Substrate Tolerance Research Articles

Crystal structures of a 6-dimethylallyltryptophan synthase, IptA: Insights into substrate tolerance and enhancement of prenyltransferase activity

Dimethylallyltryptophan synthases (DMATSs) catalyze the prenyl transfer reaction from dimethylallyl pyrophosphate (DMAPP) to an indole ring. IptA, a member of the DMATS family, is involved in biosynthesis of 6-dimethylallylindole-3-carbaldehyde in Streptomyces sp. SN-593 and catalyzes the C6-prenylation of l-Trp. The enzyme exhibits prenyl acceptor promiscuity and can accept various Trp derivatives, as observed in several other DMATS family members. Although many crystal structures of DMATS have been determined to date, the structural basis of substrate promiscuity and the acceptance of alternatives to indole-containing natural substrates remain to be clarified. In this study, we determined the crystal structures of the ternary l-Trp derivative (5-methyl-, 6-methyl-, and Nα-methyl-l-Trp) -DMSPP (dimethylallyl S-thiolopyrophosphate; stable analog of DMAPP) -enzyme complex of IptA, in addition to the substrate-free IptA and ternary l-Trp-DMSPP-IptA complex crystal structures. The overall structure of IptA exhibited a typical ABBA-fold, which is commonly found in DMATS family members, while l-Trp and DMSPP are found in a tunnel located inside the ABBA barrel. The crystal structure of the ternary l-Trp-DMSPP-enzyme complex can explain the electrophilic substitution at the C6 atom of l-Trp, which is assisted by Glu84 and His294, as previously suggested for other DMATSs. Although l-Trp snugly fitted into the active site pocket and the unoccupied space around l-Trp is very limited in the l-Trp-DMSPP-IptA complex structure, the enzyme can accommodate 5-methyl- and 6-methyl-l-Trp by slight relocation of the substrate indole ring and adjacent side chain in the active site, resulting in a higher prenylation activity for 5-methyl-l-Trp and C7 prenylation of 6-methyl-l-Trp. Like many other DMATSs, IptA cannot utilize prenyl donors larger than DMAPP. To enlarge the prenyl donor-binding pocket, the W154A mutation was introduced. As expected, this mutant produced prenylated l-Trp from l-Trp and geranyl- and farnesyl pyrophosphate.

Electrochemical Sulfonylation-Induced Lactonization of Alkenes: Synthesis of Sulfonyl Phthalides.

An electrochemical cascade sulfonylation and lactonization process of alkenes and a most widely used arylsulfonylation reagent─sulfonyl hydrazines─was developed for the first time. This electrochemical sulfonyl lactonization avoided the use of toxic metal catalysts or stoichiometric oxidants and was carried out under mild conditions. The target product γ-sulfonylated phthalides with broad and excellent substrate tolerance were achieved.

The identification and application of a robust ω-transaminase with high tolerance towards substrates and isopropylamine from a directed soil metagenome

A robust ω-transaminase, ATA1012, identified from amidogen-enriched environments. The enzyme showed excellent thermostability, high substrate tolerance, and low equivalents of amine donor, which efficiently transform substrate with 100% conversion.

The substrate scope of dehydratases in antibiotic biosynthesis and their application in kinetic resolutions.

Nine dehydratases involved in the biosynthesis of secondary metabolites in addition to FabZ from fatty acid biosynthesis were investigated for their substrate scope using a panel of N-acetylcysteamine (SNAC) thioesters. The best performing enzyme BorDH2 was applied in kinetic resolutions.

Structure-function relationship of terpenoid glycosyltransferases from plants.

Covering: up to 2021Terpenoids are physiologically active substances that are of great importance to humans. Their physicochemical properties are modified by glycosylation, in terms of polarity, volatility, solubility and reactivity, and their bioactivities are altered accordingly. Significant scientific progress has been made in the functional study of glycosylated terpenes and numerous plant enzymes involved in regio- and enantioselective glycosylation have been characterized, a reaction that remains chemically challenging. Crucial clues to the mechanism of terpenoid glycosylation were recently provided by the first crystal structures of a diterpene glycosyltransferase UGT76G1. Here, we review biochemically characterized terpenoid glycosyltransferases, compare their functions and primary structures, discuss their acceptor and donor substrate tolerance and product specificity, and elaborate features of the 3D structures of the first terpenoid glycosyltransferases from plants.

