Steady-state Kinetic Parameters Research Articles

Rapid aqueous preparation of carbon quantum dots and their application as nanozyme for the detection of hydrogen peroxide and uric acid

A green and rapid method was developed to synthesize a series of carbon quantum dots (Fe,N/CQDs, Fe,S/CQDs, S,N/CQDs and Fe,S,N/CQDs) through thermal decomposition using persimmon frost as a carbon source. All the synthesized CQDs have peroxidase-like activity, and the comparision of their steady-state kinetic parameters inferred that the peroxidase-like activity of Fe,S,N/CQDs is superior to those of Fe,S/CQDs and Fe,N/CQDs. Furthermore, Fe,S,N/CQDs exhibited good affinity towards 3,3′,5,5′-Tetramethylbenzidine (TMB) and H2O2 compared to natural horseradish peroxidase (HRP) and other nanozymes. In theory, uric acid (UA) can produce H2O2 with the catalysis by uricase. Fe,S,N/CQDs could catalyze the breakdown of H2O2 to produce reactive oxygen species (ROS), which can oxidize TMB (colorless) to oxTMB (blue). Based on the nanozyme-catalyzed oxidation of TMB, a simple and sensitive colorimetric sensor was developed for the quantitative analysis of H2O2 and UA. Low limit of detection (0.73 μM), wide linear range (1–80 μM) and good repeatability were obtained. The sensor was also demonstrated for the determination of UA in biological samples and satisfactory results were obtained.

Role of amino acid 159 in carbapenem and temocillin hydrolysis of OXA-933, a novel OXA-48 variant.

OXA-48 has rapidly disseminated worldwide and become one of the most common carbapenemases in many countries with more than 45 variants reported with, in some cases, significant differences in their hydrolysis profiles. The R214 residue, located in the ß5-ß6 loop, is crucial for the carbapenemase activity, as it stabilizes carbapenems in the active site and maintains the shape of the active site through interactions with D159. In this study, we have characterized a novel variant of OXA-48, OXA-933 with a single D159N change. To evaluate the importance of this residue, point mutations were generated (D159A, D159G, D159K, and D159W), kinetic parameters of OXA-933, OXA-48 D159G, and OXA-48 D159K were determined and compared to those of OXA-48 and OXA-244. The blaOXA-933 gene was borne on Tn2208, a 2,696-bp composite transposon made of two IS1 elements surrounded by 9 bp target site duplications and inserted into a non-self-transmissible plasmid pOXA-933 of 7,872 bp in size. Minimal inhibitory concentration values of E. coli expressing the blaOXA-933 gene or of its point mutant derivatives were lower for carbapenems (except for D159G) as compared to those expressing the blaOXA-48 gene. Steady-state kinetic parameters revealed lower catalytic efficiencies for expanded spectrum cephalosporins and carbapenems. A detailed structural analysis confirmed the crucial role of D159 in shaping the active site of OXA-48 enzymes by interacting with R214. Our work further illustrates the remarkable propensity of OXA-48-like carbapenemases to evolve through mutations at positions outside the β5-β6 loop, but interacting with key residues of it.

Emergence of a clinical Salmonella enterica serovar 1,4,[5], 12: i:-isolate, ST3606, in China with susceptibility decrease to ceftazidime-avibactam carrying a novel blaCTX-M-261 variant and a blaNDM-5.

