Oxyanion Hole Research Articles

Characterization of a Novel Moderately Thermophilic Solvent-Tolerant Esterase Isolated From a Compost Metagenome Library.

A novel esterase, EstCS1, was isolated from a compost metagenomics library. The EstCS1 protein, which consists of 309 amino acid residues with an anticipated molecular mass of 34 kDa, showed high amino acid sequence identities to predicted esterases and alpha/beta hydrolases (59%) from some cultured bacteria and to predicted lipases/esterases from uncultured bacteria. The phylogenetic analysis suggested that the EstCS1 belongs to the hormone-sensitive lipase family of lipolytic enzyme classification and contains a catalytic triad including Ser155–Asp255–His285. The Ser155 residue of the catalytic triad in the EstCS1 was located in the consensus active-site motif, GXSXG. Besides, a conserved HGGG motif placed in an oxyanion hole of the hormone-sensitive lipase family was discovered, too. The EstCS1 demonstrated the highest activity toward p-nitrophenyl propionate (C3) and caproate (C6) and was normally stable up to 60°C with optimal activity at 50°C. In addition, an optimal activity was observed at pH 8, and the EstCS1 possessed its stability within the pH range between 5 and 10. Interestingly, EstCS1 had an outstanding stability in up to 30% (v/v) organic solvents and activity over 50% in the presence of 50% (v/v) acetone, ethanol, dimethyl sulfoxide (DMSO), and N,N-dimethylformamide. The EstCS1 hydrolyzed sterically hindered tertiary alcohol esters of t-butyl acetate and linalyl acetate. Considering the properties, such as the moderate thermostability, stability against organic solvents, and activity toward esters of tertiary alcohols, the EstCS1 will be worthwhile to be used for organic synthesis and related industrial applications.

YqfB protein from Escherichia coli: an atypical amidohydrolase active towards N4-acylcytosine derivatives

Human activating signal cointegrator homology (ASCH) domain-containing proteins are widespread and diverse but, at present, the vast majority of those proteins have no function assigned to them. This study demonstrates that the 103-amino acid Escherichia coli protein YqfB, previously identified as hypothetical, is a unique ASCH domain-containing amidohydrolase responsible for the catabolism of N4-acetylcytidine (ac4C). YqfB has several interesting and unique features: i) it is the smallest monomeric amidohydrolase described to date, ii) it is active towards structurally different N4-acylated cytosines/cytidines, and iii) it has a high specificity for these substrates (kcat/Km up to 2.8 × 106 M−1 s−1). Moreover, our results suggest that YqfB contains a unique Thr-Lys-Glu catalytic triad, and Arg acting as an oxyanion hole. The mutant lacking the yqfB gene retains the ability to grow, albeit poorly, on N4-acetylcytosine as a source of uracil, suggesting that an alternative route for the utilization of this compound exists in E. coli. Overall, YqfB ability to hydrolyse various N4-acylated cytosines and cytidines not only sheds light on the long-standing mystery of how ac4C is catabolized in bacteria, but also expands our knowledge of the structural diversity within the active sites of amidohydrolases.

High-resolution structure of intramolecularly proteolyzed human mucin-1 SEA domain

SEA domains are ubiquitous in large proteins associated with highly glycosylated environments. Certain SEA domains undergo intramolecular proteolysis involving a nucleophilic attack of a serine hydroxyl group on the preceding glycine carbonyl. The mucin-1 (MUC1) SEA domain has been extensively investigated as a model of intramolecular proteolysis. Since neither a general base, a general acid, nor an oxyanion hole could be identified in MUC1 SEA, it has been suggested that proteolysis is accelerated by a non-planarity of the scissile peptide bond imposed by protein folding. A reactant distorted peptide bond has been also invoked to explain the autoproteolysis of several unrelated proteins. However, the only evidence of peptide distortion in MUC1 SEA stems from molecular dynamic simulations of the reactant modeled upon a single NMR structure of the cleaved product. We report the first high-resolution X-ray structure of cleaved MUC1 SEA. Structural comparison with uncleaved SEA domains suggests that the number of residues evolutionarily inserted in the cleaved loop of MUC1 SEA precludes the formation of a properly hydrogen-bonded beta turn. By sequence analysis, we show that this conformational frustration is shared by all known cleaved SEA domains. In addition, alternative conformations of the uncleaved precursor could be modeled in which the scissile peptide bond is planar. The implications of these structures for autoproteolysis are discussed in the light of the previous research on autoproteolysis.