Stereoselective synthesis of tri-substituted tetrahydrothiophenes and their in silico binding against mycobacterial protein tyrosine phosphatase B.

A facile approach to tri-substituted tetrahydrothiophenes via thia-Michael/aldol has been developed. The cascade reaction was carried out in the presence of 5 mol% of DABCO in ethyl acetate to afford diversely functionalized tetrahydrothiophenes (THTs) with excellent diastereoselectivity. The present methodology has broad substrate tolerance. Gram-scale reaction proceeds with equal efficiency. Functional group transformations further highlight the synthetic potential of the THTs. An asymmetric version of the cascade reaction has also been investigated and a maximum of 72% ee was observed with cinchonidine derived squaramide. Moreover, in silico based molecular docking followed by deep learning based affinity prediction and molecular dynamics simulation analysis indicate the synthesized THT derivatives can act as potent competitive inhibitors of MptpB at low micromolar to nanomolar concentrations. In silico ADME analysis further suggests the plausibility of these compounds to act as future anti-mycobacterial therapeutic leads.

Towards understanding oxygen heterocycle-forming biocatalysts: a selectivity study of the pyran synthase PedPS7.

Intramolecular oxa-Michael addition-catalysing cyclases are widespread in polyketide biosynthetic pathways. Although they have significant potential in biotechnology and chemoenzymatic synthesis of chiral heterocycles, they have only scarcely been studied. Here, we present detailed investigations on the selectivity profile of the pyran synthase PedPS7 showing that it combines broad substrate tolerance with high selectivity for the formation of up to two new stereocentres and relaxed precursor stereoisomer discrimination. Two of the four possible tetrahydropyran stereoisomers are reliably accessible by this enzyme. The results indicate fundamental differences between the individual subtypes of intramolecular oxa-Michael addition-catalysing cyclases.

Decatungstate Catalyzed Synthesis of Trifluoromethylthioesters from Aldehydes via a Radical Process.

Here we report a mild and general method for the trifluoromethylthiolation of aldehydes using N-trifluoromethylthiosaccharin as the CF3S radical source and sodium decatungstate (NaDT) as the photocatalyst. This reaction proceeds via hydrogen atom abstraction by photoactivated DT and features good functional groups and substrate tolerance. Generally, electron-rich aldehydes demonstrate better reactivity than electron-deficient ones and good selectivity is observed for the trifluoromethylthiolation of aldehydic C-H bonds over tertiary and benzylic C-H bonds. Preliminary mechanistic studies have shown that a free radical process is involved.

Recyclization of 3-formylchromone with phenacyl thiocyanate: fast and efficient access to thiophenes and 2-aminothiophenes bearing o-acylphenol moiety

A variety of substituted derivatives of thiophene and 2-aminothiophene bearing o-acylphenol moieties were obtained via the reaction between 3-formylchromone with phenacyl thiocyanate in the presence of Et3N. This metal-free procedure for the synthesis of thiophene skeletons revealed a good efficiency, higher diversity, and wide substrate tolerance. These compounds have structural similarities to biologically active compounds reported in the literature. Besides, they can be used as a suitable candidate for the synthesis of fused-thiophene heterocycles through C–H activation processes.

Direct Difluoromethylation of Heterocycles through Photosensitized Electron Transfer

AbstractDifluoromethylation is of prime importance for its applicability in functionalizing diverse fluorine‐containing heterocycles, which are core groups in diverse biologically and pharmacologically active ingredients. Herein, we report a novel transition metal‐ and oxidant‐free visible light‐photoinduced protocol for direct C(sp2)‐H difluoromethylation of heterocycles. The reaction afforded difluoromethyl heterocycles without using colored organic dyes and metal catalysts in good yields and showed a broad substrate tolerance. Moreover, the representative products exhibited potential drug activity, and one product showed good antifungal activities against Rhizoctorzia solani (62.7 %).

Structure and in silico simulations of a cold-active esterase reveals its prime cold-adaptation mechanism.