The detection rate of Salmonella enterica serovar 1,4,[5], 12: i: - (S. 1,4,[5], 12: i: -) has increased as the most common serotype globally. A S. 1,4,[5], 12: i: - strain named ST3606 (sequence type 34), isolated from a fecal specimen of a child with acute diarrhea hospitalized in a tertiary hospital in China, was firstly reported to be resistant to carbapenem and ceftazidime-avibactam. The aim of this study was to characterize the whole-genome sequence of S. 1,4,[5], 12: i: - isolate, ST3606, and explore its antibiotic resistance genes and their genetic environments. The genomic DNA of S. 1,4,[5], 12: i: - ST3606 was extracted and performed with single-molecule real-time sequencing. Resistance genes, plasmid replicon type, mobile elements, and multilocus sequence types (STs) of ST3606 were identified by ResFinder 3.2, PlasmidFinder, OriTfinder database, ISfinder database, and MLST 2.0, respectively. The conjugation experiment was utilized to evaluate the conjugation frequency of pST3606-2. Protein expression and enzyme kinetics experiments of CTX-M were performed to analyze hydrolytic activity of a novel CTX-M-261 enzyme toward several antibiotics. Single-molecule real-time sequencing revealed the coexistence of a 109-kb IncI1-Iα plasmid pST3606-1 and a 70.5-kb IncFII plasmid pST3606-2. The isolate carried resistance genes, including blaNDM-5, sul1, qacE, aadA2, and dfrA12 in pST3606-1, blaTEM-1B, aac(3)-lld, and blaCTX-M-261, a novel blaCTX-M-1 family member, in pST3606-2, and aac(6')-Iaa in chromosome. The blaCTX-M-261 was derived from blaCTX-M-55 by a single-nucleotide mutation 751G>A leading to amino acid substitution of Val for Met at position 251 (Val251Met), which conferred CTX-M increasing resistance to ceftazidime verified by antibiotics susceptibility testing of transconjugants carrying pST3606-2 and steady-state kinetic parameters of CTX-M-261. pST3606-1 is an IncI1-α incompatibility type that shares homology with plasmids of pC-F-164_A-OXA140, pE-T654-NDM-5, p_dm760b_NDM-5, and p_dmcr749c_NDM-5. The conjugation experiment demonstrated that pST3606-2 was successfully transferred to the Escherichia coli recipient C600 with four modules of OriTfinder. Plasmid-mediated horizontal transfer plays an important role in blaNDM-5 and blaCTX-M-261 dissemination, which increases the threat to public health due to the resistance to most β-lactam antibiotics. This is the first report of blaCTX-M-261 and blaNDM-5 in S. 1,4,[5], 12: i: -. The work provides insights into the enzymatic function and demonstrates the ongoing evolution of CTX-M enzymes and confirms urgency to control resistance of S. 1,4,[5], 12: i: -.

Bielectrode Strategy for Determination of CYP2E1 Catalytic Activity: Electrodes with Bactosomes and Voltammetric Determination of 6-Hydroxychlorzoxazone.

We describe a bielectrode system for evaluation of the electrocatalytic activity of cytochrome P450 2E1 (CYP2E1) towards chlorzoxazone. One electrode of the system was employed to immobilize Bactosomes with human CYP2E1, cytochrome P450 reductase (CPR), and cytochrome b5 (cyt b5). The second electrode was used to quantify CYP2E1-produced 6-hydroxychlorzoxazone by its direct electrochemical oxidation, registered using square-wave voltammetry. Using this system, we determined the steady-state kinetic parameters of chlorzoxazone hydroxylation by CYP2E1 of Bactosomes immobilized on the electrode: the maximal reaction rate (Vmax) was 1.64 ± 0.08 min-1, and the Michaelis constant (KM) was 78 ± 9 μM. We studied the electrochemical characteristics of immobilized Bactosomes and have revealed that electron transfer from the electrode occurs both to the flavin prosthetic groups of CPR and the heme iron ions of CYP2E1 and cyt b5. Additionally, it has been demonstrated that CPR has the capacity to activate CYP2E1 electrocatalytic activity towards chlorzoxazone, likely through intermolecular electron transfer from the electrochemically reduced form of CPR to the CYP2E1 heme iron ion.

Bimetallic Cu-Zn Zeolitic Imidazolate Frameworks as Peroxidase Mimics for the Detection of Hydrogen Peroxide: Electrochemical and Spectrophotometric Evaluation.

