Large Active Site Research Articles

Nickel-cobalt alloy oxide nanoparticles wrapped by carbon black for efficient oxygen evolution electrocatalysis

The search for the simple and convenient design method of a non-precious metal oxygen evolution catalyst is still an emerging strategy today. Usually, nano-alloy oxide can enhance the catalytic activity for large specific surface area and active site, which have been widely studied for the purpose of developing active electrocatalysts for water splitting, while, nanoparticles are fundamentally less stable and prone to substantial size growth in long-term operations. Therefore, a framework of hierarchical porous composite microsphere with NiCoOx nanoparticles wrapped by Carbon Black (NiCoOx@CB) was fabricated successfully through spray-drying in this paper. Benefitting from the porous nanostructure and 0 D nano-alloy oxide active site, a remarkable OER performance with a low overpotential for 241 mV at 10 mA·cm−2 in alkaline media was exhibited. And, from the support of wrapped structure, NiCoOx@CB showed a superior durability with 90 h for nanoparticles were prevented to reunion during the reaction. Also, the heterogeneous interfaces with strong charge transfers between CB and NiCoOx nanoparticles could enhance OER performance (NiCoOx showed 265 mV at 10 mA·cm−2). The proposed method in this paper work provide a facile route to construct the porous composite microsphere with excellent OER electrocatalytic activity based on non-noble metal oxide wrapped by CB.

Imidazolium based Ionic liquid Anchored over Dendritic Mesoporous Silica Nanospheres possessing a Magnetic Core as an Efficient and Recyclable Base Catalyst

AbstractIn this study, Magnetic core‐shell dendritic mesoporous silica nanospheres immobilized with imidazolium‐based di‐cationic ionic liquid (Fe3O4@SiO2@DSiO2‐Im‐IL) possessing a large surface area and numerous active site for adsorption of various molecules to carry out organic transformations have been successfully developed. The synthesized nanocatalyst was found to be easily recyclable by applying external magnetic force. The structure, morphology, thermal stability, and magnetic behavior of the synthesized magnetic nanocatalyst were confirmed by different physiochemical techniques like FT‐IR, P‐XRD, FE‐SEM, TGA, VSM, BET, and TEM analysis. The efficiency of the developed heterogeneous nanocatalyst has been investigated for the green synthesis of numerous benzopyran derivatives by employing different aldehydes and active methylene compounds. The catalyst exhibits marvellous activity and efficiency in terms of high product yield, less catalyst use, and wide substrate scope even at room temperature. Finally, after the successful formation of the product, the catalyst was regenerated by utilizing an external magnetic field. The catalyst has been reused multiple times without any noteworthy loss in its activity which confirms the stability of the developed catalyst. The preparation of the catalyst is cost‐effective, and shows atom economy with the utilization of green solvent and without any waste production, which authenticates that it could be a greener pathway for the synthesis of the benzopyran derivatives.

Novel graphene-like layer-by-layer stacked carbon nanotube-supported Fe-Ni alloy for oxygen evolution and oxygen reduction reactions

In this study, we prepared a novel graphene-like stacked carbon nanotube-supported Fe-Ni alloy nanometer composite material. The cubic structure of nickel dimethylglyoxime was synthesized as the base material. Under hydrothermal condition, FePc was adsorbed on the surface of the nickel dimethylglyoxime cube. In addition, hydrogen peroxide not only decomposes FePc into Pc and Fe2+, but also reduces Fe2+ to Fe. On the other hand, hydrogen peroxide was first found could decompose and peel the cube of nickel dimethylglyoxime into a novel type of nanotubes with graphene-like nanosheet stacking structure. After the final calcination, the Fe2+ and Ni2+ were converted to FeNi alloy, and the dimethylglyoxime carrier with its loaded Pc was converted to carbon material. The novel structure has a large BET, which is conducive to electron transport, product transport and provides a large active site for Fe-Ni3 alloy. The composite has high electro-catalysis performance for OER and ORR, which provides a new idea for the study of new energy reserves, energy conversion and reserve materials. This study provides a new idea for preparing novel carbon nanotube-loaded transition metal composites in the field of electrocatalysis.

Distribution of Aromatic Amino Acid Residues in Substrate-Binding Regions Modulates Substrate Specificity of Microbial Debranching Enzymes.

