Steady-state Kinetic Analysis Research Articles

Kinetic analysis of F 420 ‐dependent NADP + oxidoreductase (Fno) variants reveal interrupted inner subunit communication

F420-dependent NADP+ oxidoreductase (Fno) from Archaeoglobus fulgidus catalyzes the reversible reduction of NADP+ to NADPH within the cell. The catalyzed reaction involves the transfer of a hydride from the C-5 position of the reduced F420 cofactor to the C4 position of NADP+, producing NADPH (Figure 1). Our previous studies on wild-type Fno has determined that the enzyme participates in negative cooperativity and half-site reactivity, suggesting that Fno is a regulatory enzyme and capable of inner subunit communication. Several amino acids have been identified at the interface of the two subunits that are believed to aid in subunit communication. These residues include, R186, H133, T192, T09 and S190. We have created a library of Fno variants to probe the role of these amino acids in subunit communication. We have conducted fluorescence binding experiments, as well as steady-state and pre steady-state kinetic analysis of these Fno variants. Our results have shown that R186, S190 and T192 are involved in subunit communication, switching from negative cooperativity to no cooperativity behavior, unlike H133. Our results are reported here.

Steady-state kinetic analysis of halogenase-supporting flavin reductases BorF and AbeF reveals different kinetic mechanisms

The short-chain flavin reductases BorF and AbeF reduce FAD to FADH2, which is then used by flavin-dependent halogenases (BorH and AbeH respectively) to regioselectively chlorinate tryptophan in the biosynthesis of indolotryptoline natural products. Recombinant AbeF and BorF were overexpressed and purified as homodimers from E. coli, and copurified with substoichiometric amounts of FAD, which could be easily removed. AbeF and BorF can reduce FAD, FMN, and riboflavin in vitro and are selective for NADH over NADPH. Initial velocity studies in the presence and absence of inhibitors showed that BorF proceeds by a sequential ordered kinetic mechanism in which FAD binds first, while AbeF follows a random-ordered sequence of substrate binding. Fluorescence quenching experiments verified that NADH does not bind BorF in the absence of FAD, and that both AbeF and BorF bind FAD with higher affinity than FADH2. pH-rate profiles of BorF and AbeF were bell-shaped with maximum kcat at pH 7.5, and site-directed mutagenesis of BorF implicated His160 and Arg38 as contributing to the catalytic activity and the pH dependence.

Target-induced mimic enzyme deactivation based on mixed-node metal-organic frameworks for colorimetric assay of hydrogen sulfide

Accurate detection of hydrogen sulfide (H2S) is of great significance for environmental monitoring and protection. We propose a colorimetric method for the detection of H2S by the use of mixed-node Cu-Fe metal organic frameworks (Cu-Fe MOFs) as highly efficient mimic enzymes for target-induced deactivation. The Cu-Fe MOFs were synthesized by a simple solvothermal method and could catalyze the H2O2 mediated oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to oxTMB with a blue color. The presence of dissolved H2S would deactivate the mimic enzymes, and then the blue color disappeared. The mechanism of the sensor was discussed by steady-state kinetic analysis. The designed assay was highly sensitive for H2S detection with a linear range of 0−80 μmol/L and a detection limit of 1.6 μmol/L. Moreover, some potential substances in the water samples had no interference. This method with the advantages of low cost, high sensitivity, selectivity, and visual readout with the naked eye was successfully applied to the determination of H2S in industrial wastewater samples.

Distinct kinetic mechanisms of H3K4 methylation catalyzed by MLL3 and MLL4 core complexes