A copper incorporated zeolitic imidazolate framework-8 (ZIF-8) has been synthesized and demonstrated to be a potential material for a peroxidase mimic. The resultant bimetallic Cu-Zn incorporated MOF is used for the dual mode sensing of hydrogen peroxide by following electrochemical as well as spectrophotometric methods. Using 3,3',5,5'-tetramethylbenzidine (TMB) as a chromogenic substrate, spectrophotometric studies are carried out, and the steady state kinetic parameters are determined for two different concentrations of Cu incorporated ZIF-8 (viz Cu@ZIF-8-1 and Cu@ZIF-8-2). It is found that both Cu@ZIF-8-1 and Cu@ZIF-8-2 exhibit more affinity toward the TMB substrate than the horseradish peroxidase (HRP) enzyme as indicated by the low Km values obtained for the substrate. Also, as the concentration of incorporated Cu increases, Vmax values are also found to be enhanced. Electrochemically, the Cu@ZIF-8 modified glassy carbon electrode (GCE) showed a good response for peroxide detection in the concentration range from 0.5 mM to 5 mM at a working potential of -0.25 V in PBS (pH 7.0) with a limit of detection (LOD) value of 0.46 mM and a sensitivity of 20.25 μA/mM. Further, the chromogenic substrate TMB is successfully immobilized on the electrode surface and subsequently used for the peroxide detection along with Cu@ZIF-8. Here, TMB acts as a mediator and shifted the working potential to 0.1 V in acetate buffer (pH 5.0) in the concentration range from 0.5 mM to 5 mM with an LOD value of 0.499 mM and a sensitivity of 0.097 μA/mM. Interestingly, the same electrode in PBS of pH 7.0 showed a response to peroxide at a working potential of -0.1 V in the concentration range from 0.5 mM to 5 mM with an LOD value of 0.143 mM and a sensitivity of 0.33 μA/mM. Moreover, the applicability of this material for peroxide sensing is evaluated using milk samples, and the proposed material is able to recover the peroxide present in milk. Thus, the bimetallic Cu-Zn MOF can be utilized for the dual mode sensing of peroxide and can be extended for various real time applications.

Genomic characterization of an NDM-9-producing Acinetobacter baumannii clinical isolate and role of Glu152Lys substitution in the enhanced cefiderocol hydrolysis of NDM-9.

Here, we characterized the first French NDM-9-producing Acinetobacter baumannii isolate. A. baumannii 13A297, which belonged to the STPas25 (international clone IC7), was highly resistant to β-lactams including cefiderocol (MIC >32 mg/L). Whole genome sequencing (WGS) using both Illumina and Oxford Nanopore technologies revealed a 166-kb non-conjugative plasmid harboring a blaNDM-9 gene embedded in a Tn125 composite transposon. Complementation of E. coli DH5α and A. baumannii CIP70.10 strains with the pABEC plasmid carrying the blaNDM-1 or blaNDM-9 gene, respectively, resulted in a significant increase in cefiderocol MIC values (16 to >256-fold), particularly in the NDM-9 transformants. Interestingly, steady-state kinetic parameters, measured using purified NDM-1 and NDM-9 (Glu152Lys) enzymes, revealed that the affinity for cefiderocol was 3-fold higher for NDM-9 (Km = 53 μM) than for NDM-1 (Km = 161 μM), leading to a 2-fold increase in catalytic efficiency for NDM-9 (0.13 and 0.069 μM-1.s-1, for NDM-9 and NDM-1, respectively). Finally, we showed by molecular docking experiments that the residue 152 of NDM-like enzymes plays a key role in cefiderocol binding and resistance, by allowing a strong ionic interaction between the Lys152 residue of NDM-9 with both the Asp223 residue of NDM-9 and the carboxylate group of the R1 substituent of cefiderocol.