Structural Basis for Substrate Specificity and Carbapenemase Activity of OXA-48 Class D β-Lactamase.

Carbapenem-hydrolyzing class D β-lactamases (CHDLs) are a diverse family of enzymes that are rapidly becoming the predominant cause of bacterial resistance against β-lactam antibiotics in many regions of the world. OXA-48, an atypical member of CHDLs, is one of the most frequently observed in the clinic and exhibits a unique substrate profile. We applied X-ray crystallography to OXA-48 complexes with multiple β-lactam antibiotics to elucidate this enzyme's carbapenemase activity and its preference of imipenem over meropenem and other substrates such as cefotaxime. In particular, we obtained acyl-enzyme complexes of OXA-48 with imipenem, meropenem, faropenem, cefotaxime, and cefoxitin, and a product complex with imipenem. Importantly, the product complex captures a key reaction milestone with the newly generated carboxylate group still in the oxyanion hole, and represents the first such complex with a wild-type serine β-lactamase. A potential hydrogen bond is observed between the two carboxylate groups from the product and the carbamylated Lys73, representing the stage immediately after the breakage of the acyl-enzyme bond where the product carboxylate would be neutral. The placement of the product carboxylate also illustrates the approximate transient location of the deacylation water that has long eluded structural characterization in class D β-lactamases. Additionally, comparing the product complex with the acyl-enzyme intermediates provides new insights into the various mechanisms by which specific side chain groups hinder the access of the deacylation water to the acyl-enzyme linkage, especially in meropenem. Taken together, these data offer valuable information on the substrate specificity of OXA-48 and the catalytic mechanism of CHDLs.

Role of the I16-D194 ionic interaction in the trypsin fold

Activity in trypsin-like proteases is the result of proteolytic cleavage at R15 followed by an ionic interaction that ensues between the new N terminus of I16 and the side chain of the highly conserved D194. This mechanism of activation, first proposed by Huber and Bode, organizes the oxyanion hole and primary specificity pocket for substrate binding and catalysis. Using the clotting protease thrombin as a relevant model, we unravel contributions of the I16-D194 ionic interaction to Na+ binding, stability of the transition state and the allosteric E*-E equilibrium of the trypsin fold. The I16T mutation abolishes the I16-D194 interaction and compromises the architecture of the oxyanion hole. The D194A mutation also abrogates the I16-D194 interaction but, surprisingly, has no effect on the architecture of the oxyanion hole that remains intact through a new H-bond established between G43 and G193. In both mutants, loss of the I16-D194 ionic interaction compromises Na+ binding, reduces stability of the transition state, collapses the 215–217 segment into the primary specific pocket and abrogates the allosteric E*-E equilibrium in favor of a rigid conformation that binds ligand at the active site according to a simple lock-and-key mechanism. These findings refine the structural role of the I16-D194 ionic interaction in the Huber-Bode mechanism of activation and reveal a functional linkage with the allosteric properties of the trypsin fold like Na+ binding and the E*-E equilibrium.

Studies on the interactions between nicosulfuron and degradation enzymes

The interactions between nicosulfuron and two degradation enzymes (vegetative catalase 1 and manganese ABC transporter) from the Bacillus subtilis YB1 strain were studied and molecular docking simulations and surface plasmon resonance (SPR) were used to research their specific interaction patterns and affinities. The results showed that vegetative catalase 1 and manganese ABC transporter bound specifically to nicosulfuron and that the former binding ability was stronger than that of the latter. The manganese ABC transporter mainly interacted with nicosulfuron by strong hydrophobic interactions and hydrogen bonds (oxyanion hole), while vegetative catalase 1 formed a strong hydrophobic interaction with nicosulfuron in its main channel and hydrogen bond with nicosulfuron in the side chains. Vegetative catalase 1 and manganese ABC transporter catalyze nicosulfuron degradation, and molecular docking simulations and SPR are good methods for studying molecular interactions, which could make a foundation for the study of degradation mechanisms of enzymes.

Structure and mechanism of piperideine-6-carboxylate dehydrogenase from Streptomyces clavuligerus.

