The first comprehensive phylogenetic and biochemical analysis of NADH diphosphatases reveals that the enzyme from Tuber melanosporum is highly active towards NAD+

AbstractNudix (for nucleoside diphosphatases linked to other moieties, X) hydrolases are a diverse family of proteins capable of cleaving an enormous variety of substrates, ranging from nucleotide sugars to NAD+-capped RNAs. Although all the members of this superfamily share a common conserved catalytic motif, the Nudix box, their substrate specificity lies in specific sequence traits, which give rise to different subfamilies. Among them, NADH pyrophosphatases or diphosphatases (NADDs) are poorly studied and nothing is known about their distribution. To address this, we designed a Prosite-compatible pattern to identify new NADDs sequences. In silico scanning of the UniProtKB database showed that 3% of Nudix proteins were NADDs and displayed 21 different domain architectures, the canonical architecture (NUDIX-like_zf-NADH-PPase_NUDIX) being the most abundant (53%). Interestingly, NADD fungal sequences were prominent among eukaryotes, and were distributed over several Classes, including Pezizomycetes. Unexpectedly, in this last fungal Class, NADDs were found to be present from the most common recent ancestor to Tuberaceae, following a molecular phylogeny distribution similar to that previously described using two thousand single concatenated genes. Finally, when truffle-forming ectomycorrhizal Tuber melanosporum NADD was biochemically characterized, it showed the highest NAD+/NADH catalytic efficiency ratio ever described.

Download Full-text

Characterization of the First OXA-10 Natural Variant with Increased Carbapenemase Activity

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01817-18 ◽

2018 ◽

Vol 63 (1) ◽

Cited By ~ 4

Author(s):

Stathis D. Kotsakis ◽

Carl-Fredrik Flach ◽

Mohammad Razavi ◽

D. G. Joakim Larsson

Keyword(s):

Carbapenem Resistance ◽

Catalytic Efficiency ◽

Resistance Mechanisms ◽

Broad Host Range ◽

Evolutionary Potential ◽

Class D ◽

Steady State Kinetic ◽

Natural Variant ◽

Hydrolyzing Enzymes ◽

Catalytic Motif

ABSTRACTWhile carbapenem resistance in Gram-negative bacteria is mainly due to the production of efficient carbapenemases, β-lactamases with a narrower spectrum may also contribute to resistance when combined with additional mechanisms. OXA-10-type class D β-lactamases, previously shown to be weak carbapenemases, could represent such a case. In this study, two novel OXA-10 variants were identified as the sole carbapenem-hydrolyzing enzymes in meropenem-resistant enterobacteria isolated from hospital wastewater and found by next-generation sequencing to express additional β-lactam resistance mechanisms. The new variants, OXA-655 and OXA-656, were carried by two related IncQ1 broad-host-range plasmids. Compared to the sequence of OXA-10, they both harbored a Thr26Met substitution, with OXA-655 also bearing a leucine instead of a valine in position 117 of the SAV catalytic motif. Susceptibility profiling of laboratory strains replicating the naturalblaOXAplasmids and of recombinant clones expressing OXA-10 and the novel variants in an isogenic background indicated that OXA-655 is a more efficient carbapenemase. The carbapenemase activity of OXA-655 is due to the Val117Leu substitution, as shown by steady-state kinetic experiments, where thekcatof meropenem hydrolysis was increased 4-fold. In contrast, OXA-655 had no activity toward oxyimino-β-lactams, while its catalytic efficiency against oxacillin was significantly reduced. Moreover, the Val117Leu variant was more efficient against temocillin and cefoxitin. Molecular dynamics indicated that Val117Leu affects the position 117-Leu155 interaction, leading to structural shifts in the active site that may alter carbapenem alignment. The evolutionary potential of OXA-10 enzymes toward carbapenem hydrolysis combined with their spread by promiscuous plasmids indicates that they may pose a future clinical threat.

Download Full-text

Engineered unnatural ubiquitin for optimal detection of deubiquitinating enzymes

10.1101/2020.01.30.926881 ◽

2020 ◽

Cited By ~ 1

Author(s):

Wioletta Rut ◽

Mikołaj Żmudziński ◽

Scott J. Snipas ◽

Miklos Bekes ◽

Tony T. Huang ◽

...