Debranching enzymes (DBEs) directly hydrolyze α-1,6-glucosidic linkages in glycogen, starch, and related polysaccharides, making them important in the starch processing industry. However, the ambiguous substrate specificity usually restricts synergistic catalysis with other amylases for improving starch utilization. Herein, a glycogen-debranching enzyme from Saccharolobus solfataricus (SsGDE) and two isoamylases from Pseudomonas amyloderamosa (PaISO) and Chlamydomonas reinhardtii (CrISO) were used to investigate the molecular mechanism of substrate specificity. Along with the structure-based computational analysis, the aromatic residues in the substrate-binding region of DBEs played an important role in binding substrates. The aromatic residues in SsGDE appeared clustered, contributing to a small substrate-binding region. In contrast, the aromatic residues in isoamylase were distributed dispersedly, forming a large active site. The distinct characteristics of substrate-binding regions in SsGDE and isoamylase might explain their substrate preferences for maltodextrin and amylopectin, respectively. By modulating the substrate-binding region of SsGDE, variants Y323F and V375F were obtained with significantly enhanced activities, and the activities of Y323F and V375F increased by 30 and 60% for amylopectin, and 20 and 23% for DE4 maltodextrin, respectively. This study revealed the molecular mechanisms underlying the substrate specificity for SsGDE and isoamylases, providing a route for engineering enzymes to achieve higher catalytic performance.

Hierarchical 3D Snowflake-like Iron Diselenide: A Robust Electrocatalyst for Furaltadone Detection.

An electrocatalyst with a large active site is critical for the development of a high-performance electrochemical sensor. This work demonstrates the fabrication of an iron diselenide (FeSe2)-modified screen-printed carbon electrode (SPCE) for the electrochemical determination of furaltadone (FLD). It has been prepared by the facile method and systematically characterized with various microscopic/spectroscopic approaches. Due to advantageous physiochemical properties, the FeSe2/SPCE showed a low charge-transfer resistance value of 200 Ω in 5.0 mM [Fe(CN)6]3-/4- containing 0.1 M KCl. More importantly, the FeSe2/SPCE exhibited superior catalytic performance compared to the bare SPCE for FLD sensing based on the electrochemical response in terms of a peak potential of -0.44 V (vs Ag/AgCl (sat. KCl)) and cathodic response current of -22.8 μA. Operating at optimal conditions, the FeSe2-modified electrode showed wide linearity from 0.01 to 252.2 μM with a limit of detection of 0.002 μM and sensitivity of 1.15 μA μM-1 cm-2. The analytical performance of the FeSe2-based platform is significantly higher than many previously reported FLD electrochemical sensors. Furthermore, the FeSe2/SPCE also has a promising platform for FLD detection with high sensitivity, good selectivity, excellent stability, and robust reproducibility. Thus, the finding above shows that the FeSe2/SPCE is a highly suitable candidate for the electrochemical determination of glucose levels for real-time applications such as in human urine and river water samples.

C-H Insertion in Dirhodium Tetracarboxylate-Catalyzed Reactions despite Dynamical Tendencies toward Fragmentation: Implications for Reaction Efficiency and Catalyst Design.

Rh-catalyzed C-H insertion reactions to form β-lactones suffer from post-transition state bifurcations, with the same transition states leading to ketones and ketenes via fragmentation in addition to β-lactones. In such a circumstance, traditional transition state theory cannot predict product selectivity, so we employed ab initio molecular dynamics simulations to do so and provide a framework for rationalizing the origins of said selectivity. Weak interactions between the catalyst and substrate were studied using energy decomposition and noncovalent interaction analyses, which unmasked an important role of the 2-bromophenyl substituent that has been used in multiple β-lactone-forming C-H insertion reactions. Small and large catalysts were shown to behave differently, with the latter providing a means of overcoming dynamically preferred fragmentation by lowering the barrier for the recombination of the product fragments in the grip of the large catalyst active site cavity.