Here we determined the structure of a cold active family IV esterase (EstN7) cloned from Bacillus cohnii strain N1. EstN7 is a dimer with a classical α/β hydrolase fold. It has an acidic surface that is thought to play a role in cold-adaption by retaining solvation under changed water solvent entropy at lower temperatures. The conformation of the functionally important cap region is significantly different to EstN7's closest relatives, forming a bridge-like structure with reduced helical content providing greater access to the active site through more than one substrate access tunnel. However, dynamics do not appear to play a major role in cold adaption. Molecular dynamics at different temperatures, rigidity analysis, normal mode analysis and geometric simulations of motion confirm the flexibility of the cap region but suggest that the rest of the protein is largely rigid. Rigidity analysis indicates the distribution of hydrophobic tethers is appropriate to colder conditions, where the hydrophobic effect is weaker than in mesophilic conditions due to reduced water entropy. Thus, it is likely that increased substrate accessibility and tolerance to changes in water entropy are important for of EstN7's cold adaptation rather than changes in dynamics.

Oleaginous yeasts- substrate preference and lipid productivity: a view on the performance of microbial lipid producers

BackgroundOleaginous yeasts are promising microbial platforms for sustainable, bio-based production of biofuels and oleochemical building blocks. Bio-based residues provide sustainable and cost-effective carbon sources for fermentative yeast oil production without land-use change. Considering the regional abundancy of different waste streams, we chose complex biomass residue streams of marine origin; macroalgae hydrolysate, and terrestrial origin; wheat straw hydrolysate in the presence, and absence of corn steep liquor as a complex nitrogen source. We investigated the biomass and lipid yields of an array of well-described oleaginous yeasts; R. glutinis, T. asahii, R. mucilaginosa, R. toruloides, C. oleaginosus growing on these hydrolysates. Furthermore, their sugar utilization, fatty acid profile, and inhibitory effect of the hydrolysates on yeast growth were compared. For correlative reference, we initially performed comparative growth experiments for the strains on individual monomeric sugars separately. Each of these monomeric sugars was a dominant carbon source in the complex biomass hydrolysates evaluated in this study. In addition, we evaluated N-acetylglucosamine, the monomeric building block of chitin, as a low-cost nitrogen and carbon source in yeast fermentation.ResultsC. oleaginosus provided the highest biomass and lipid yields. In the wheat straw and brown algae hydrolysates, this yeast strain gained 7.5 g/L and 3.8 g/L lipids, respectively. Cultivation in algae hydrolysate resulted in a higher level of unsaturated fatty acids in the lipids accumulated by all yeast strains. R. toruloides and C. oleaginosus were able to effectively co-utilize mannitol, glucose, and xylose. Growth rates on wheat straw hydrolysate were enhanced in presence of corn steep liquor.ConclusionsAmong the yeast strains investigated in this study, C. oleaginosus proved to be the most versatile strain in terms of substrate utilization, productivity, and tolerance in the complex media. Various fatty acid profiles obtained on each substrate encourage the manipulation of culture conditions to achieve the desired fatty acid composition for each application. This could be accomplished by combining the element of carbon source with other formerly studied factors such as temperature and oxygen. Moreover, corn steep liquor showed promise for enhancement of growth in the oleaginous strains provided that carbon substrate is available.

A Bifunctional B,N‐Based Asymmetric Catalytic Nitrostyrene‐Michael Addition Acting through a 10‐Membered Ring Cyclic Transition State

AbstractThe B,N‐bifunctional catalyst homoboroproline has been applied to a catalytic asymmetric nitroalkene‐Michael addition to β‐nitrostyrene analogues, showing broad substrate tolerance, high conversions and moderate to good asymmetric induction. The ability of homoboroproline to act as an efficient catalyst based on enamine‐formation of the secondary amine, coupled with intramolecular Lewis‐acid chelation of the nitro function, in a non‐FLP manner, to effect efficient and enantioselective catalysis via a proposed large 10‐membered ring transition state is remarkable and reinforced by theoretical calculations.

Silica Supported Synthesis and Quorum Quenching Ability of Isoxazolones Against Both Gram Positive and Gram Negative Bacterial Pathogens

AbstractIsoxazolones are synthesised using easily available, economical and reusable silica (TLC grade) catalyst. High yields are obtained in aqueous reaction system with substrate tolerance without affecting the yield. This multigram scale, purification free method exhibits prospects for a large scale use in future. These compounds were screened for their potential application as quorum quenchers in pathogenic bacteria. The prepared compounds were analysed for quorum quenching (QQ) activity on Gram negative bioreporter strain Chromobacterium violaceum by inhibiting its pigment production i. e. violacein production in a concentration dependent manner. Some Isoxazolone derivatives showed very good quorum quenching activity. Moreover these derivatives also showed very good antibiofilm activity against both Gram negative (Pseudomonas aeruginosa ATCC 27853) and Gram positive (Staphylococcus aureus ATCC 6538) bacterial human pathogens through Quorum Quenching mechanism. Isoxazolones thus have enormous potential to be exploited in the medical field against bacterial infections caused by both Gram negative and Gram positive bacterial human pathogens.