Keyword(s):

Amino Acids ◽

Neurodegenerative Disorders ◽

Unnatural Amino Acids ◽

Biochemical Analysis ◽

Deubiquitinating Enzymes ◽

Fluorogenic Substrates ◽

Protein Conjugates ◽

Highly Active ◽

Selective Chemical

AbstractDeubiquitinating enzymes (DUBs) are responsible for removing ubiquitin (Ub) from its protein conjugates. DUBs have been implicated as attractive therapeutic targets in the treatment of viral diseases, neurodegenerative disorders and cancer. The lack of selective chemical tools for the exploration of these enzymes significantly impairs the determination of their roles in both normal and pathological states. Commercially available fluorogenic substrates are based on the C-terminal Ub motif or contain Ub coupled to a fluorophore (Z-LRGG-AMC, Ub-AMC); therefore, these substrates suffer from lack of selectivity. By using a hybrid combinatorial substrate library (HyCoSuL) and a defined P2 library containing a wide variety of nonproteinogenic amino acids, we established a full substrate specificity profile for two DUBs—MERS PLpro and human UCH-L3. Based on these results, we designed and synthesized Ub-based substrates and activity-based probes (ABPs) containing selected unnatural amino acids located in the C-terminal Ub motif. Biochemical analysis and cell-based experiments confirmed the activity and selectivity of engineered Ub-based substrates and probes. Using this approach, we propose that for any protease that recognizes Ub and Ub-like substrates, a highly active and selective unnatural substrate or probe can be engineered.

Download Full-text

Known Evolutionary Paths Are Accessible to Engineered ß-Lactamases Having Altered Protein Motions at the Timescale of Catalytic Turnover

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2020.599298 ◽

2020 ◽

Vol 7 ◽

Author(s):

Lorea Alejaldre ◽

Claudèle Lemay-St-Denis ◽

Carles Perez Lopez ◽

Ferran Sancho Jodar ◽

Victor Guallar ◽

...

Keyword(s):

Molecular Evolution ◽

Synergistic Effect ◽

Protein Dynamics ◽

Catalytic Efficiency ◽

Specific Sequence ◽

Protein Motions ◽

Protein Energy ◽

Protein Functions ◽

Directed Molecular Evolution

The evolution of new protein functions is dependent upon inherent biophysical features of proteins. Whereas, it has been shown that changes in protein dynamics can occur in the course of directed molecular evolution trajectories and contribute to new function, it is not known whether varying protein dynamics modify the course of evolution. We investigate this question using three related ß-lactamases displaying dynamics that differ broadly at the slow timescale that corresponds to catalytic turnover yet have similar fast dynamics, thermal stability, catalytic, and substrate recognition profiles. Introduction of substitutions E104K and G238S, that are known to have a synergistic effect on function in the parent ß-lactamase, showed similar increases in catalytic efficiency toward cefotaxime in the related ß-lactamases. Molecular simulations using Protein Energy Landscape Exploration reveal that this results from stabilizing the catalytically-productive conformations, demonstrating the dominance of the synergistic effect of the E014K and G238S substitutions in vitro in contexts that vary in terms of sequence and dynamics. Furthermore, three rounds of directed molecular evolution demonstrated that known cefotaximase-enhancing mutations were accessible regardless of the differences in dynamics. Interestingly, specific sequence differences between the related ß-lactamases were shown to have a higher effect in evolutionary outcomes than did differences in dynamics. Overall, these ß-lactamase models show tolerance to protein dynamics at the timescale of catalytic turnover in the evolution of a new function.

Download Full-text

Highly Active and Stable Large Catalase Isolated from a Hydrocarbon Degrading Aspergillus terreus MTCC 6324

Enzyme Research ◽

10.1155/2016/4379403 ◽

2016 ◽

Vol 2016 ◽

pp. 1-8 ◽

Cited By ~ 6

Author(s):

Preety Vatsyayan ◽

Pranab Goswami

Keyword(s):