Combustion-driven synthesis route for bimetallic Ag–Bi nanoparticle-anchored carbon nanotube electrodes for high-performance supercapacitors

Bimetallic nanostructures within carbon-based materials can overcome the fundamental limits of energy materials, which cannot be obtained using a single material. However, their synthesis involves time-consuming and complex processes that cause phase/interface segregation and non-uniformly distributed metal elements. Herein, we report a facile combustion-driven synthesis for bimetallic Ag–Bi nanoparticle (NP)-anchored carbon nanotube (CNT) electrodes. One-step combustion wave passing through freestanding films comprising Ag2O and Bi powders, nitrocellulose layers within CNTs enables high-density thermochemical reactions in seconds. The rapid heating-cooling rates induce the formation of liquefied Ag–Bi and trapping of metastable Ag–Bi phases at the carbon surfaces, thereby synthesizing homogeneously mixed bimetallic Ag–Bi NPs anchored on the CNTs, along with smaller diameters (∼20 nm) and high distribution density. A supercapacitor electrode employing them exhibits outstanding specific capacitance and retention (1372-1093 Fg-1 at 2-5 mVs−1, and 101.3% of the stabilized capacitance after 10,000 cycles at 100 mVs−1). This was attributed to the large active site surface area from the small diameters and high distribution density of the bimetallic Ag–Bi NPs by low surface energy, and highly stable adhesion to the CNTs. The synthesis strategy can be extended to a scalable fabrication method of various multi-metallic nanostructures for versatile electrochemical electrodes and catalysts.

Three candidate 2-(2-phenylethyl)chromone-producing type III polyketide synthases from Aquilaria sinensis (Lour.) Gilg have multifunctions synthesizing benzalacetones, quinolones and pyrones

2-(2-Phenylethyl)chromones (PECs), with extensive pharmacological activities, have been found in only few plant species and are the characteristic components of agarwood which is an expensive fragrant and resinous wood. However, little is known about PEC biosynthesis pathway. Here, three type III polyketide synthases (PKSs), AsPKS3, AsPKS4, and AsPKS5, highly expressed in the agarwood layer were isolated from Aquilaria sinensis and characterized. The qRT–PCR results showed that the expression of them was strongly induced by salt, salicylic acid (SA), or methyl jasmonate (MeJA). In addition, their expression levels were consistent with PEC accumulation in calli under salt treatment. AsPKS proteins exhibited multiple activities in vitro, including catalyzing the synthesis of p-hydroxybenzalacetone, feruloylmethane, 4-hydroxy-N-methyl-2(1H)-quinolone, and 1-methyl-2-phenethylquinolin-4(1H)-one (which has a structure similar to those of PECs). Moreover, AsPKS4 or AsPKS5 could also catalyze the production of pyrones in vitro. The pH and temperature optima for the p-hydroxybenzalacetone or 4-hydroxy-N-methyl-2(1H)-quinolone production of AsPKSs were found to be different. Protein structure prediction showed that the three AsPKSs have large active site entrances allowing binding of bulky substrates such as N-methylanthraniloyl-CoA. Additionally, the three AsPKS proteins were localized in the cytoplasm and nucleus. This study not only facilitates the synthesis of structurally diverse benzalacetones, quinolones, and pyrones but also indicates prime enzymes involved in the biosynthesis of PECs for future studies.

BACE1: A Key Regulator in Alzheimer's Disease Progression and Current Development of its Inhibitors.

Alzheimer's disease (AD) is a chronic neurodegenerative disease with no specific disease-modifying treatment. β-secretase (BACE1) is considered the potential and rationale target because it is involved in the rate-limiting step, which produces toxic Aβ42 peptides that leads to deposits in the form of amyloid plaques extracellularly, resulting in AD. This study aims to discuss the role and implications of BACE1 and its inhibitors in the management of AD. We have searched and collected the relevant quality work from PubMed using the following keywords "BACE1", BACE2", "inhibitors", and "Alzheimer's disease". In addition, we included the work which discusses the role of BACE1 in AD and the recent work on its inhibitors. In this review, we have discussed the importance of BACE1 in regulating AD progression and the current development of BACE1 inhibitors. However, the development of a BACE1 inhibitor is very challenging due to the large active site of BACE1. Nevertheless, some of the BACE1 inhibitors have managed to enter advanced phases of clinical trials, such as MK-8931 (Verubecestat), E2609 (Elenbecestat), AZD3293 (Lanabecestat), and JNJ-54861911 (Atabecestat). This review also sheds light on the prospect of BACE1 inhibitors as the most effective therapeutic approach in delaying or preventing AD progression. BACE1 is involved in the progression of AD. The current ongoing or failed clinical trials may help understand the role of BACE1 inhibition in regulating the Aβ load and cognitive status of AD patients.

In Silico Analysis of the L-2-Hydroxyglutarate Dehydrogenase Gene Mutations and Their Biological Impact on Disease Etiology.