Processive kinetics in the three-step lanosterol 14α-demethylation reaction catalyzed by human cytochrome P450 51A1

Cytochrome P450 (P450, CYP) family 51 enzymes catalyze the 14α-demethylation of sterols, leading to critical products used for membranes and the production of steroids, as well as signaling molecules. In mammals, P450 51 catalyzes the 3-step, 6-electron oxidation of lanosterol to form (4β,5α)-4,4-dimethyl-cholestra-8,14,24-trien-3-ol (FF-MAS). P450 51A1 can also use24,25-dihydrolanosterol (a natural substrate in the Kandutsch-Russell cholesterol pathway). 24,25-Dihydrolanosterol and the corresponding P450 51A1 reaction intermediates, the 14α-alcohol and -aldehyde derivatives of dihydrolanosterol, were synthesized to study the kinetic processivity of the overall 14α-demethylation reaction of human P450 51A1. A combination of steady-state kinetic parameters, steady-state binding constants, dissociation rates of P450-sterol complexes, and kinetic modeling of the time course of oxidation of a P450-dihydrolanosterol complex showed that the overall reaction is highly processive, withkoffrates of P450 51A1-dihydrolanosterol and the 14α-alcohol and 14α-aldehyde complexes being 1 to 2 orders of magnitude less than the forward rates of competing oxidations. epi-Dihydrolanosterol (the 3α-hydroxy analog) was as efficient as the common 3β-hydroxy isomer in the binding and formation of dihydro FF-MAS. The common lanosterol contaminant dihydroagnosterol was found to be a substrate of human P450 51A1, with roughly one-half the activity of dihydrolanosterol. Steady-state experiments with 14α-methyl deuterated dihydrolanosterol showed no kinetic isotope effect, indicating that C-14αC-H bond breaking is not rate-limiting in any of the individual steps. The high processivity of this reaction generates higher efficiency and also renders the reaction less sensitive to inhibitors.

Electrochemical biosensor for trypsin activity assay based on cleavage of immobilized tyrosine-containing peptide

In this work, we proposed a biosensor for trypsin proteolytic activity assay using immobilization of model peptides on screen-printed electrodes (SPE) modified with gold nanoparticles (AuNPs) prepared by electrosynthetic method. Sensing of proteolytic activity was based on electrochemical oxidation of tyrosine residues of peptides. We designed peptides containing N-terminal cysteine residue for immobilization on an SPE, modified with gold nanoparticles, trypsin-specific cleavage site and tyrosine residue as a redox label. The peptides were immobilized on SPE by formation of chemical bonds between mercapto groups of the N-terminal cysteine residues and AuNPs. After the incubation with trypsin, time-dependent cleavage of the immobilized peptides was observed by decline in tyrosine electrochemical oxidation signal. The kinetic parameters of trypsin, such as the catalytic constant (kcat), the Michaelis constant (KM) and the catalytic efficiency (kcat/KM), toward the CGGGRYR peptide were determined as 0.33 ± 0.01 min−1, 198 ± 24 nM and 0.0016 min−1 nM−1, respectively. Using the developed biosensor, we demonstrated the possibility of analysis of trypsin specificity toward the peptides with amino acid residues disrupting proteolysis. Further, we designed the peptides with proline or glutamic acid residues after the cleavage site (CGGRPYR and CGGREYR), and trypsin had reduced activity toward both of them according to the existing knowledge of the enzyme specificity. The developed biosensor system allows one to perform a comparative analysis of the protease steady-state kinetic parameters and specificity toward model peptides with different amino acid sequences.

A bacterial cold-active dye-decolorizing peroxidase from an Antarctic Pseudomonas strain.

