engineer an enzyme to have both enhanced activity and stability. Here, we have attempted to reconstruct the dynamics correlation network involved with active center to improve both activity and stability of a 2,3-butanediol dehydrogenase (2,3-BDH) by introducing inter-chain disulfide bonds. A computational strategy was first applied to evaluate the effect of introducing inter-chain disulfide bond on activity and stability of three 2,3-BDHs, and the N258C mutation of 2,3-BDH from Corynebacterium glutamicum (CgBDH) was proved to be effective in improving both activity and stability. In the results, CgBDH-N258C showed a different unfolding curve from the wild type, with two melting temperatures (Tm) of 68.3 °C and 50.8 °C, 19.7 °C and 2 °C higher than 48.6 °C of the wild type. Its half-life was also improved by 14.8-fold compared to the wild type. Catalytic efficiency (kcat/Km) of the mutant was increased by 7.9-fold toward native substrate diacetyl and 8.8-fold toward non-native substrate 2,5-hexanedione compared to the wild type. Molecular dynamics simulations revealed that an interaction network formed by Cys258, Arg162, Ala144 and the catalytic residues was reconstructed in the mutant and the dynamics change caused by the disulfide bond could be propagated through the interactions network. This improved the enzyme stability and activity by decreasing the flexibility and locking more “reactive” pose, respectively. Further construction of mutations including A144G showing a 44-fold improvement in catalytic efficiency toward meso-2,3-BD confirmed the role of modifying dynamics correlation network in tunning enzyme activity and selectivity. This study provided important insights into the relationship among dynamics, enzyme catalysis and stability, and will be useful in the designing new enzymes with co-evolution of stability, activity and selectivity.

Efficient activity screening of new glucuronoyl esterases using a pNP-based assay

Glucuronoyl esterases (CE15, EC 3.1.1.117) catalyze the hydrolysis of ester bonds between lignin and carbohydrates in lignocellulose. They are widespread within fungi and bacteria, and are subjects to research interest due to their potential applicability in lignocellulose processing. Identifying new and relevant glucuronoyl esterase candidates is challenging because available model substrates poorly represent the natural substrate, which leads to inefficient screening for the activity. In this study, we demonstrate how fifteen novel, fungal, putative glucuronoyl esterases from family CE15 were expressed and screened for activity towards a commercially available, colorimetric assay based on the methyl-ester of 4-O-methyl-aldotriuronic acid linked to para-nitrophenol (methyl ester-UX-β-pNP) and coupled with the activity of GH67 (α-glucuronidase) and GH43 (β-xylosidase) activity. The assay provides easy means for accurately establishing activity and determining specific activity of glucuronoyl esterases. Out of the fifteen expressed CE15 proteins, seven are active and were purified to determine their specific activity. The seven active enzymes originate from Auricularia subglabra (3 proteins), Ganoderma sinensis (2 proteins) and Neocallimastix californiae (2 proteins). Among the CE15 proteins not active towards the screening substrate (methyl ester-UX-β-pNP) were proteins originating from Schizophyllum commune, Podospora anserina, Trametes versicolor, and Coprinopsis cinerea. It is unexpected that CE15 proteins from such canonical lignocellulose degraders do not have the anticipated activity, and these observations call for deeper investigations.

The Promiscuous Flavin-Dependent Monooxygenase PboD from Aspergillus ustus Increases the Structural Diversity of Hydroxylated Pyrroloindoline Diketopiperazines.

The potential of natural products as pharmaceutical and agricultural agents is based on their large structural diversity, resulting in part from modifications of the backbone structure by tailoring enzymes during biosynthesis. Flavin-dependent monooxygenases (FMOs), as one such group of enzymes, play an important role in the biosynthesis of diverse natural products, including cyclodipeptide (CDP) derivatives. The FMO PboD was shown to catalyze C-3 hydroxylation at the indole ring of cyclo-l-Trp-l-Leu in the biosynthesis of protubonines, accompanied by pyrrolidine ring formation. PboD substrate promiscuity was investigated in this study by testing its catalytic activity toward additional tryptophan-containing CDPs in vitro and biotransformation in Aspergillus nidulans transformants bearing a truncated protubonine gene cluster with pboD and two acetyltransferase genes. High acceptance of five CDPs was detected for PboD, especially of those with a second aromatic moiety. Isolation and structure elucidation of five pyrrolidine diketopiperazine products, with two new structures, proved the expected stereospecific hydroxylation and pyrrolidine ring formation. Determination of kinetic parameters revealed higher catalytic efficiency of PboD toward three CDPs consisting of aromatic amino acids than of its natural substrate cyclo-l-Trp-l-Leu. In the biotransformation experiments with the A. nidulans transformant, modest formation of hydroxylated and acetylated products was also detected.