The L-2-hydroxyglutarate dehydrogenase (L2HGDH) gene encodes an important mitochondrial enzyme. However, its altered activity results in excessive levels of L-2-hydroxyglutarate, which results in diverse psychiatric features of intellectual disability. In the current study, we executed an in-silico analysis of all reported L2HGDH missense and nonsense variants in order to investigate their biological significance. Among the superimposed 3D models, the highest similarity index for a wild-type structure was shown by the mutant Glu336Lys (87.26%), while the lowest similarity index value was shown by Arg70* (10.00%). Three large active site pockets were determined using protein active site prediction, in which the 2nd largest pocket was shown to encompass the substrate L-2-hydroxyglutarate (L2HG) binding residues, i.e., 89Gln, 195Tyr, 402Ala, 403Gly and 404Val. Moreover, interactions of wild-type and mutant L2HGDH variants with the close functional interactor D2HGDH protein resulted in alterations in the position, number and nature of networking residues. We observed that the binding of L2HG with the L2HGDH enzyme is affected by the nature of the amino acid substitution, as well as the number and nature of bonds between the substrate and protein molecule, which are able to affect its biological activity.

Cluster Model Study into the Catalytic Mechanism of α-Ketoglutarate Biodegradation by the Ethylene-Forming Enzyme Reveals Structural Differences with Nonheme Iron Hydroxylases

Ethylene is an important signaling molecule in plants that triggers the growth of leaves, flowers, and fruits. One of the enzymes involved in the biosynthesis of ethylene is the ethylene-forming enzyme (EFE), which is an usual nonheme iron enzyme that biodegrades α-ketoglutarate into three CO2 molecules and ethylene. As the detailed mechanism of EFE in the biosynthesis of ethylene remains controversial and particularly the function of the co-substrate l-arginine, we decided to pursue a density functional theory study on the possible pathways of the enzyme leading to ethylene biosynthesis and test many possible pathways and mechanisms. A large active site cluster model of 322 atoms was created, which contains all the features of the first- and second-coordination sphere of the active site and substrate (α-ketoglutarate) binding pockets. The calculations identify a persuccinate intermediate that triggers a bifurcation pathway in the enzyme and either react with a molecule of CO2 to form a carbonate or forms a high-valent iron(IV)-oxo species through heterolytic dioxygen bond cleavage. Our studies show that both the bifurcation pathways converge to the same intermediate again and can lead to ethylene products, although the two pathways have different kinetics. Interestingly, our studies also show that the iron(IV)-oxo itself can form a carbonate and ethylene but through much higher barriers. As a matter of fact, these barriers are higher in energy than the typical aliphatic hydroxylation barriers and may not be competitive with arginine hydroxylation. Inclusion of the l-arginine co-substrate into the model leads to minor changes in the structure and fold, but its charge and dipole moment does not seem to affect the first stage of the catalytic cycle. Moreover, the key activation barriers seem less affected by the inclusion of l-arginine into the model. We, therefore, believe that the role of l-arginine is to lock α-ketoglutarate and its products into a tight binding pocket to enable its degradation and to prevent early release of CO2. Our studies show that due to the distinct differences in α-ketoglutarate positioning between different arginine activating nonheme iron dioxygenases in the co-substrate binding pocket and its tighter binding in EFE, we predict that the release of CO2 is prevented in the first stage of the oxygen activation mechanism. This enables attack of CO2 on a persuccinate complex to form carbonate products, leading to ethylene formation. This work gives suggestions on the engineering of EFE into a hydroxylase or improving the ethylene biosynthesis.

Stepwise binding of inhibitors to human cytochrome P450 17A1 and rapid kinetics of inhibition of androgen biosynthesis