The methyltransferases MLL3 and MLL4 primarily catalyze the monomethylation of histone H3 lysine 4 (H3K4) on enhancers to regulate cell-type-specific gene expression and cell fate transition. MLL3 and MLL4 share almost identical binding partners and biochemical activities, but perform specific and nonredundant functions. The features and functions that distinguish MLL3 and MLL4 remain elusive. Here, we characterize the kinetic mechanisms of MLL3 and MLL4 ternary complexes containing the catalytic SET domain from MLL3 or MLL4 (MLL3SET or MLL4SET), the SPRY domain of ASH2L (ASH2LSPRY), and a short fragment of RBBP5 (RBBP5AS-ABM) to search for possible explanations. Steady-state kinetic analyses and inhibition studies reveal that the MLL3 complex catalyzes methylation in a random sequential bi–bi mechanism. In contrast, the MLL4 complex adopts an ordered sequential bi–bi mechanism, in which the cofactor S-adenosylmethionine (AdoMet) binds to the enzyme prior to the H3 peptide, and the methylated H3 peptide dissociates from the enzyme before S-adenosylhomocysteine (AdoHcy) detaches after methylation. Substrate-binding assays using fluorescence polarization (FP) confirm that AdoMet binding is a prerequisite for H3 binding for the MLL4 complex but not for the MLL3 complex. Molecular dynamic simulations reveal that the binding of AdoMet exclusively induces conformational constraints on the AdoMet-binding groove and the H3 substrate-binding pocket of MLL4, therefore stabilizing a specific active conformation to ease entry of the substrate H3. The distinct kinetic mechanisms and conformational plasticities provide important insights into the differential functions of MLL3 and MLL4 and may also guide the development of selective inhibitors targeting MLL3 or MLL4.

Local interactions with the Glu166 base and the conformation of an active site loop play key roles in carbapenem hydrolysis by the KPC-2 β-lactamase

The Klebsiella pneumoniae carbapenemase-2 (KPC-2) is a common source of antibiotic resistance in Gram-negative bacterial infections. KPC-2 is a class A β-lactamase that exhibits a broad substrate profile and hydrolyzes most β-lactam antibiotics including carbapenems owing to rapid deacylation of the covalent acyl-enzyme intermediate. However, the features that allow KPC-2 to deacylate substrates more rapidly than non-carbapenemase enzymes are not clear. The active-site residues in KPC-2 are largely conserved in sequence and structure compared with non-carbapenemases, suggesting that subtle alterations may collectively facilitate hydrolysis of carbapenems. We utilized a nonbiased genetic approach to identify mutants deficient in carbapenem hydrolysis but competent for ampicillin hydrolysis. Subsequent pre–steady-state enzyme kinetics analyses showed that the substitutions slow the rate of deacylation of carbapenems. Structure determination via X-ray diffraction indicated that a F72Y mutant forms a hydrogen bond between the tyrosine hydroxyl group and Glu166, which may lower basicity and impair the activation of the catalytic water for deacylation, whereas several mutants impact the structure of the Q214-R220 active site loop. A T215P substitution lowers the deacylation rate and drastically alters the conformation of the loop, thereby disrupting interactions between the enzyme and the carbapenem acyl-enzyme intermediate. Thus, the environment of the Glu166 general base and the precise placement and conformational stability of the Q214-R220 loop are critical for efficient deacylation of carbapenems by the KPC-2 enzyme. Therefore, the design of carbapenem antibiotics that interact with Glu166 or alter the Q214-R220 loop conformation may disrupt enzyme function and overcome resistance.

1445. Deciphering the Role of the Y221H Ω-loop Substitution in Pseudomonas-derived Cephalosporinase (PDC) in Cephalosporin Resistance