The core of β-lactam antibiotics originates from amino acids of primary metabolism in certain microorganisms. β-Lactam-producing bacteria, including Streptomyces clavuligerus, synthesize the precursor of the amino acid α-aminoadipic acid by the catabolism of lysine in two steps. The second reaction, the oxidation of piperideine-6-carboxylate (or its open-chain form α-aminoadipate semialdehyde) to α-aminoadipic acid, is catalysed by the NAD+-dependent enzyme piperideine-6-carboxylate dehydrogenase (P6CDH). This structural study, focused on ligand binding and catalysis, presents structures of P6CDH from S. clavuligerus in its apo form and in complexes with the cofactor NAD+, the product α-aminoadipic acid and a substrate analogue, picolinic acid. P6CDH adopts the common aldehyde dehydrogenase fold, consisting of NAD-binding, catalytic and oligomerization domains. The product binds in the oxyanion hole, close to the catalytic residue Cys299. Clear density is observed for the entire cofactor, including the nicotinamide riboside, in the binary complex. NAD+ binds in an extended conformation with its nicotinamide ring overlapping with the binding site of the carboxylate group of the product, implying that the conformation of the cofactor may change during catalysis. The binding site of the substrate analogue overlaps with that of the product, suggesting that the cyclic form of the substrate, piperideine-6-carboxylate, may be accepted as a substrate by the enzyme. The catalytic mechanism and the roles of individual residues are discussed in light of these results.

Gene cloning, expression and homology modeling of first fibrinolytic enzyme from mushroom (Cordyceps militaris)

Fibrinolytic enzymes are important thrombolytic agents for blood-clotting disorders like cardiovascular diseases. Availability of novel recombinant fibrinolytic enzymes can overcome the shortcomings of current thrombolytic drugs. With the objective of facilitating their cost-effective production for therapeutic applications and for gaining deeper insight into their structure-function, we have cloned and expressed the first fibrinolytic protease gene from Cordyceps militaris. Cordyceps militaris fibrinolytic enzyme (CmFE) has one open reading frame of 759 bp encoding “pre-pro-protein” of 252 amino acids. Recombinant CmFE was expressed as 28 kDa extracellular enzyme in Pichia pastoris which was capable of degrading fibrin clot. A structure homology model of CmFE was developed using urokinase-type plasminogen activator. The active site contains catalytic triad His41, Asp83, Ser177 and consensus sequence of GDSGG. The substrate binding residues are Asp (171), Gly (194) and Ser (192). Its trypsin-like specificity is determined by the critical Asp171 in S1 subsite. The “oxyanion hole” is formed by backbone amide hydrogen atoms of Gly-175 and Ser-177. CmFE contains six conserved cysteines forming three disulfide linkages. This is the first study describing cloning, expression and prediction of structure-function relationship of a mushroom fibrinolytic protease. Hence it has great relevance in application of fibrinolytic enzymes as thrombolytic agents.

Aspectos estructurales y funcionales de la N-Succinil-L, L-diaminopimelato desuccinilasa, una enzima clave para el crecimiento bacteriano y un blanco para el control antimicrobiano

La N-Succinil-L, L-diaminopimelato desuccinilasa (DapE) es una amidohidrolasa dependiente de iones de zinc, homodimérica estricta, que cataliza la descomposición del N-succinil-L, L-2,6-diaminopimelato (NSDAP), en succinato y diaminopimelato (DAP). Reacción que constituye la única fuente de meso-diaminopimelato (mDAP) y L-Lys en la mayoría de las bacterias. DapE es esencial para el crecimiento bacteriano y un blanco farmacológico antimicrobiano. El desarrollo de los inhibidores anti-DapE debe tener en cuenta las propiedades dinámicas de la enzima. Se buscan compuestos que interfieran con la formación del agujero del oxianión, en donde participan grupos de ambas subunidades del dímero, que se acomoda en posición catalítica mediante el cambio conformacional de la enzima de un estado abierto a uno cerrado, después de la unión del sustrato; estabilizando a los intermediarios de reacción y produciendo un descenso en la energía de activación. Con base en el análisis cristalográfico y el acoplamiento del sustrato en DapE que se presenta en este trabajo, se discute el papel de la flexibilidad conformacional de la enzima en la hidrólisis del sustrato. Se observa que tanto el grupo carbonilo del sustrato es susceptible al ataque como una molécula de agua ubicada en el sitio activo y se encuentran cercanos a la trayectoria de ataque, en el ángulo de Bürgi-Dunitz.