Cytochrome P450 (P450) 17A1 catalyzes the 17α-hydroxylation of progesterone and pregnenolone as well as the subsequent lyase cleavage of both products to generate androgens. However, the selective inhibition of the lyase reactions, particularly with 17α-hydroxy pregnenolone, remains a challenge for the treatment of prostate cancer. Here, we considered the mechanisms of inhibition of drugs that have been developed to inhibit P450 17A1, including ketoconazole, seviteronel, orteronel, and abiraterone, the only approved inhibitor used for prostate cancer therapy, as well as clotrimazole, known to inhibit P450 17A1. All five compounds bound to P450 17A1 in a multistep process, as observed spectrally, over a period of 10 to 30 s. However, no lags were observed for the onset of inhibition in rapid-quench experiments with any of these five compounds. Furthermore, the addition of substrate to inhibitor–P450 17A1 complexes led to an immediate formation of product, without a lag that could be attributed to conformational changes. Although abiraterone has been previously described as showing slow-onset inhibition (t1/2 = 30 min), we observed rapid and strong inhibition. These results are in contrast to inhibitors of P450 3A4, an enzyme with a larger active site in which complete inhibition is not observed with ketoconazole and clotrimazole until the changes are completed. Overall, our results indicate that both P450 17A1 reactions—17α-hydroxylation and lyase activity—are inhibited by the initial binding of any of these inhibitors, even though subsequent conformational changes occur.

Nano cobalt ferrite encapsulated‐silica particles bearing melamine as an easily recyclable catalyst for the synthesis of dihydropyrano[2,3‐c]pyrazoles under green conditions

AbstractIn this research, it was displayed an efficient method for synthesis of 6‐amino‐2,4‐dihydropyrano[2,3‐c] pyrazol‐5‐carbonitrile derivatives by using CoFe2O4 silica supported bearing melamine as a magnetic nanocatalyst. This nanocomposite was prepared in several steps and characterized by XRD, FT‐IR, SEM, EDX, VSM, and TGA techniques. The catalyst has a large active site that has functionalized on the surface of nanostructure. The advantages of this magnetic nanocatalyst are simple achievable, effective heterogeneous nanocatalyst, non‐toxic, small amount of catalyst used, resulted products with high yields and short reaction times, easy work up, and reusability. The central core nanoparticles are excellent chemical and magnetic stability. Therefore, the magnetic moment of cobalt ferrite relaxes much slower than the magnetite with the similar particle size.

Density Functional Theory Study into the Reaction Mechanism of Isonitrile Biosynthesis by the Nonheme Iron Enzyme ScoE

The nonheme iron enzyme ScoE catalyzes the biosynthesis of an isonitrile substituent in a peptide chain. To understand details of the reaction mechanism we created a large active site cluster model of 212 atoms that contains substrate, the active oxidant and the first- and second-coordination sphere of the protein and solvent. Several possible reaction mechanisms were tested and it is shown that isonitrile can only be formed through two consecutive catalytic cycles that both use one molecule of dioxygen and α-ketoglutarate. In both cycles the active species is an iron(IV)-oxo species that in the first reaction cycle reacts through two consecutive hydrogen atom abstraction steps: first from the N–H group and thereafter from the C–H group to desaturate the NH-CH2 bond. The alternative ordering of hydrogen atom abstraction steps was also tested but found to be higher in energy. Moreover, the electronic configurations along that pathway implicate an initial hydride transfer followed by proton transfer. We highlight an active site Lys residue that is shown to donate charge in the transition states and influences the relative barrier heights and bifurcation pathways. A second catalytic cycle of the reaction of iron(IV)-oxo with desaturated substrate starts with hydrogen atom abstraction followed by decarboxylation to give isonitrile directly. The catalytic cycle is completed with a proton transfer to iron(II)-hydroxo to generate the iron(II)-water resting state. The work is compared with experimental observation and previous computational studies on this system and put in a larger perspective of nonheme iron chemistry.

Time-dependent growth of the dendritic silver prepared using square wave voltammetry technique for methylene blue photodegradation

Abstract Silver (Ag) particle is a promising photocatalyst material with relatively high catalytic activity and good absorption in the visible light region. A dendritic structure of Ag has been studied in the purpose to enhance photocatalytic activity due to a large surface area and active site number of the metallic Ag particles. In this work, the Ag dendritic structure was synthesized from a surfactant-free electrolyte using the square wave voltammetry technique. The time-dependent growth of the Ag dendrites and their photocatalytic activity on methylene blue (MB) photodegradation are reported. Morphological analysis exhibits the fractal dendritic structure of Ag was found to continuously grow by increasing the deposition time. The Ag dendrites showed a low charge transfer resistance (366.21 Ω) and high specific capacitance (2.09 F/g). A high rate of MB degradation (45.57%) under ultraviolet irradiation indicated that the Ag dendrites produced using this technique are effective for the photocatalytic degradation of MB dye.