DyP (dye-decolorizing peroxidase) enzymes are hemeproteins that catalyze the H2O2-dependent oxidation of various molecules and also carry out lignin degradation, albeit with low activity. We identified a dyp gene in the genome of an Antarctic cold-tolerant microbe (Pseudomonas sp. AU10) that codes for a class B DyP. The recombinant protein (rDyP-AU10) was produced using Escherichia coli as a host and purified. We found that rDyP-AU10 is mainly produced as a dimer and has characteristics that resemble psychrophilic enzymes, such as high activity at low temperatures (20 °C) when using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and H2O2 as substrates, thermo-instability, low content of arginine, and a catalytic pocket surface larger than the DyPs from some mesophilic and thermophilic microbes. We also report the steady-state kinetic parameters of rDyP-AU10 for ABTS, hydroquinone, and ascorbate. Stopped-flow kinetics revealed that Compound I is formed with a rate constant of (2.07 ± 0.09) × 106 M-1 s-1 at pH 5 and that this is the predominant species during turnover. The enzyme decolors dyes and modifies kraft lignin, suggesting that this enzyme may have potential use in bioremediation and in the cellulose and biofuel industries. KEY POINTS: • An Antarctic Pseudomonas strain produces a dye-decolorizing peroxidase. • The recombinant enzyme (rDyP-AU10) was produced in E. coli and purified. • rDyP-AU10 showed high activity at low temperatures. •rDyP-AU10 is potentially useful for biotechnological applications.

Structural and Biochemical Features of OXA-517: a Carbapenem and Expanded-Spectrum Cephalosporin Hydrolyzing OXA-48 Variant.

OXA-48-producing Enterobacterales have now widely disseminated throughout the world. Several variants have now been reported, differing by just a few amino-acid substitutions or deletions, mostly in the region of the loop β5-β6. As OXA-48 hydrolyzes carbapenems but lacks significant expanded-spectrum cephalosporin (ESC) hydrolytic activity, ESCs were suggested as a therapeutic option. Here, we have characterized OXA-517, a natural variant of OXA-48- with an Arg214Lys substitution and a deletion of Ile215 and Glu216 in the β5-β6 loop, capable of hydrolyzing at the same time ESC and carbapenems. MICs values of E. coli expressing blaOXA-517 gene revealed reduced susceptibility to carbapenems (similarly to OXA-48) and resistance to ESCs. Steady-state kinetic parameters revealed high catalytic efficiencies for ESCs and carbapenems. The blaOXA-517 gene was located on a ca. 31-kb plasmid identical to the prototypical IncL blaOXA-48-carrying plasmid except for an IS1R-mediated deletion of 30.7-kb in the tra operon. The crystal structure of OXA-517, determined to 1.86 Å resolution, revealed an expanded active site compared to that of OXA-48, which allows for accommodation of the bulky ceftazidime substrate. Our work illustrates the remarkable propensity of OXA-48-like carbapenemases to evolve through mutation/deletion in the β5-β6 loop to extend its hydrolysis profile to encompass most β-lactam substrates.

Detection and quantification of UV-transparent keto-androgens by dinitrophenylhydrazine derivatization for discontinuous kinetic assays.

Kinetic assays with recombinant enzymes are critical to determine the steady state kinetic parameters for androgen conversion to understand their role in androgen biosynthesis and metabolism. Detection and quantification of 5α-reduced androgens remain difficult to assay because they are UV-transparent compounds. Therefore, radioactive isotopic versions of these compounds are often required to conduct steady-state kinetics. Here we developed a derivatization protocol with dinitrophenylhydrazine (DNPH) to form hydrazones on the ketones of androgens enabling them to be detected by UV-reverse phase high performance liquid chromatography (RP-HPLC). We determined the kinetic parameters for the conversion of 5α-androstane-3,17-dione (5AD) to 5α-dihydrotestosterone (DHT), 11-keto-5α-androstane-3,17-dione (11K-5AD) to 11-keto-5α-dihydrotestosterone (11K-DHT), and 11β-hydroxy-5α-androstane-3,17-dione (11β-OH-5AD) to 11β-hydroxy-5α-dihydrotestosterone (11β-OH-DHT) catalyzed by recombinant aldo-keto reductase 1C3 (AKR1C3) as measured by product formation post DNPH derivatization.