Influence of pH and temperature on the performance and microbial community during the production of medium-chain carboxylic acids using winery effluents as substrate.

Winery effluents containing high ethanol concentrations and diverse organic matter are ideal substrates for producing medium-chain carboxylic acids via fermentation and chain elongation. However, the process needs to be better understood. This study presents novel insights into the bioconversion mechanisms of medium-chain carboxylic acids by correlating fermentation and chain elongation kinetic profiles with the study of microbial communities at different pH (5 to 7) conditions and temperatures (30 to 40°C). It was found that high productivities of MCCA were obtained using a native culture and winery effluents as a natural substrate. Minor pH variations significantly affected the metabolic pathway of the microorganisms for MCCA production. The maximal productivities of hexanoic (715mg/L/d) and octanoic (350mg/L/d) acids were found at pH 6 and 35°C. Results evidence that the presence of Clostridium, Bacteroides, and Negativicutespromotes the high productions of MCCA. The formation of heptanoic acid was favor when Mogibacterium and Burkholderia were present.

Spectroscopic, electrochemical, and kinetic trends in Fe(III)–thiolate disproportionation near physiologic pH

In addition to its primary oxygen-atom-transfer function, cysteamine dioxygenase (ADO) exhibits a relatively understudied anaerobic disproportionation reaction (ADO-Fe(III)-SR → ADO-Fe(II) + ½ RSSR) with its native substrates. Inspired by ADO disproportionation reactivity, we employ [Fe(tacn)Cl3] (tacn = 1,4,7-triazacyclononane) as a precursor for generating Fe(III)–thiolate model complexes in buffered aqueous media. A series of Fe(III)–thiolate model complexes are generated in situ using aqueous [Fe(tacn)Cl3] and thiol-containing ligands cysteamine, penicillamine, mercaptopropionate, cysteine, cysteine methyl ester, N-acetylcysteine, and N-acetylcysteine methyl ester. We observe trends in UV–Vis and electron paramagnetic resonance (EPR) spectra, disproportionation rate constants, and cathodic peak potentials as a function of thiol ligand. These trends will be useful in rationalizing substrate-dependent Fe(III)–thiolate disproportionation reactions in metalloenzymes.Graphical abstract

Wafer reconstitution: embedded multi-die III-V and silicon co-integration platform

Abstract Co-integration of III-V and silicon on the same substrate can enable many applications in photonics, RF, imaging, and sensing. Wafer reconstitution (WARE) is an embedded multi-die integration platform that allows for the integration of heterogenous materials on 200/300 mm Si substrates. This paper describes an integration flow for InP dies attached to 200 mm silicon. Electrical measurements on InGaAs diodes fabricated on WARE wafers confirm that the performance is similar to devices on native InP substrates.

Substrate recruitment via eIF2γ enhances catalytic efficiency of a holophosphatase that terminates the integrated stress response

Dephosphorylation of pSer51 of the α subunit of translation initiation factor 2 (eIF2αP) terminates signaling in the integrated stress response (ISR). A trimeric mammalian holophosphatase comprised of a protein phosphatase 1 (PP1) catalytic subunit, the conserved C-terminally located ~70 amino acid core of a substrate-specific regulatory subunit (PPP1R15A/GADD34 or PPP1R15B/CReP) and G-actin (an essential cofactor) efficiently dephosphorylate eIF2αP in vitro. Unlike their viral or invertebrate counterparts, with whom they share the conserved 70 residue core, the mammalian PPP1R15s are large proteins of more than 600 residues. Genetic and cellular observations point to a functional role for regions outside the conserved core of mammalian PPP1R15A in dephosphorylating its natural substrate, the eIF2 trimer. We have combined deep learning technology, all-atom molecular dynamics simulations, X-ray crystallography, and biochemistry to uncover binding of the γ subunit of eIF2 to a short helical peptide repeated four times in the functionally important N terminus of human PPP1R15A that extends past its conserved core. Binding entails insertion of Phe and Trp residues that project from one face of an α-helix formed by the conserved repeats of PPP1R15A into a hydrophobic groove exposed on the surface of eIF2γ in the eIF2 trimer. Replacing these conserved Phe and Trp residues with Ala compromises PPP1R15A function in cells and in vitro. These findings suggest mechanisms by which contacts between a distant subunit of eIF2 and elements of PPP1R15A distant to the holophosphatase active site contribute to dephosphorylation of eIF2αP by the core PPP1R15 holophosphatase and to efficient termination of the ISR in mammals.