BackgroundAntimicrobial resistance is a major global health threat. Pseudomonas aeruginosa is a leading cause of nosocomial infections and a key opportunistic pathogen in cystic fibrosis. Multidrug resistant strains are classified as a “serious threat” by the CDC. Pseudomonas-derived cephalosporinase (PDC) is largely responsible for β-lactam antibiotic resistance in P. aeruginosa. Single amino acid substitutions in the essential Ω-loop region (e.g. Y221H by structural alignment-based numbering of class C β-lactamases) have been shown to enhance hydrolysis of ceftazidime (CAZ) and ceftolozane (TOL), limiting therapeutic options for P. aeruginosa.MethodsWe undertook detailed studies to explore the mechanisms by which Y221H enhances CAZ and TOL MICs. MIC measurements were performed per CLSI guidelines using MH Agar. Thermal stability was determined by circular dichroism. Enzyme kinetic properties were determined using spectrophotometric techniques. Molecular dynamics techniques were used to predict structural changes.Results E. coli expressing blaPDC-3-Y221H is less susceptible to CAZ (MIC 0.5 mg/L WT → 8 mg/L Y221H) and TOL (MIC 2 mg/L WT → 16 mg/L Y221H). Using steady-state kinetic analysis, Y221H was found to hydrolyze CAZ with a KM = 585 µM, a kcat = 3.4 sec-1, and kcat/KM = 0.0058 µM-1s-1. With cephalothin, a good PDC substrate, we observed KM = 26.6 µM, kcat = 70.1 s-1, and kcat/KM = 2.6 µM-1 s-1 for Y221H. Using Electrospray ionization mass spectrometry (ESI-MS), CAZ was detected covalently bound to WT, but not Y221H when incubated at 1000-fold molar excess. Avibactam (AVI) inhibited Y223H (Ki = 70 nM vs. 19 nM for WT). Y221H thermal stability decreased by 5°C (Tm = 47°C vs 52°C WT). AVI at 10-fold molar excess does not increase Tm in Y221H or WT. WT-MetaDynamics (WT MDS) predicts the opening of a hidden pocket by repositioning residue 221 (Figure 1).).Figure 1: (Left) We carried out enhanced sampling metadynamics simulations to generate free-energy landscapes as a function of the dihedral angles of residue 221. This identifies the differences in the dynamics of the tyrosyl side chains in the wild type Y221 and the imidazole ring of the H221 variant. (Right) The rotation of the side chain in H221 opens a cryptic pocket (green mesh), which is occluded in the wild type. The Ω-loop is colored red. ConclusionPDC-3 Y221H increases CAZ & TOL MICs and alters catalytic activity, primarily by a change in kcat. Our modelling analyses suggest altered conformational flexibility and structure-function relationships in the Ω-loop. These results help to advance our understanding of PDC and will inform development of novel antibiotics and inhibitors.DisclosuresRobert A. Bonomo, MD, Entasis, Merck, Venatorx (Research Grant or Support)

Unlocking the potential of the formate pathway in the photo-assisted Sabatier reaction

Although solar irradiation has demonstrated beneficial synergy for catalytic CO2 hydrogenation, it has long been debated as to whether the observed photo-assisted catalytic enhancement has a photo-activation component or is driven solely by the heating effect. Here, we confirm that surface adsorbates play a critical role in photo-assisted CO2 activation. We isolate the rate-determining intermediate species in the photo-assisted Sabatier reaction by in situ isotopic spectroscopy. Specifically, we reveal that photo-activation of the formate adsorbate (HCO2*) offers an eightfold photo-enhancement on NiOx/La2O3@TiO2. Concurrently, the active La2O3@TiO2 support facilitates the adsorption of CO2, which may contribute to the sustained HCO2* formation and conversion. The findings are crucial as they provide surface intermediary insights in the design of catalysts for photo-assisted CO2 conversion. Ultimately, the understanding may unlock the solar-driven hydrogenation catalytic pathway for other reactions involving HCO2* such as the synthesis of methanol. Although the light-promoted Sabatier reaction has great potential for CO2 remediation strategies, its mechanism is not fully understood. Now, a steady-state isotopic kinetic analysis coupled with infrared spectroscopy reveals important aspects of this hydrogenation process over a NiOx/La2O3@TiO2 composite catalyst.

Kinetic analysis of inhibition of α-glucosidase by leaf powder from Morus australis and its component iminosugars.

1-DNJ: 1-deoxynojirimycin; GAL-DNJ: 2-O-α-D-galactopyranosyl-DNJ.

ZIF-67-derived Co3O4 hollow nanocage with efficient peroxidase mimicking characteristic for sensitive colorimetric biosensing of dopamine.

Co3O4 hollow nanocages (Co3O4 HNCs) were prepared by simple calcination with ZIF-67 as the precursor. Compared with ordinary nano-sized Co3O4, skeletal Co3O4 HNCs have a larger specific surface area and porosity, lead to better dispersion, which can expose more catalytic active sites, and obtain higher catalytic activity. Experiments indicate that Co3O4 HNCs are used as a catalyst to make H2O2 generate O2. At the same time, Co3O4 HNCs act as bridge to accelerate the electrons transfer from the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) to the dissolved oxygen and efficiently obtain blue oxidized TMB (oxTMB) at low concentration of H2O2. Steady-state kinetic analysis shows a lower Km and a higher Vmax value than other materials, indicating its excellent affinity and high catalytic efficiency. Based on the inhibitory effect of dopamine (DA) on TMB oxidation in the system, a sensitive, visual colorimetric biosensing method is developed. The calibration curve of DA has a good linear response at both high and low concentrations. Compared with other system, it has the unique advantage of very low detection limit, while retaining a wide detection range, and realizes the accurate detection of actual samples with different concentrations.