Derivation of steady-state first-order rate constant equations for enzyme-substrate complex dissociation, as well as zero-order rate constant equations in relation to background assumptions

The maximum velocity (Vmax) of catalysis and the substrate concentration ([ST]) at half the Vmax, the KM, are regarded as steady-state (SS) parameters even though they are the outcomes of zero-order kinetics (ZOK). The research was aimed at disputing such a claim with the following objectives: To: 1) carry out an overview of issues pertaining to the validity of assumptions; 2) derive the needed steady-state (SS) equations distinct from Michaelian equations that can be fitted to both experimental variables and kinetic parameters; 3) calculate the SS first-order rate constant for the dissociation of enzyme-substrate complex (ES) to free substrate, S and enzyme, E; 4) derive the equation of rate constant as a function of the reciprocal of the duration of each catalytic event in the reaction pathway. The experimental values of the data were generated by Bernfeld and Lineweaver-Burk methods. The calculated SS 1st order-order rate constant was « the zero-order Michaelian value, and the difference is ≈ 97.59 % of the zero-order value; the SS catalytic rate differed from the zero-order catalytic rate by ≈ 76.41 % of the latter value; and it was ≈ 93.87 % with respect to the 2nd order rate constant for the formation of enzyme-substrate complex. The equations of time-dependent rate constants, KM, and dissociation constants were derived. The magnitude of [ST] must be > the concentration ([E0]) of the E for the quasi-steady-state assumption (or approximation) to hold. The SS kinetic parameters are not equivalent to zero-order parameters.

Identification of small molecules targeting homoserine acetyl transferase from Mycobacterium tuberculosis and Staphylococcus aureus

There is an urgent need to validate new drug targets and identify small molecules that possess activity against both drug-resistant and drug-sensitive bacteria. The enzymes belonging to amino acid biosynthesis have been shown to be essential for growth in vitro, in vivo and have not been exploited much for the development of anti-tubercular agents. Here, we have identified small molecule inhibitors targeting homoserine acetyl transferase (HSAT, MetX, Rv3341) from M. tuberculosis. MetX catalyses the first committed step in L-methionine and S-adenosyl methionine biosynthesis resulting in the formation of O-acetyl-homoserine. Using CRISPRi approach, we demonstrate that conditional repression of metX resulted in inhibition of M. tuberculosis growth in vitro. We have determined steady state kinetic parameters for the acetylation of L-homoserine by Rv3341. We show that the recombinant enzyme followed Michaelis–Menten kinetics and utilizes both acetyl-CoA and propionyl-CoA as acyl-donors. High-throughput screening of a 2443 compound library resulted in identification of small molecule inhibitors against MetX enzyme from M. tuberculosis. The identified lead compounds inhibited Rv3341 enzymatic activity in a dose dependent manner and were also active against HSAT homolog from S. aureus. Molecular docking of the identified primary hits predicted residues that are essential for their binding in HSAT homologs from M. tuberculosis and S. aureus. ThermoFluor assay demonstrated direct binding of the identified primary hits with HSAT proteins. Few of the identified small molecules were able to inhibit growth of M. tuberculosis and S. aureus in liquid cultures. Taken together, our findings validated HSAT as an attractive target for development of new broad-spectrum anti-bacterial agents that should be effective against drug-resistant bacteria.

Kinetic and stability study on the immobilized enzymatic step of one‐pot dimerization of 2‐[2‐(dimethylamino)ethoxy]ethanol

Tertiary amine catalysts (TACs) are essential organic molecules used in polyurethane manufacturing. Synthesis of these chemicals involves the use of catalytic transition metals at high temperatures and high pressures. Simple amine systems react easily with transition metals; however, amine systems with multiple hydroxyl groups and methylamine complexes produce significant byproducts. In this study, synthesis that forgo transition metals is explored. Enzymes can provide a clean industrial alternative to transition metal use. 2‐[2‐(dimethylamino)ethoxy]ethanol (DMAEE) is the monomer used in the synthesis of a model TAC. When DMAEE is first enzymatically oxidized, the aldehyde can serve as an electrophile for 2‐(methylamino)ethanol (MMEA), forming the DMAEE dimer. Choline oxidase catalysis is the first step of this pathway. Steady state kinetic parameters of choline oxidase with choline and DMAEE as substrates, optimum pH, and temperature stability are reported in this study. The effect of MMEA on the enzymatic reaction is examined as well. In an industrial application, immobilized enzymes can enhance stability over a broad range of temperature and pH. Therefore, choline oxidase was immobilized on CNBr‐activated Sepharose beads, and the effects of immobilization are reported in this study.