Antennae-specific carboxylesterase genes from Indian meal moth: Identification, tissue distribution and the response to semiochemicals

In the olfactory recognition system of insects, carboxylesterases (CXEs) in insect antennae play crucial roles in inactivating the chemical signals. Identification of antennae-specific CXEs provides the molecular basis of odorant degradation and signal inactivation in insects through which potential pest control strategies can be further studied. Although pheromone-based attract-and-kill has been proved to be an effective method to monitor and control Plodia interpunctella, very few reports have focused on the degradation of pheromone as well as the host volatiles and environmental odorants. In the present study, we identified 28 candidate P. interpunctella carboxylesterases (PintCXEs) from our previously reported antennal transcriptome data. Almost all full-length PintCXEs displayed the characteristics of conserved motifs found in other insect CXEs, such as the oxyanion hole residues (Gly-Gly-Ala), the catalytic triad (Ser-Glu-His), and the conserved pentapeptide Gly-X-Ser-X-Gly. Through phylogenetic analysis, two major groups of PintCXEs were identified, including the moth antennal esterases and mitochondrial and cytosolic esterases. Further gene expression profiling revealed that 13 PintCXEs (PintCXE2, PintCXE4, PintCXE9, PintCXE11, PintCXE13, PintCXE15-21, PintCXE25) were highly expressed in antennae, indicating that these PintCXEs were potentially involved in odorant degradation. Among these PintCXEs, PintCXE2, PintCXE4, PintCXE11-12, PintCXE15 and PintCXE17 were male-specific, while PintCXE9, PintCXE13, PintCXE20 and PintCXE25 were highly expressed in female antennae compared with the male antennae. The expressions of PintCXE19 and PintCXE25 were significantly up-regulated by ester treatment, suggesting that they could be involved in degradation of sex pheromone and environment esters. Our results lay a solid foundation for better understanding the olfactory inactivation system of insects.

The modular structure of α/β-hydrolases.

The α/β-hydrolase fold family is highly diverse in sequence, structure and biochemical function. To investigate the sequence-structure-function relationships, the Lipase Engineering Database (https://led.biocatnet.de) was updated. Overall, 280638 protein sequences and 1557 protein structures were analysed. All α/β-hydrolases consist of the catalytically active core domain, but they might also contain additional structural modules, resulting in 12 different architectures: core domain only, additional lids at three different positions, three different caps, additional N- or C-terminal domains and combinations of N- and C-terminal domains with caps and lids respectively. In addition, the α/β-hydrolases were distinguished by their oxyanion hole signature (GX-, GGGX- and Y-types). The N-terminal domains show two different folds, the Rossmann fold or the β-propeller fold. The C-terminal domains show a β-sandwich fold. The N-terminal β-propeller domain and the C-terminal β-sandwich domain are structurally similar to carbohydrate-binding proteins such as lectins. The classification was applied to the newly discovered polyethylene terephthalate (PET)-degrading PETases and MHETases, which are core domain α/β-hydrolases of the GX- and the GGGX-type respectively. To investigate evolutionary relationships, sequence networks were analysed. The degree distribution followed a power law with a scaling exponent γ=1.4, indicating a highly inhomogeneous network which consists of a few hubs and a large number of less connected sequences. The hub sequences have many functional neighbours and therefore are expected to be robust toward possible deleterious effects of mutations. The cluster size distribution followed a power law with an extrapolated scaling exponent τ=2.6, which strongly supports the connectedness of the sequence space of α/β-hydrolases. DATABASE: Supporting data about domains from other proteins with structural similarity to the N- or C-terminal domains of α/β-hydrolases are available in Data Repository of the University of Stuttgart (DaRUS) under doi: https://doi.org/10.18419/darus-458.

Arabidopsis immunity regulator EDS1 in a PAD4/SAG101-unbound form is a monomer with an inherently inactive conformation

In plant innate immunity, enhanced disease susceptibility 1 (EDS1) integrates all pathogen-induced signals transmitted by TIR-type NLR receptors. Driven by an N-terminal α/β-hydrolase-fold domain with a protruding interaction helix, EDS1 assembles with two homologs, phytoalexin-deficient 4 (PAD4) and senescence-associated gene 101 (SAG101). The resulting heterodimers are critical for EDS1 function and structurally well characterized. Here, we resolve solution and crystal structures of unbound Arabidopsis thaliana EDS1 (AtEDS1) using nanobodies for crystallization. These structures, together with gel filtration and immunoprecipitation data, show that PAD4/SAG101-unbound AtEDS1 is stable as a monomer and does not form the homodimers recorded in public databases. Its PAD4/SAG101 anchoring helix is disordered unless engaged in protein/protein interactions. As in the complex with SAG101, monomeric AtEDS1 has a substrate-inaccessible esterase triad with a blocked oxyanion hole and without space for a covalent acyl intermediate. These new structures suggest that the AtEDS1 monomer represents an inactive or pre-activated ground state.