Molecular Mass ◽

Aspergillus Terreus ◽

Specific Activity ◽

Catalytic Efficiency ◽

Highly Active ◽

Peptide Mass ◽

Tandem Mass Spectroscopy ◽

Isoelectric Ph ◽

Alkaline Ph Stability ◽

High Level

A hydrocarbon degrading Aspergillus terreus MTCC 6324 produces a high level of extremely active and stable cellular large catalase (CAT) during growth on n-hexadecane to combat the oxidative stress caused by the hydrocarbon degrading metabolic machinery inside the cell. A 160-fold purification with specific activity of around 66 × 105 U mg−1 protein was achieved. The native protein molecular mass was 368 ± 5 kDa with subunit molecular mass of nearly 90 kDa, which indicates that the native CAT protein is a homotetramer. The isoelectric pH (pI) of the purified CAT was 4.2. BLAST aligned peptide mass fragments of CAT protein showed its highest similarity with the catalase B protein from other fungal sources. CAT was active in a broad range of pH 4 to 12 and temperature 25°C to 90°C. The catalytic efficiency (Kcat/Km) of 4.7 × 108 M−1 s−1 within the studied substrate range and alkaline pH stability (half-life, t1/2 at pH 12~15 months) of CAT are considerably higher than most of the extensively studied catalases from different sources. The storage stability (t1/2) of CAT at physiological pH 7.5 and 4°C was nearly 30 months. The haem was identified as haem b by electrospray ionization tandem mass spectroscopy (ESI-MS/MS).

Download Full-text

Mesoporous bimetallic Fe/Co as highly active heterogeneous Fenton catalyst for the degradation of tetracycline hydrochlorides

Scientific Reports ◽

10.1038/s41598-019-52013-y ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Junchao Li ◽

Xuhua Li ◽

Jindong Han ◽

Fansheng Meng ◽

Jinyuan Jiang ◽

...

Keyword(s):

Bimetallic Catalyst ◽

Removal Rate ◽

Catalytic Efficiency ◽

Nitrogen Adsorption ◽

Catalytic Performance ◽

Operating Parameters ◽

Co Catalyst ◽

Highly Active ◽

Fenton Catalyst ◽

Potential Applications

Abstract Mesoporous bimetallic Fe/Co was prepared as a Fenton-like catalyst to degrade the tetracycline hydrochlorides (TC). The nanocasting strategy with KIT-6 as a hard template was carried out to synthesize the mesoporous bimetallic catalyst. The mesoporous bimetallic Fe/Co catalyst was characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), nitrogen adsorption-desorption isotherms, and Brunauer-Emmett-Teller (BET) method. The results showed that the catalyst has significant nanofeatures; the surface area, pore size, and particle size were 113.8 m2g−1, 4 nm, and 10 nm, respectively. In addition, the effects of the operating parameters, such as the iron-to-cobalt ratio, pH, H2O2, and initial TC concentrations on its catalytic performance were investigated. The best operating parameters were as follows: iron-to-cobalt ratio = 2:1 to 1:1, pH = 5–9, H2O2: 30 mmol, initial TC less than 30 mg/L. Furthermore, the mesoporous bimetallic Fe/Co showed a good performance for degrading TC, achieving a removal rate of 86% of TC after 3 h under the reaction conditions of H2O2 = 30 mmol, mesoporous bimetallic Fe/Co = 0.6 g/L, TC = 30 mg/L, pH = 7.0, and temperature = 25.5 °C. The mesoporous bimetallic Fe/Co catalyst shows good stability and reusability. This work demonstrated that mesoporous bimetallic Fe/Co has excellent catalytic efficiency, smaller amounts of leached ions, and wider pH range, which enhance its potential applications.

Download Full-text

Highly Active Hydrogenation Catalysts Based on Pd Nanoparticles Dispersed along Hierarchical Porous Silica Covered with Polydopamine as Interfacial Glue

Catalysts ◽

10.3390/catal10040449 ◽

2020 ◽

Vol 10 (4) ◽

pp. 449

Author(s):

Miguel Ródenas ◽

Jamal El Haskouri ◽

José Vicente Ros-Lis ◽

M. Dolores Marcos ◽

Pedro Amorós ◽

...