Cross-dehydrogenative Coupling of N-Aryl Tetrahydroisoquinolines Catalyzed by an Anthraquinone-containing Polymeric Photosensitizer.

This work reports the photocatalytic application of an anthraquinone-containing polymeric photosensitizer (AQ-PHEMA) in the visible light-induced cross-dehydrogenative-coupling of N-aryl tetrahydroisoquinolines with several nucleophiles, including nitromethane, 1-methyl-2-alkyl ketone and dialkyl (aryl) phosphine oxide. The results revealed that the reaction could be catalyzed by AQ-PHEMA efficiently to afford a series of 1-substituted-2-aryl-1,2,3,4-tetrahydroisoquinolines in good to excellent yields with nice substrate tolerance under aerobic conditions at room temperature. The practical application potential was also showcased by a gram-scale synthesis. More importantly, the utilization of AQ-PHEMA as a heterogeneous photosensitizer also showed nice recyclability and reusability of the catalyst, whereas AQ-PHEMA can be easily separated and reused for at least 8 times without significant loss of photocatalytic activity.

Copper pyrithione (CuPT)-catalyzed/mediated amination and thioarylation of (hetero)aryl halides: A competition

A facile method for the synthesis of N-arylheterocycles and di(hetero)aryl sulfides under mild condition is described. In these transformations, copper pyrithione (CuPT) was used as the copper catalyst for C─N coupling, while served as catalyst and coupled partner for C─S coupling with high yields and broad substrate tolerance. The S-arylation process was also utilized for the construction of valuable bioactive 2-sulfonylpyridine 1-oxide derivatives.

Phosphine-Catalyzed Synthesis of 3-Allyl-4-pyrones by the Tandem Reaction of Diynones and Allylic Alcohols.

A simple and effective tandem reaction of diynones and allylic alcohols was developed to afford functionalized 3-allyl-4-pyrones in moderate to excellent yields. This protocol underwent a Michael addition─Claisen rearrangement─O-cyclization process, which exhibited broad substrate tolerance, high regioselectivity, and atom economy under a metal-free condition. Moreover, functional transformation of the products was also further studied.

The Renaissance of Electro‐Organic Synthesis for the Difunctionalization of Alkenes and Alkynes: A Sustainable Approach

AbstractElectro‐organic reactions are now considered as one of the most efficient and environmentally benign methodologies to synthesize highly functionalized motifs like difunctionalized unsaturated compounds from readily available substrates. Excellent regioselectivity, functional group tolerance and broad range of substrates are the main advantages of electrochemical difunctionalization reactions. Alkenes and alkynes readily accept radical or ionic derivatives which makes it vital precursors for the electrochemical synthesis of industrially relevant and biologically active molecules through difunctionalization. This review aims to provide the readers an excellent coverage of the different electrochemical difunctionalization of alkenes and alkynes such as 1,2‐homodifunctionalization, 1,2‐heterodifunctionalization, rearrangement, ipso‐migration, cyclization and dehydrogenative annulation reactions.

Potassium tert-Butoxide Promoted Synthesis of Dihydroquinazolinones.

We herein report an efficient synthetic protocol to access heterocyclic dihydroquinazolinones by a transition-metal-free process, involving the reaction of 2-aminobenzonitriles with aldehydes in the presence of KOtBu. The method is compatible with aromatic ketones providing 2,2-disubstituted dihydroquinazolinones in high yields. This reaction proceeds feasibly at room temperature and features a broad substrate scope and tolerance to a range of functional groups. The mechanism follows a radical pathway.

Direct Csp2−H difluoromethylation of heterocycle by K2S2O8

We reveal here a novel and efficient transition-metal-free C(sp2)–H oxidative difluoromethylation of heterocycle. It uses stable and nontoxic NaSO2CF2H as difluoromethyl source and potassium persulfate as oxidant without the need for metal catalyst. Significantly, the reaction affords difluoromethyl heterocycles in moderate to good yields with a broad substrate tolerance.