Cloning and Expression of Genes for Biodegrading Nodularin by Sphingopyxis sp. USTB-05.

Biodegradation is efficient for removing cyanobacterial toxins, such as microcystins (MCs) and nodularin (NOD). However, not all the microbial strains with the microcystin-biodegrading enzymes MlrA and MlrC could biodegrade NOD. Studies on genes and enzymes for biodegrading NOD can reveal the function and the biodegradation pathway of NOD. Based on successful cloning and expression of the USTB-05-A and USTB-05-C genes from Sphingopyxis sp. USTB-05, which are responsible for the biodegradation of MCs, the pathway for biodegrading NOD by these two enzymes was investigated in this study. The findings showed that the enzyme USTB-05-A converted cyclic NOD (m/z 825.4516) into its linear type as the first product by hydrolyzing the arginine and Adda peptide bond, and that USTB-05-C cut off the Adda and glutamic acid peptide bond of linearized NOD (m/z 843.4616) and produced dimeric Adda (m/z 663.4377) as the second product. Further, based on the homology modeling of enzyme USTB-05-A, site-directed mutants of USTB-05-A were constructed and seven crucial sites for enzyme USTB-05-A activity were found. A complete enzymatic mechanism for NOD biodegradation by USTB-05-A in the first step was proposed: glutamic acid 172 and histidine 205 activate a water molecule facilitating a nucleophilic attack on the arginine and Adda peptide bond of NOD; tryptophan 176 and tryptophan 201 contact the carboxylate side chain of glutamic acid 172 and accelerate the reaction rates; and histidine 260 and asparagine 264 function as an oxyanion hole to stabilize the transition states.

MD simulations reveal alternate conformations of the oxyanion hole in the Zika virus NS2B/NS3 protease.

Recent crystallography studies have shown that the binding site oxyanion hole plays an important role in inhibitor binding, but can exist in two conformations (active/inactive). We have undertaken molecular dynamics (MD) calculations to better understand oxyanion hole dynamics and thermodynamics. We find that the Zika virus (ZIKV) NS2B/NS3 protease maintains a stable closed conformation over multiple 100-ns conventional MD simulations in both the presence and absence of inhibitors. The S1, S2, and S3 pockets are stable as well. However, in two of eight simulations, the A132-G133 peptide bond in the binding pocket of S1' spontaneously flips to form a 310 -helix that corresponds to the inactive conformation of the oxyanion hole, and then maintains this conformation until the end of the 100-ns conventional MD simulations without inversion of the flip. This conformational change affects the S1' pocket in ZIKV NS2B/NS3 protease active site, which is important for small molecule binding. The simulation results provide evidence at the atomic level that the inactive conformation of the oxyanion hole is more favored energetically when no specific interactions are formed between substrate/inhibitor and oxyanion hole residues. Interestingly, however, transition between the active and inactive conformation of the oxyanion hole can be observed by boosting the valley potential in accelerated MD simulations. This supports a proposed induced-fit mechanism of ZIKV NS2B/NS3 protease from computational methods and provides useful direction to enhance inhibitor binding predictions in structure-based drug design.

Structural and functional analyses of the lipase CinB from Enterobacter asburiae

Lipases are widely present in various plants, animals and microorganisms, constituting a large category of enzymes. They have the ability to catalyze the cleavage of ester bonds. The lipase CinB from Enterobacter asburiae (E. asburiae) is an acetyl esterase. The primary amino acid sequence suggests that the EaCinB protein belongs to the α/β-hydrolase (ABH) superfamily of the esterase/lipase superfamily. However, its molecular functions have not yet been determined. Here, we report the crystal structure of E. asburiae CinB at a 1.45 Å resolution. EaCinB contains a signal peptide, cap domain and catalytic domain. The active site of EaCinB contains the catalytic triad (Ser180-His307-Asp277) on the catalytic domain. The oxyanion hole is composed of Gly106 and Gly107 within the conserved sequence motif HGGG (amino acid residues 106–109). The substrate is accessible between the α1 and α2 helices or the α1 helix and catalytic domain. Narrow substrate pockets are formed by the α2 helix of the cap domain. Site-directed mutagenesis showed that EaCinB-W208H exhibits a higher catalytic ability than EaCinB-WT by approximately nine times. Our results provide insight into the molecular function of EaCinB.