Characterisation of the interaction of guanine nucleotides with ribosomal GTPase Lsg1

Ribosome biogenesis in eukaryotes requires the participation of several transactivation factors that are involved in the modification, assembly, transport and quality control of the ribosomal subunits. One of these factors is the Large subunit GTPase 1 (Lsg1), a protein that acts as the release factor for the export adaptor named Nonsense-mediated mRNA decay 3 protein (Nmd3) and facilitates the incorporation of the last structural protein uL16 into the 60S subunit. Here, we characterised the recombinant yeast Lsg1 and studied its catalysis and binding properties for guanine nucleotides. We described the interaction of Lsg1 with guanine nucleotides alone and in the presence of the complex Nmd3•60S using fluorescence spectroscopy. Lsg1 has a greater affinity for GTP than for GDP suggesting that in the cell cytoplasm it exists mainly bound to the former. In the presence of 60S subunits loaded with Nmd3, the affinity of Lsg1 for both nucleotides increases but to a larger extent towards GTP. From this observation together with the excess of GTP present in the cytoplasm of exponentially growing cells over that of GDP, we can infer that the pre-ribosomal particle composed by Nmd3•60S acts as a GTP Stabilising Factor for Lsg1. Additionally, Lsg1 undergoes different conformational changes depending on its binding partner or the guanine nucleotides it interacts with. Steady-state kinetic analysis of free Lsg1 indicated slow GTP hydrolysis with values of kcat 1 min−1 and Km of 34 μM.

Structure and function of a flavin-dependent S-monooxygenase from garlic (Allium sativum)

Allicin is a component of the characteristic smell and flavor of garlic (Allium sativum). A flavin-containing monooxygenase (FMO) produced by A. sativum (AsFMO) was previously proposed to oxidize S-allyl-l-cysteine (SAC) to alliin, an allicin precursor. Here, we present a kinetic and structural characterization of AsFMO that suggests a possible contradiction to this proposal. Results of steady-state kinetic analyses revealed that AsFMO exhibited negligible activity with SAC; however, the enzyme was highly active with l-cysteine, N-acetyl-l-cysteine, and allyl mercaptan. We found that allyl mercaptan with NADPH was the preferred substrate-cofactor combination. Rapid-reaction kinetic analyses showed that NADPH binds tightly (KD of ∼2 μm) to AsFMO and that the hydride transfer occurs with pro-R stereospecificity. We detected the formation of a long-wavelength band when AsFMO was reduced by NADPH, probably representing the formation of a charge-transfer complex. In the absence of substrate, the reduced enzyme, in complex with NADP+, reacted with oxygen and formed an intermediate with a spectrum characteristic of C4a-hydroperoxyflavin, which decays several orders of magnitude more slowly than the kcat The presence of substrate enhanced C4a-hydroperoxyflavin formation and, upon hydroxylation, oxidation occurred with a rate constant similar to the kcat The structure of AsFMO complexed with FAD at 2.08-Å resolution features two domains for binding of FAD and NADPH, representative of class B flavin monooxygenases. These biochemical and structural results are consistent with AsFMO being an S-monooxygenase involved in allicin biosynthesis through direct formation of sulfenic acid and not SAC oxidation.

Structural and biochemical analysis of phosphoethanolamine methyltransferase from the pine wilt nematode Bursaphelenchus xylophilus

In free-living and parasitic nematodes, the methylation of phosphoethanolamine to phosphocholine provides a key metabolite to sustain phospholipid biosynthesis for growth and development. Because the phosphoethanolamine methyltransferases (PMT) of nematodes are essential for normal growth and development, these enzymes are potential targets of inhibitor design. The pine wilt nematode (Bursaphelenchus xylophilus) causes extensive damage to trees used for lumber and paper in Asia. As a first step toward testing BxPMT1 as a potential nematicide target, we determined the 2.05 Å resolution x-ray crystal structure of the enzyme as a dead-end complex with phosphoethanolamine and S-adenosylhomocysteine. The three-dimensional structure of BxPMT1 served as a template for site-directed mutagenesis to probe the contribution of active site residues to catalysis and phosphoethanolamine binding using steady-state kinetic analysis. Biochemical analysis of the mutants identifies key residues on the β1d-α6 loop (W123F, M126I, and Y127F) and β1e-α7 loop (S155A, S160A, H170A, T178V, and Y180F) that form the phosphobase binding site and suggest that Tyr127 facilitates the methylation reaction in BxPMT1.