Keyword(s):

Porous Silica ◽

Catalytic Efficiency ◽

Pd Nanoparticles ◽

Hydrogenation Catalysts ◽

Highly Active ◽

Hierarchical Porous ◽

Similar Materials ◽

Comprehensive Comparison ◽

Palladium Content

New catalysts based on Pd(0) nanoparticles (Pd NPs) on a bimodal porous silica of the UVM-7/polydopamine (PDA) support have been synthesized following two preparative strategies based on the sequential or joint incorporation of two components of the composite (Pd and PDA). We analyzed the role played by the PDA as ‘interfacial glue’ between the silica scaffold and the Pd NPs. The catalysts were tested for the hydrogenation of 4-nitrophenol using (NEt4)BH4 as the hydrogenating agent. In addition to the palladium content, the characterization of the catalysts at the micro and nanoscale has highlighted the importance of different parameters, such as the size and dispersion of the Pd NPs, as well as their accessibility to the substrate (greater or lesser depending on their entrapment level in the PDA) on the catalytic efficiency. Staged sequential synthesis has led to better catalytic results. The most active Pd(0) centers seem to be Pd NPs of less than 1 nm on the PDA surface. The efficiency of the catalysts obtained is superior to that of similar materials without PDA. A comprehensive comparison has been made with other catalysts based on Pd NPs in a wide variety of supports. The TOF values achieved are among the best described in the literature.

Download Full-text

Abstract P276: Tissue Primacy of Chymase as an Angiotensin II-forming Enzyme from Angiotensin-(1-12)

Hypertension ◽

10.1161/hyp.68.suppl_1.p276 ◽

2016 ◽

Vol 68 (suppl_1) ◽

Author(s):

Sarfaraz Ahmad ◽

Jasmina Varagic ◽

Che P Cheng ◽

James F Collawn ◽

Louis J Dell’Italia ◽

...

Keyword(s):

Angiotensin Ii ◽

Left Ventricle ◽

Rat Heart ◽

Kinetic Data ◽

Plasma Membranes ◽

Catalytic Efficiency ◽

Heart Tissue ◽

Ang Ii ◽

Efficiency Ratio

Breaking the prevailing acceptance of ACE primacy as the Ang II-forming enzyme, we have demonstrated that cardiac Ang II production in human and rat heart tissues are primarily mediated by chymase. In this study, we compared the affinity of cardiac chymase to generate Ang II from Ang-(1-12) or Ang I in plasma membranes (PMs) isolated from the diseased left atria of humans and SHR left ventricle. PMs (50-100 μg) were exposed to increasing concentrations of either Ang-(1-12) or Ang I substrate (0-300 μM) for 30 min at 37 o C in the presence of lisinopril (200 μM). The K m and V max of human cardiac chymase (Mean ± SE) were 29 ± 0.9 vs 87 ± 8.8 μM and 57 ± 1.4 vs 145 ± 3.7 μM/min/mg for Ang-(1-12) and Ang I substrates, respectively. Similarly, the K m and V max of rat cardiac chymase were 64 ± 6.3 vs 142 ± 17 μM and 13.2 ± 1.3 vs 1.9 ± 0.2 μM/min/mg for Ang-(1-12) and Ang I substrate, respectively. These data suggest that cardiac chymase has a higher affinity for Ang-(1-12) substrate compared to Ang I in both human and rat heart tissues. Further, our kinetic data show that the catalytic efficiency (ratio of V max /K m ) of human and rat chymase were 1.2 and 15.4-fold higher for Ang-(1-12) substrate compared to Ang I. Overall, our findings suggest that Ang-(1-12), rather than Ang I, is the preferred substrate for chymase in the generation of Ang II by human and rat heart tissue.

Download Full-text

MnO2 nanowires anchored on amine functionalized graphite nanosheets: highly active and reusable catalyst for organic oxidation reactions

RSC Advances ◽

10.1039/c5ra17777d ◽

2015 ◽

Vol 5 (112) ◽

pp. 92585-92595 ◽

Cited By ~ 10

Author(s):

A. Chakravarty ◽

D. Sengupta ◽

B. Basu ◽

A. Mukherjee ◽

G. De

Keyword(s):

Catalytic Efficiency ◽

Reusable Catalyst ◽

Oxidation Reactions ◽

Graphite Nanosheets ◽

Highly Active ◽

Amine Functionalized ◽

Graphite Nanosheet ◽

Prepared Composite ◽

Organic Oxidation ◽

Mno2 Nanowires

MnO2 nanowires were synthesized on amine functionalized graphite nanosheet and excellent catalytic efficiency of as-prepared composite towards organic oxidation reactions was demonstrated.