Recognition of the β-lactam carboxylate triggers acylation of Neisseria gonorrhoeae penicillin-binding protein 2

Resistance of Neisseria gonorrhoeae to extended-spectrum cephalosporins (ESCs) has become a major threat to human health. The primary mechanism by which N. gonorrhoeae becomes resistant to ESCs is by acquiring a mosaic penA allele, encoding penicillin-binding protein 2 (PBP2) variants containing up to 62 mutations compared with WT, of which a subset contribute to resistance. To interpret molecular mechanisms underpinning cephalosporin resistance, it is necessary to know how PBP2 is acylated by ESCs. Here, we report the crystal structures of the transpeptidase domain of WT PBP2 in complex with cefixime and ceftriaxone, along with structures of PBP2 in the apo form and with a phosphate ion bound in the active site at resolutions of 1-7-1.9 Å. These structures reveal that acylation of PBP2 by ESCs is accompanied by rotation of the Thr-498 side chain in the KTG motif to contact the cephalosporin carboxylate, twisting of the β3 strand to form the oxyanion hole, and rolling of the β3-β4 loop toward the active site. Recognition of the cephalosporin carboxylate appears to be the key trigger for formation of an acylation-competent state of PBP2. The structures also begin to explain the impact of mutations implicated in ESC resistance. In particular, a G545S mutation may hinder twisting of β3 because its side chain hydroxyl forms a hydrogen bond with Thr-498. Overall, our data suggest that acylation is initiated by conformational changes elicited or trapped by binding of ESCs and that these movements are restricted by mutations associated with resistance against ESCs.

Multiscale Computational Study on the Catalytic Mechanism of the Nonmetallo Amidase Maleamate Amidohydrolase (NicF).

Maleamate amidohydrolase (NicF) is a key enzyme in vitamin B3 metabolism that catalyzes the hydrolysis of maleamate to produce maleic acid and ammonia. Unlike most members from the amidohydrolase superfamily it does not require a metal ion. Here, we use multiscale computational enzymology to investigate the catalytic mechanism, substrate binding, oxyanion hole, and roles of key active site residues of NicF from Bordetella bronchiseptica. In particular, molecular dynamics (MD) simulations, quantum mechanics/molecular mechanics (QM/MM) and QTAIM methods have been applied. The mechanism of the NicF-catalyzed reaction proceeds by a nucleophilic addition-elimination sequence involving the formation of a thioester enzyme intermediate (IC2 in stage 1) followed by hydrolysis of the thioester bond to form the products (stage 2). Consequently, the formation of IC2 in stage 1 is the rate-limiting step with a barrier of 88.8 kJ·mol-1 relative to the reactant complex, RC. Comparisons with related metal-dependent enzymes, particularly the zinc-dependent nicotinamidase from Streptococcus pneumonia (SpNic), have also been made to further illustrate unique features of the present mechanism. Along with -NH- donor groups of the oxyanion hole (i.e., HN-Thr146, HN-Cys150), the active site β-hydroxyl of threonine (HO-βThr146) is concluded to play a role in stabilizing the carbonyl oxygen of maleamate during the mechanism.

Synthesis, molecular docking, and biological evaluation of 3-oxo-2-tolylhydrazinylidene-4,4,4-trifluorobutanoates bearing higher and natural alcohol moieties as new selective carboxylesterase inhibitors