The Siderophore Synthetase IucA of the Aerobactin Biosynthetic Pathway Uses an Ordered Mechanism.

Biosynthesis of the hydroxamate siderophore aerobactin requires the activity of four proteins encoded within the iuc operon. Recently, we biochemically reconstituted the biosynthetic pathway and structurally characterized IucA and IucC, two enzymes that sequentially couple N6-acetyl-N6-hydroxylysine to the primary carboxylates of citrate. IucA and IucC are members of a family of non-ribosomal peptide synthetase-independent siderophore (NIS) synthetases that are involved in the production of other siderophores, including desferrioxamine, achromobactin, and petrobactin. While structures of several members of this family were solved previously, there is limited mechanistic insight into the reaction catalyzed by NIS synthetases. Therefore, we performed a terreactant steady-state kinetic analysis and herein provide evidence for an ordered mechanism in which the chemistry is preceded by the formation of the quaternary complex. We further probed two regions of the active site with site-directed mutagenesis and identified several residues, including a conserved motif that is present on a dynamic loop, that are important for substrate binding and catalysis.

Heterologous Expression and Functional Characterization of Novel CYP2C9 Variants Identified in the Alaska Native People.

CYP2C9 is a major form of human liver cytochrome P450 that is responsible for the oxidative metabolism of several widely used low-therapeutic index drugs, including (S)-warfarin and phenytoin. In a cohort of Alaska Native people, ultrarare or novel CYP2C9 protein variants, M1L (rs114071557), N218I (rs780801862), and P279T (rs182132442, CYP2C9*29), are expressed with higher frequencies than the well characterized CYP2C9*2 and CYP2C9*3 alleles. We report here on their relative expression in lentivirus-infected HepG2 cells and the functional characterization of purified reconstituted enzyme variants expressed in Escherichia coli toward (S)-warfarin, phenytoin, flurbiprofen, and (S)-naproxen. In the infected HepG2 cells, robust mRNA and protein expression were obtained for wild-type, N218I, and P279T variants, but as expected, the M1L variant protein was not translated in this liver-derived cell line. His-tagged wild-type protein and the N218I and P279T variants, but not M1L, expressed well in E. coli and were highly purified after affinity chromatography. Upon reconstitution with cytochrome P450 oxidoreductase and cytochrome b5, the N218I and P279T protein variants metabolized (S)-warfarin, phenytoin, flurbiprofen, and (S)-naproxen to the expected monohydroxylated or O-demethylated metabolites. Steady-state kinetic analyses revealed that the relative catalytic efficiency ratios of (S)-warfarin metabolism by the P279T and N218I variants were 87% and 24%, respectively, of wild-type CYP2C9 protein. A similar rank ordering was observed for metabolism of phenytoin, flurbiprofen, and (S)-naproxen. We conclude that carriers of the variant N218I and, especially, the M1L alleles would be at risk of exacerbated therapeutic effects from drugs that rely on CYP2C9 for their metabolic clearance. SIGNIFICANCE STATEMENT: Novel gene variants of CYP2C9-M1L, and N218I, along with P279T (CYP2C9*29)-are expressed in Alaska Native people at relatively high frequencies. In vitro characterization of their functional effects revealed that each variant confers reduced catalytic efficiency toward several substrates, including the low-therapeutic index drugs (S)-warfarin and phenytoin. These data provide the first functional information for new, common CYP2C9 variants in this understudied population. The data may help guide dose adjustments in allele carriers, thus mitigating potential healthcare disparities.