Colorimetric assay for the detection of dopamine using bismuth ferrite oxide (Bi2Fe4O9) nanoparticles as an efficient peroxidase-mimic nanozyme

This work describes the preparation of ternary bismuth ferrite oxide nanoparticles (Bi2Fe4O9 NPs) with an enzyme mimetic activity for dopamine (DA) qualitative and quantitative detection. Bi2Fe4O9 NPs were prepared using a facile, low cost, and one-pot hydrothermal treatment. The chemical composition, morphology, and optical properties of Bi2Fe4O9 nanozyme were characterized using different techniques such as Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction pattern (XRD), X-ray photoelectron spectroscopy (XPS), thermo-gravimetric analysis (TGA), dynamic light scattering (DLS), field-emission scanning electron microscopy (FESEM) imaging, FESEM-energy dispersive X-ray spectroscopy (EDS), UV–vis absorption, and fluorescence emission spectroscopy. Bi2Fe4O9 NPs were utilized to catalyze the oxidation of a typical chromogenic peroxidase substrate, 3,3′,5,5′-tetramethylbenzidine (TMB), to form the blue-colored oxidized product (oxTMB), in the presence of hydrogen peroxide (H2O2). All reactions occurred in acetate buffer solution (pH 3.5) to generate hydroxyl radicals (•OH) and the kinetics were followed by UV–vis absorbance at 654 nm. The steady-state kinetic parameters were obtained from the Michaelis-Menten equation and exhibited a good catalytic efficiency of Bi2Fe4O9 NPs as enzyme mimetics. Michaelis–Menten constant (Km) values were estimated as 0.07 and 0.73 mM for TMB and H2O2, respectively. The presented method is efficient, rapid, cost-effective, and sensitive for the colorimetric detection of dopamine with a linear range (LR) from 0.15 to 50 μM and a detection limit (LOD) of 51 nM. The proposed colorimetric sensor was successfully applied for the detection of different concentrations of dopamine in spiked fetal bovine serum (FBS) and horse serum (HS) samples. It is anticipated that Bi2Fe4O9 nanozyme holds great potential in biomedical analysis and diagnostic applications of dopamine-related diseases.

Biochemical characterization of OXA-244, an emerging OXA-48 variant with reduced β-lactam hydrolytic activity.

OXA-48-producing Enterobacterales have widely disseminated globally with an increasing number of variants identified. Among them, OXA-244 is increasingly reported, despite detection difficulties. To determine the steady-state kinetic parameters of OXA-244. The blaOXA-244 gene was amplified, cloned into plasmids p-TOPO and pET41b+, and transformed into Escherichia coli TOP10 for MIC determination and E. coli BL21 DE3 for purification. Steady-state kinetic parameters and IC50s of clavulanic acid, tazobactam and NaCl were determined using purified OXA-244. Molecular modelling was also performed. A reduction in MICs of temocillin and carbapenems was observed in E. coli expressing OXA-244 as compared with OXA-48. The kinetic parameters revealed a reduced carbapenemase activity of OXA-244 as compared with OXA-48, especially for imipenem, which was 10-fold lower. Similarly, catalytic efficiency (kcat/Km) was reduced by 4-fold and 20-fold for ampicillin and temocillin, respectively. Kinetic parameters for cephalosporins were, however, similar. Molecular modelling studies evidenced the key role of R214 in OXA-48, establishing salt bridges with D159 and with the carboxylate group of the R1 substituent of temocillin. These interactions are not possible with G214 in OXA-244, explaining the reduced affinity of temocillin for this enzyme. The R214G mutation in OXA-244 is also likely to induce changes in the active site's water network that would explain the decrease in the hydrolysis rate of carbapenems. Our data confirm that the R214G mutation (present in OXA-244) results in reduced carbapenem- and temocillin-hydrolysing activity, confirming the crucial role of residue 214 in the hydrolysis of these substrates by OXA-48-like β-lactamases.