Mechanism of Oxidative Ring‐Closure as Part of the Hygromycin Biosynthesis Step by a Nonheme Iron Dioxygenase

AbstractNonheme iron dioxygenases catalyze vital reactions for biosystems including the biosynthesis of antibiotics. One such enzyme, namely the hygromycin biosynthesis enzyme (HygX), performs an oxidative ring‐closure reaction to form an ortho−ester product, which is a relevant reaction step for drug synthesis and biotechnology. To understand the selective reaction mechanism of oxidative ring‐closure to form ortho−ester products in HygX, we investigated its catalytic reaction mechanism leading to various products. Large active site cluster models were set‐up and various pathways for substrate activation have been calculated. The work identifies a high‐valent iron(IV)‐oxo species in the quintet spin state as the active oxidant that selectively abstracts a proton of an alcohol group of the substrate, which is followed by a hydrogen atom abstraction from a tertiary C−H group and rapid electron transfer. The latter‐formed biradical intermediate rearranges to form the desaturated ring‐closed product. The calculations show that an active site Lys residue donates positive charge to the metal−oxo group and guides the reaction to a chemoselective desaturation pathway.

Glutarate Hydroxylation by the Carbon Starvation-Induced Protein D: A Computational Study into the Stereo- and Regioselectivities of the Reaction.

The carbon starvation-induced protein D (CsiD) is a recently characterized iron(II)/α-ketoglutarate-dependent oxygenase that activates a glutarate molecule as substrate at the C2 position to exclusively produce (S)-2-hydroxyglutarate products. This selective hydroxylation reaction by CsiD is an important component of the lysine biodegradation pathway in Escherichia coli; however, little is known on the details and the origin of the selectivity of the reaction. So far, experimental studies failed to trap and characterize any short-lived catalytic cycle intermediates. As no computational studies have been reported on this enzyme either, we decided to investigate the chemical reaction mechanism of glutarate activation by an iron(IV)-oxo model of the CsiD enzyme. In this work, we present a density functional theory study on a large active site cluster model of CsiD and investigate the glutarate hydroxylation pathways by a high-valent iron(IV)-oxo species leading to (S)-2-hydroxyglutarate, (R)-2-hydroxyglutarate, and 3-hydroxyglutarate. In agreement with experimental observation, the favorable product channel leads to pro-S C2-H hydrogen atom abstraction to form (S)-2-hydroxyglutarate. The reaction is stepwise with a hydrogen atom abstraction by an iron(IV)-oxo species followed by OH rebound from a radical intermediate. The work presented in this paper shows that despite the fact that the C-H bond strengths at the C2 and C3 positions of glutarate are similar in the gas phase, substrate binding and positioning guide the reaction to an enantioselective reaction process by destabilizing the hydrogen atom abstraction pathways for the pro-R C2-H and C3-H positions. Our studies predict the chemical properties of the iron(IV)-oxo species and its rate constants with glutarate and deuterated-glutarate. Moreover, the work shows little protein motions during the catalytic process, while the substrate entrance into the substrate binding pocket appears to be guided by three active site arginine residues that position the substrate for pro-S C2-H hydrogen atom abstraction. Finally, the calculations show that irrespective of the position of the substrate and what C-H bond is closest to the metal center, the lowest energy pathway is for a selective pro-S C2-H hydrogen atom abstraction.

Revealing Donor Substrate-Dependent Mechanistic Control on DXPS, an Enzyme in Bacterial Central Metabolism.

The thiamin diphosphate-dependent enzyme 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) catalyzes the formation of DXP from pyruvate (donor) and d-glyceraldehyde 3-phosphate (d-GAP, acceptor). DXPS is essential in bacteria but absent in human metabolism, highlighting it as a potential antibacterial drug target. The enzyme possesses unique structural and mechanistic features that enable development of selective inhibition strategies and raise interesting questions about DXPS function in bacterial pathogens. DXPS distinguishes itself within the ThDP enzyme class by its exceptionally large active site and random sequential mechanism in DXP formation. In addition, DXPS displays catalytic promiscuity and relaxed acceptor substrate specificity, yet previous studies have suggested a preference for pyruvate as the donor substrate when d-GAP is the acceptor substrate. However, such donor specificity studies are potentially hindered by a lack of knowledge about specific, alternative donor–acceptor pairs. In this study, we exploited the promiscuous oxygenase activity of DXPS to uncover alternative donor substrates for DXPS. Characterization of glycolaldehyde, hydroxypyruvate, and ketobutyrate as donor substrates revealed differences in stabilization of enzyme-bound intermediates and acceptor substrate usage, illustrating the influence of the donor substrate on reaction mechanism and acceptor specificity. In addition, we found that DXPS prevents abortive acetyl-ThDP formation from a DHEThDP carbanion/enamine intermediate, similar to transketolase, supporting the potential physiological relevance of this intermediate on DXPS. Taken together, these results offer clues toward alternative roles for DXPS in bacterial pathogen metabolism.