Role of UDP-N-acetylmuramic acid in the regulation of MurA activity revealed by molecular dynamics simulations.

The peptidoglycan biosynthesis pathway plays a vital role in bacterial cells, and facilitates peptidoglycan layer formation, a fundamental structural component of the bacterial cell wall. The enzymes in this pathway are candidates for antibiotic development, as most do not have mammalian homologues. The UDP-N-acetylglucosamine (UNAG) enolpyruvyl transferase enzyme (MurA) in the peptidoglycan pathway cytoplasmic step is responsible for the phosphoenolpyruvate (PEP)-UNAG catalytic reaction, forming UNAG enolpyruvate and inorganic phosphate. Reportedly, UDP-N-acetylmuramic acid (UNAM) binds tightly to MurA forming a dormant UNAM-PEP-MurA complex and acting as a MurA feedback inhibitor. MurA inhibitors are complex, owing to competitive binding interactions with PEP, UNAM, and UNAG at the MurA active site. We used computational methods to explore UNAM and UNAG binding. UNAM showed stronger hydrogen-bond interactions with the Arg120 and Arg91 residues, which help to stabilize the closed conformation of MurA, than UNAG. Binding free energy calculations using end-point computational methods showed that UNAM has a higher binding affinity than UNAG, when PEP is attached to Cys115. The unbinding process, simulated using τ-random acceleration molecular dynamics, showed that UNAM has a longer relative residence time than UNAG, which is related to several complex dissociation pathways, each with multiple intermediate metastable states. This prevents the loop from opening and exposing the Arg120 residue to accommodate UNAG and potential new ligands. Moreover, we demonstrate the importance of Cys115-linked PEP in closed-state loop stabilization. We provide a basis for evaluating novel UNAM analogues as potential MurA inhibitors. PUBLIC SIGNIFICANCE: MurA is a critical enzyme involved in bacterial cell wall biosynthesis and is involved in antibiotic resistance development. UNAM can remain in the target protein's active site for an extended time compared to its natural substrate, UNAG. The prolonged interaction of this highly stable complex known as the 'dormant complex' comprises UNAM-PEP-MurA and offers insights into antibiotic development, providing potential options against drug-resistant bacteria and advancing our understanding of microbial biology.

Identifying Residues for Substrate Recognition in Human GPAT4 by Molecular Dynamics Simulations.

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the first step in triacylglycerol synthesis. Understanding its substrate recognition mechanism may help to design drugs to regulate the production of glycerol lipids in cells. In this work, we investigate how the native substrate, glycerol-3-phosphate (G3P), and palmitoyl-coenzyme A (CoA) bind to the human GPAT isoform GPAT4 via molecular dynamics simulations (MD). As no experimentally resolved GPAT4 structure is available, the AlphaFold model is employed to construct the GPAT4-substrate complex model. Using another isoform, GPAT1, we demonstrate that once the ligand binding is properly addressed, the AlphaFold complex model can deliver similar results to the experimentally resolved structure in MD simulations. Following the validated protocol of complex construction, we perform MD simulations using the GPAT4-substrate complex. Our simulations reveal that R427 is an important residue in recognizing G3P via a stable salt bridge, but its motion can bring the ligand to different binding hotspots on GPAT4. Such high flexibility can be attributed to the flexible region that exists only on GPAT4 and not on GPAT1. Our study reveals the substrate recognition mechanism of GPAT4 and hence paves the way towards designing GPAT4 inhibitors.

Computational mechanistic investigation of the kinetic resolution of α-methyl-phenylacetaldehyde by norcoclaurine synthase

Norcoclaurine synthase from Thalictrum flavum (TfNCS) demonstrated high stereospecificity and yield in catalyzing the Pictet-Spengler reaction of dopamine with chiral aldehydes, achieving kinetic resolution of aldehydes. However, the mechanism and the factors contributing to the stereoselectivity remain unclear. Herein, by using quantum chemical calculations, the mechanisms of TfNCS-catalyzed reactions of dopamine with both enantiomers of α-methyl-phenylacetaldehyde are studied. The calculations reveal a mechanism mirroring the reaction of natural substrates, for which the deprotonation of the C5−H of the cyclized intermediate is rate-limiting. The calculated overall barriers are 20.1 kcal mol-1 and 21.6 kcal mol-1 for the reactions of (R)- and (S)-α-methyl-phenylacetaldehyde, respectively. The M97 and L72 residues are proposed to be the key residues contributing to the stereospecificity. The obtained detailed information is helpful for designing new variants of TfNCS with extended substrate scope, and also advancing our understanding of TfNCS reactions for potential applications.