The unique interplay between copper and zinc during catalytic carbon dioxide hydrogenation to methanol

In spite of numerous works in the field of chemical valorization of carbon dioxide into methanol, the nature of high activity of Cu/ZnO catalysts, including the reaction mechanism and the structure of the catalyst active site, remains the subject of intensive debate. By using high-pressure operando techniques: steady-state isotope transient kinetic analysis coupled with infrared spectroscopy, together with time-resolved X-ray absorption spectroscopy and X-ray powder diffraction, and supported by electron microscopy and theoretical modeling, we present direct evidence that zinc formate is the principal observable reactive intermediate, which in the presence of hydrogen converts into methanol. Our results indicate that the copper–zinc alloy undergoes oxidation under reaction conditions into zinc formate, zinc oxide and metallic copper. The intimate contact between zinc and copper phases facilitates zinc formate formation and its hydrogenation by hydrogen to methanol.

Impact of 1,N6-ethenoadenosine, a damaged ribonucleotide in DNA, on translesion synthesis and repair

Incorporation of ribonucleotides into DNA can severely diminish genome integrity. However, how ribonucleotides instigate DNA damage is poorly understood. In DNA, they can promote replication stress and genomic instability and have been implicated in several diseases. We report here the impact of the ribonucleotide rATP and of its naturally occurring damaged analog 1,N6-ethenoadenosine (1,N6-ϵrA) on translesion synthesis (TLS), mediated by human DNA polymerase η (hpol η), and on RNase H2-mediated incision. Mass spectral analysis revealed that 1,N6-ϵrA in DNA generates extensive frameshifts during TLS, which can lead to genomic instability. Moreover, steady-state kinetic analysis of the TLS process indicated that deoxypurines (i.e. dATP and dGTP) are inserted predominantly opposite 1,N6-ϵrA. We also show that hpol η acts as a reverse transcriptase in the presence of damaged ribonucleotide 1,N6-ϵrA but has poor RNA primer extension activities. Steady-state kinetic analysis of reverse transcription and RNA primer extension showed that hpol η favors the addition of dATP and dGTP opposite 1,N6-ϵrA. We also found that RNase H2 recognizes 1,N6-ϵrA but has limited incision activity across from this lesion, which can lead to the persistence of this detrimental DNA adduct. We conclude that the damaged and unrepaired ribonucleotide 1,N6-ϵrA in DNA exhibits mutagenic potential and can also alter the reading frame in an mRNA transcript because 1,N6-ϵrA is incompletely incised by RNase H2.

Structure and Function of the Cytochrome P450 Monooxygenase Cinnamate 4-hydroxylase from Sorghum bicolor.

Cinnamate 4-hydroxylase (C4H; CYP73A) is a cytochrome P450 monooxygenase associated externally with the endoplasmic reticulum of plant cells. The enzyme uses NADPH-cytochrome P450 reductase as a donor of electrons and hydroxylates cinnamic acid to form 4-coumaric acid in phenylpropanoid metabolism. In order to better understand the structure and function of this unique class of plant P450 enzymes, we have characterized the enzyme C4H1 from lignifying tissues of sorghum (Sorghum bicolor), encoded by Sobic.002G126600 Here we report the 1.7 Å resolution crystal structure of CYP73A33. The obtained structural information, along with the results of the steady-state kinetic analysis and the absorption spectroscopy titration, displays a high degree of similarity of the structural and functional features of C4H to those of other P450 proteins. Our data also suggest the presence of a putative allosteric substrate-binding site in a hydrophobic pocket on the enzyme surface. In addition, comparing the newly resolved structure with those of well-investigated cytochromes P450 from mammals and bacteria enabled us to identify those residues of critical functional importance and revealed a unique sequence signature that is potentially responsible for substrate specificity and catalytic selectivity of C4H.

Steady-State Kinetics of Enzyme-Catalyzed Hydrolysis of Echothiophate, a P-S Bonded Organophosphorus as Monitored by Spectrofluorimetry.