L-Cysteine as an Irreversible Inhibitor of the Peroxidase-Mimic Catalytic Activity of 2-Dimensional Ni-Based Nanozymes

The ability to modulate the catalytic activity of inorganic nanozymes is of high interest. In particular, understanding the interactions of inhibitor molecules with nanozymes can bring them one step closer to the natural enzymes and has thus started to attract intense interest. To date, a few reversible inhibitors of the nanozyme activity have been reported. However, there are no reports of irreversible inhibitor molecules that can permanently inhibit the activity of nanozymes. In the current work, we show the ability of L-cysteine to act as an irreversible inhibitor to permanently block the nanozyme activity of 2-dimensional (2D) NiO nanosheets. Determination of the steady state kinetic parameters allowed us to obtain mechanistic insights into the catalytic inhibition process. Further, based on the irreversible catalytic inhibition capability of L-cysteine, we demonstrate a highly specific sensor for the detection of this biologically important molecule.

Thermostability Engineering of a Class II Pyruvate Aldolase from Escherichia coli by in Vivo Folding Interference

The use of enzymes in industrial processes is often limited by the unavailability of biocatalysts with prolonged stability. Thermostable enzymes allow increased process temperature and thus higher substrate and product solubility, reuse of expensive biocatalysts, resistance against organic solvents, and better “evolvability” of enzymes. In this work, we have used an activity-independent method for the selection of thermostable variants of any protein in Thermus thermophilus through folding interference at high temperature of a thermostable antibiotic reporter protein at the C-terminus of a fusion protein. To generate a monomeric folding reporter, we have increased the thermostability of the moderately thermostable Hph5 variant of the hygromycin B phosphotransferase from Escherichia coli to meet the method requirements. The final Hph17 variant showed 1.5 °C higher melting temperature (Tm) and 3-fold longer half-life at 65 °C compared to parental Hph5, with no changes in the steady-state kinetic parameters. Additionally, we demonstrate the validity of the reporter by stabilizing the 2-keto-3-deoxy-l-rhamnonate aldolase from E. coli (YfaU). The most thermostable multiple-mutated variants thus obtained, YfaU99 and YfaU103, showed increases of 2 and 2.9 °C in Tm compared to the wild-type enzyme but severely lower retro-aldol activities (150- and 120-fold, respectively). After segregation of the mutations, the most thermostable single variant, Q107R, showed a Tm 8.9 °C higher, a 16-fold improvement in half-life at 60 °C and higher operational stability than the wild-type, without substantial modification of the kinetic parameters.

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.

Measurement of Nucleotide Hydrolysis Using Fluorescent Biosensors for Phosphate.

Assays for the detection of inorganic phosphate (Pi) are widely used to measure the activity of nucleotide hydrolyzing enzymes, such as ATPases and GTPases. The fluorescent biosensors for Pi, described here, are based on fluorescently labeled versions of E. coli phosphate-binding protein (PBP), which translates Pi binding into a large change in fluorescence intensity. In comparison with other Pi-detection systems, these biosensors are characterized by a high sensitivity (sub-micromolar Pi concentrations) and high time resolution (tens of milliseconds), and they are therefore particularly well suited for measurements of phosphate ester hydrolysis in real time. In this chapter, it is described how the Pi biosensors can be used to measure kinetics of ATPase and GTPase reactions, both under steady state and pre-steady state conditions. An example protocol is given for determining steady state kinetic parameters, Km and kcat, of the ATP-dependent chromatin remodeler Chd1, in a plate reader format. In addition, the measurement of Pi release kinetics under pre-steady state conditions is described, including a detailed experimental procedure for a singleturnover measurement of ATP hydrolysis by the ABC-type ATPase SufBC using rapid mixing.