Download Full-text

The Assessment of Cholinesterase from the Liver ofPuntius Javanicusas Detection of Metal Ions

The Scientific World JOURNAL ◽

10.1155/2014/571094 ◽

2014 ◽

Vol 2014 ◽

pp. 1-9 ◽

Cited By ~ 9

Author(s):

Mohd Khalizan Sabullah ◽

Mohd Rosni Sulaiman ◽

Mohd Yunus Abd Shukor ◽

Mohd Arif Syed ◽

Nor Aripin Shamaan ◽

...

Keyword(s):

Metal Ions ◽

Room Temperature ◽

Optimum Parameter ◽

Catalytic Efficiency ◽

Storage Condition ◽

Maximal Velocity ◽

Optimal Temperature ◽

Efficiency Ratio ◽

Single Exposure ◽

Significant Difference

Crude extract of ChE from the liver ofPuntius javanicuswas purified using procainamide-sepharyl 6B. S-Butyrylthiocholine iodide (BTC) was selected as the specific synthetic substrate for this assay with the highest maximal velocity and lowest biomolecular constant at 53.49 µmole/min/mg and 0.23 mM, respectively, with catalytic efficiency ratio of 0.23. The optimum parameter was obtained at pH 7.5 and optimal temperature in the range of 25 to 30°C. The effect of different storage condition was assessed where ChE activity was significantly decreased after 9 days of storage at room temperature. However, ChE activity showed no significant difference when stored at 4.0, 0, and −25°C for 15 days. Screening of heavy metals shows that chromium, copper, and mercury strongly inhibitedP. javanicusChE by lowering the activity below 50%, while several pairwise combination of metal ions exhibited synergistic inhibiting effects on the enzyme which is greater than single exposure especially chromium, copper, and mercury. The results showed thatP. javanicusChE has the potential to be used as a biosensor for the detection of metal ions.

Download Full-text

Phenotypic and Biochemical Comparison of the Carbapenem-Hydrolyzing Activities of Five Plasmid-Borne AmpC β-Lactamases

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01762-09 ◽

2010 ◽

Vol 54 (11) ◽

pp. 4556-4560 ◽

Cited By ~ 63

Author(s):

Hedi Mammeri ◽

Hélène Guillon ◽

François Eb ◽

Patrice Nordmann

Keyword(s):

Biochemical Analysis ◽

Carbapenem Resistance ◽

Catalytic Efficiency ◽

Wild Type ◽

E Coli ◽

Genes Encoding ◽

High Level ◽

Biochemical Comparison ◽

Outer Membrane Permeability

ABSTRACT The CMY-2, ACT-1, DHA-1, ACC-1, and FOX-1 enzymes are representative of five plasmid-mediated AmpC (pAmpC) β-lactamase clusters. Resistance to imipenem has been reported in Enterobacteriaceae as a result of pAmpC expression combined with decreased outer membrane permeability. The aim of this study was to determine the role of different pAmpCs in carbapenem resistance and to define the structure/activity relationship supporting carbapenemase activity. The ampC genes encoding the five pAmpCs and the chromosomal AmpC of Escherichia coli EC6, which was used as a reference cephalosporinase, were cloned and introduced into wild-type E. coli TOP10 and OmpC/OmpF porin-deficient E. coli HB4 strains. The MICs of β-lactams for the recombinant strains revealed that CMY-2, ACT-1, and DHA-1 β-lactamases conferred a high level of resistance to ceftazidime and cefotaxime once expressed in E. coli TOP10 and reduced significantly the susceptibility to imipenem once expressed in E. coli HB4. In contrast, FOX-1 and ACC-1 enzymes did not confer resistance to imipenem. Biochemical analysis showed that CMY-2 β-lactamase and, to a lesser extent, ACT-1 exhibited the highest catalytic efficiency toward imipenem and showed low Km values. A modeling study revealed that the large R2 binding site of these two enzymes may support the carbapenemase activity. Therefore, CMY-2-type, ACT-1-type, and DHA-1-type β-lactamases may promote the emergence of carbapenem resistance in porin-deficient clinical isolates.

Download Full-text