Structures of LnmK, a Bifunctional Acyltransferase/Decarboxylase, with Substrate Analogues Reveal the Basis for Selectivity and Stereospecificity

LnmK stereospecifically accepts (2R)-methylmalonyl-CoA, generating propionyl-S-acyl carrier protein to support polyketide biosynthesis. LnmK and its homologues are the only known enzymes that carry out a decarboxylation (DC) and acyl transfer (AT) reaction in the same active site as revealed by structure-function studies. Substrate-assisted catalysis powers LnmK, as decarboxylation of (2R)-methylmalonyl-CoA generates an enolate capable of deprotonating active site Tyr62, and the Tyr62 phenolate subsequently attacks propionyl-CoA leading to a propionyl-O-LnmK acyl-enzyme intermediate. Due to the inherent reactivity of LnmK and methylmalonyl-CoA, a substrate-bound structure could not be obtained. To gain insight into substrate specificity, stereospecificity, and catalytic mechanism, we determined the structures of LnmK with bound substrate analogues that bear malonyl-thioester isosteres where the carboxylate is represented by a nitro or sulfonate group. The nitro-bearing malonyl-thioester isosteres bind in the nitronate form, with specific hydrogen bonds that allow modeling of the (2R)-methylmalonyl-CoA substrate and rationalization of stereospecificity. The sulfonate isosteres bind in multiple conformations, suggesting the large active site of LnmK allows multiple binding modes. Considering the smaller malonyl group has more conformational freedom than the methylmalonyl group, we hypothesized the active site can entropically screen against catalysis with the smaller malonyl-CoA substrate. Indeed, our kinetic analysis reveals malonyl-CoA is accepted at 1% of the rate of methylmalonyl-CoA. This study represents another example of how our nitro- and sulfonate-bearing methylmalonyl-thioester isosteres are of use for elucidating enzyme-substrate binding interactions and revealing insights into catalytic mechanism. Synthesis of a larger panel of analogues presents an opportunity to study enzymes with complicated structure-function relationships such as acyl-CoA carboxylases, trans-carboxytransferases, malonyltransferases, and β-ketoacylsynthases.

Crystal structures of a novel family IV esterase in free and substrate-bound form.

Bacterial lipolytic enzymes of family IV are homologs of the mammalian hormone-sensitive lipases (HSL) and have been successfully used for various biotechnological applications. The broad substrate specificity and ability for enantio-, regio-, and stereoselective hydrolysis are remarkable features of enzymes from this class. Many crystal structures are available for esterases and lipases, but structures of enzyme-substrate or enzyme-inhibitor complexes are less frequent although important to understand the molecular basis of enzyme-substrate interaction and to rationalize biochemical enzyme characteristics. Here, we report on the structures of a novel family IV esterase isolated from a metagenomic screen, which shows a broad substrate specificity. We solved the crystal structures in the apo form and with a bound substrate analogue at 1.35 and 1.81Å resolution, respectively. This enzyme named PtEst1 hydrolyzed more than 60 out 96 structurally different ester substrates thus being substrate promiscuous. Its broad substrate specificity is in accord with a large active site cavity, which is covered by an α-helical cap domain. The substrate analogue methyl 4-methylumbelliferyl hexylphosphonate was rapidly hydrolyzed by the enzyme leading to a complete inactivation caused by covalent binding of phosphinic acid to the catalytic serine. Interestingly, the alcohol leaving group 4-methylumbelliferone was found remaining in the active site cavity, and additionally, a complete inhibitor molecule was found at the cap domain next to the entrance of the substrate tunnel. This unique situation allowed gaining valuable insights into the role of the cap domain for enzyme-substrate interaction of esterases belonging to family IV. DATABASE: Structural data of PtEst1 are available in the worldwide protein data bank (https://www.rcsb.org) under the accession codes: 6Z68 (apo-PtEst1) and 6Z69 (PtEst1-inhibitor complex).