Enzyme-catalyzed hydrolysis of echothiophate, a P–S bonded organophosphorus (OP) model, was spectrofluorimetrically monitored, using Calbiochem Probe IV as the thiol reagent. OP hydrolases were: the G117H mutant of human butyrylcholinesterase capable of hydrolyzing OPs, and a multiple mutant of Brevundimonas diminuta phosphotriesterase, GG1, designed to hydrolyze a large spectrum of OPs at high rate, including V agents. Molecular modeling of interaction between Probe IV and OP hydrolases (G117H butyrylcholinesterase, GG1, wild types of Brevundimonas diminuta and Sulfolobus solfataricus phosphotriesterases, and human paraoxonase-1) was performed. The high sensitivity of the method allowed steady-state kinetic analysis of echothiophate hydrolysis by highly purified G117H butyrylcholinesterase concentration as low as 0.85 nM. Hydrolysis was michaelian with Km = 0.20 ± 0.03 mM and kcat = 5.4 ± 1.6 min−1. The GG1 phosphotriesterase hydrolyzed echothiophate with a high efficiency (Km = 2.6 ± 0.2 mM; kcat = 53400 min−1). With a kcat/Km = (2.6 ± 1.6) × 107 M−1min−1, GG1 fulfills the required condition of potential catalytic bioscavengers. quantum mechanics/molecular mechanics (QM/MM) and molecular docking indicate that Probe IV does not interact significantly with the selected phosphotriesterases. Moreover, results on G117H mutant show that Probe IV does not inhibit butyrylcholinesterase. Therefore, Probe IV can be recommended for monitoring hydrolysis of P–S bonded OPs by thiol-free OP hydrolases.

Highly dispersed Pt nanoparticles on ultrasmall EMT zeolite: A peroxidase-mimic nanoenzyme for detection of H2O2 or glucose

In past decade, Pt-based nanomaterials as peroxidase mimics have attracted much attention for H2O2 and glucose detection. However, easy aggregation of Pt nanoparticles (Pt NPs) greatly decreases their peroxidase-like activity. In this work, novel Pt/EMT nanocomposites were prepared by uniformly loading Pt NPs (5–8 nm) onto the support of ultrasmall EMT zeolite (15–20 nm), a kind of low-silica microporous aluminosilicate material. The hybrid Pt/EMT nanomaterials could be well dispersed in water to form a homogeneous suspension, and were then utilized as a superior peroxidase-like catalyst for oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2). The optimal catalyst of 2.6Pt/EMT nanocomposite exhibited excellent catalytic performance toward H2O2 and TMB than natural enzyme of horseradish peroxidase (HRP) by using a steady-state kinetic analysis based on the typical Michaelis-Menten kinetics theory. The peroxidase-like catalyst showed a promising activity in a wide pH and temperature range as well as the long-term stability. A facile and reliable colorimetric assay based on the peroxidase mimic of Pt/EMT nanocomposite was constructed for precise detection of H2O2 and glucose in a wide linear range, with low limits of detection of 1.1 μM and 13.2 μM, respectively. Due to high selectivity to glucose against other sugars on the catalyst, the method was demonstrated to accurately measure the concentration of glucose in real samples including human blood serum and fruit juices, indicating a potential application of the Pt/EMT nanocomposites as a robust peroxidase mimic and a reliable biosensor in the fields of clinical diagnosis, pharmaceutical, food research and so on.

Mechanistic and Crystallographic Studies of Azoreductase AzoA from Bacillus wakoensis A01.

The azoreductase AzoA from the alkali-tolerant Bacillus wakoensis A01 has been studied to reveal its structural and mechanistic details. For this, a recombinant expression system was developed which yields impressive amounts of fully active enzyme. The purified holo enzyme is remarkably solvent-tolerant and thermostable with an apparent melting temperature of 71 °C. The dimeric enzyme contains FMN as a prosthetic group and is strictly NADH dependent. While AzoA shows a negligible ability to use molecular oxygen as an electron acceptor, it is efficient in reducing various azo dyes and quinones. The kinetic and catalytic mechanism has been studied in detail using steady state kinetic analyses and stopped-flow studies. The data show that AzoA performs quinone and azo dye reductions via a two-electron transfer. Moreover, quinones were shown to be much better substrates (kcat values of 100–400 s–1 for several naphtoquinones) when compared with azo dyes. This suggests that the physiological role of AzoA and sequence-related microbial reductases is linked to quinone reductions and that they can better be annotated as quinone reductases. The structure of AzoA has been determined in complex with FMN at 1.8 Å resolution. AzoA displays unique features in the active site providing clues for explaining its catalytic and thermostability features. An uncommon loop, when compared with sequence-related reductases, forms an active site lid with Trp60 acting as an anchor. Several Trp60 mutants have been analyzed disclosing an important role of this residue in the stability of AzoA, while they retained activity. Structural details are discussed in relation to other azo and quinone reductases. This study provides new insights into the molecular functioning of AzoA and sequence-related reductases.