In vitro demetalation of central magnesium in various chlorophyll derivatives using Mg-dechelatase homolog from the chloroflexi Anaerolineae.

In the metabolic pathway of chlorophylls (Chls), an enzyme called STAY-GREEN or SGR catalyzes the removal of the central magnesium ion of Chls and their derivatives to their corresponding free bases, including pheophytins. The substrate specificity of SGR has been investigated through in vitro reactions using Chl-related molecules. However, information about the biochemical properties and reaction mechanisms of SGR and its substrate specificity remains elusive. In this study, we synthesized various Chl derivatives and investigated their in vitro dechelations using an SGR enzyme. Chl-a derivatives with the C3-vinyl group on the A-ring, which is commonly found as a substituent in natural substrates, and their analogs with ethyl, hydroxymethyl, formyl, and styryl groups at the C3-position were prepared as substrates. In vitro dechelatase reactions of these substrates were performed using an SGR enzyme derived from an Anaerolineae bacterium, allowing us to investigate their specificity. Reactivity was reduced for substrates with an electron-withdrawing formyl or sterically demanding styryl group at the C3-position. Furthermore, the Chl derivative with the C8-styryl group on the B-ring was less reactive for SGR dechelation than the C3-styryl substrate. These results indicate that the SGR enzyme recognizes substituents on the B-ring of substrates more than those on the A-ring.

Biochemical characterization of a multiple prenyltransferase from Tolypocladium inflatum

Prenylation plays a pivotal role in the diversification and biological activities of natural products. This study presents the functional characterization of TolF, a multiple prenyltransferase from Tolypocladium inflatum. The heterologous expression of tolF in Aspergillus oryzae, coupled with feeding the transformed strain with paxilline, resulted in the production of 20- and 22-prenylpaxilline. Additionally, TolF demonstrated the ability to prenylated the reduced form of paxilline, β-paxitriol. A related prenyltransferase TerF from Chaunopycnis alba, exhibited similar substrate tolerance and regioselectivity. In vitro enzyme assays using purified recombinant enzymes TolF and TerF confirmed their capacity to catalyze prenylation of paxilline, β-paxitriol, and terpendole I. Based on previous reports, terpendole I should be considered a native substrate. This work not only enhances our understanding of the molecular basis and product diversity of prenylation reactions in indole diterpene biosynthesis, but also provides insights into the potential of fungal indole diterpene prenyltransferase to alter their position specificities for prenylation. This could be applicable for the synthesis of industrially useful compounds, including bioactive compounds, thereby opening up new avenues for the development of novel biosynthetic strategies and pharmaceuticals.Key points• The study characterizes TolF as a multiple prenyltransferase from Tolypocladium inflatum.• TerF from Chaunopycnis alba shows similar substrate tolerance and regioselectivity compared to TolF.• The research offers insights into the potential applications of fungal indole diterpene prenyltransferases.Graphical abstract

Determining the Usage Properties of Some Natural Substrate Materials in submerged macrophyte Vallisneria sp. culture

In this research, which targets removing nutrients resulting from the metabolic wastes of fish and unconsumed feed from the environment in aquarium systems where fish, plants, and ground material are used in the same environment and use them as plant nutrients, it was aimed to determine the usage properties of zeolite, leonardite and diatomite materials on the growth parameters of the Vallisneria sp. plant. 
 The study was conducted in three stages for 8 months. The first step of the study was planned as the plant adaptation phase, and no fish were added to the environment. In the second step, fish was introduced. The third step was designed to determine the adsorbent mixture's plant nutrient storage capacity (effective usage period). In the experimental aquariums, no plant nutrient or fertiliser other than fish food was used at all stages. The effects and using duration on the growth of the Vallisneria sp plant of adsorbed nutrients from unconsumed feed by leonardite and zeolite mixture were investigated in three stages in aquarium condition. Trial groups were formed by adding the adsorbent mixture of the feed having a 40% protein value to the first group (G1), the adsorbent mixture of the feed having a 33% protein value to the second group (G2), and the adsorbent mixture without feed to the third group (G3).
 When the SGR values at the end of the experiment were compared, there was no statistical difference between the G1 and G2 groups, which contained 40% and 33% protein (P>0.05). On the other hand, both groups (G1, G2) showed significantly higher (P