To search for effective and selective inhibitors of carboxylesterase (CES), a series of 3-oxo-2-tolylhydrazinylidene-4,4,4-trifluorobutanoates bearing higher or natural alcohol moieties was synthesized via pre-transesterification of ethyl trifluoroacetylacetate with alcohols to isolate transesterificated oxoesters as lithium salts, which were then subjected to azo coupling with tolyldiazonium chloride. Inhibitory activity against porcine liver CES, along with two structurally related serine hydrolases, acetylcholinesterase and butyrylcholinesterase, were investigated using enzyme kinetics and molecular docking. Kinetics studies demonstrated that the tested keto-esters are reversible and selective mixed-type CES inhibitors. Analysis of X-ray crystallographic data together with our IR and NMR spectra and QM calculations indicated that the Z-isomers were the most stable. The kinetic data were well explained by the molecular docking results of the Z-isomers, which showed specific binding of the compounds in the CES catalytic active site with carbonyl oxygen atoms in the oxyanion hole and non-specific binding outside it. Some compounds were studied as inhibitors of the main human isozymes involved in biotransformation of ester-containing drugs, hCES1 and hCES2. Esters of geraniol (3d) and adamantol (3e) proved to be highly active and selective inhibitors of hCES2, inhibiting the enzyme in the nanomolar range, whereas esters of borneol (3f) and isoborneol (3g) were more active and selective against hCES1. Computational ADMET studies revealed that all test compounds had excellent intestinal absorption, medium blood-brain barrier permeability, and low hERG liability risks. Moreover, all test compounds possessed radical-scavenging properties and low acute toxicity. Overall, the results indicate that members of this novel series of esters have the potential to be good candidates as hCES1 or hCES2 inhibitors for biomedicinal applications.

Structural basis of microcystinase activity for biodegrading microcystin-LR

Microcystinase (MlrA) catalyzes the first and most important biodegradation step of hepatotoxic microcystin-LR (MC-LR) produced and released from cyanobacterial cells, and the underlying catalytic mechanism is not completely understood yet. MlrA was postulated previously to be a metalloprotease with an active site of H260AIH263NE265, a variant of the common metal-binding motif of HEXXH. Through comparison with representative modes in HEXXH-containing metalloproteases, molecular dynamics simulation, homology modeling, and docking, the active sites of MlrA involved in the MC-LR biodegradation by Sphingomonas sp. USTB-05 were predicted. Site-directed mutants of MlrA were constructed for verification then. The results show that MlrA is likely not a metalloprotease, but a glutamate protease belonging to type II CAAX prenyl endopeptidases. Combined with the biodegradation of MC-LR by MlrA and its mutants, a complete enzymatic mechanism for MC-LR biodegradation by MlrA is proposed: Glu172 and His205 activate a water molecule facilitating a nucleophilic attack on the Adda-Arg peptide bond of MC-LR; Trp176 and Trp201 contact the carboxylate side chain of Glu172and, by raising its pKa potentially, accelerate the reaction rates; His260 and Asn264 (located in the previous postulated active center of H260AIH263NE265) function as an oxyanion hole to stabilize the transition states. This study reveals the enzymatic mechanism of MlrA for catalyzing MC-LR in both the representative modes and the experiments of site-directed mutagenesis.

Human Fatty Acid Synthase: A Computational Study of the Transfer of the Acyl Moieties from MAT to the ACP Domain

AbstractHuman Fatty Acid Synthase (hFAS) is a multidomain enzyme responsible for the biosynthesis of saturated fatty acids, which are crucial for numerous biological processes. During the biosynthetic reaction, hFAS incorporates acetyl and malonyl moieties through the action of its malonyl‐acetyl transferase (MAT) domain. These precursor molecules are transferred from CoA to the acyl‐carrier protein (ACP) domain by a two‐stage ping‐pong catalytic mechanism. The first stage, during which the acyl moieties are relocated from CoA to the MAT domain, has been elucidated in a previous work of ours. In this study, we employ QM/MM calculations at the DLPNO‐CCSD(T)/CBS:AMBER level of theory to understand the catalytic machinery behind the transfer of the acyl moieties from MAT to the ACP domain. The results show that the acyl transfer to the ACP's phosphopantetheine (PNS) occurs in two sequential events: Step 3) asynchronous concerted reaction that combines the deprotonation of the PNS's thiol group and the nucleophilic attack on the acyl‐Ser581 ester carbon; Step 4) tetrahedral intermediate breakdown and regeneration of the catalytic Ser581/His683 dyad. The energy barriers of these two steps are inferior to those reported for the first catalytic stage, suggesting that the rate‐limiting step of the MAT mechanism lies on Stage 1, particularly on the concerted nucleophilic attack that starts the reaction. The oxyanion hole, formed by the backbone amines of Met499 and Leu582, was reported to transiently stabilize the tetrahedral intermediate and to play a favorable catalytic effect on the overall MAT reaction, particularly through the reduction of the energetic span. The knowledge gathered in this work complements previous experimental and theoretical studies and instigates further investigations that explore the therapeutic potential of hFAS.