Characterization of the phenylurea hydrolases A and B: founding members of a novel amidohydrolase subgroup

2009 ◽  
Vol 418 (2) ◽  
pp. 431-441 ◽  
Author(s):  
Jeevan L. Khurana ◽  
Colin J. Jackson ◽  
Colin Scott ◽  
Gunjan Pandey ◽  
Irene Horne ◽  
...  
Keyword(s):  
Metal Binding ◽  
Catalytic Mechanism ◽  
Binding Motif ◽  
Evolutionary Origins ◽  
Effects Of Temperature ◽  
Amidohydrolase Superfamily ◽  
Challenging Question ◽  
Active Site Sequence

Mycobacterium brisbanense strain JK1, a bacterium capable of degrading the herbicide diuron, was isolated from herbicide-exposed soil. A gene/enzyme system with diuron hydrolase activity was isolated from this strain and named PUH (phenylurea hydrolase) B (puhB/PuhB) because of its close similarity to the previously characterized PUH A (puhA/PuhA). Both PUHs were heterologously expressed, purified and characterized. The PUHs were found to oligomerize as hexamers in solution, with each monomer containing a mononuclear Zn2+ active site. Sequence analysis showed that these enzymes belong to the metal-dependent amidohydrolase superfamily, although they contain a hitherto unreported Asn-X-His metal-binding motif and appear to form a novel sub-group within this superfamily. The effects of temperature and solvent on the enzymes were characterized. Determination of the kinetic parameters of the PUHs was used alongside Brønsted plots to develop a plausible catalytic mechanism, which is similar to that used by urease. In addition to the primary PUH activity, both enzymes are catalytically promiscuous, efficiently hydrolysing esters, carbamates and phosphotriesters. In fact, an analogue of diuron, in which the C–N bond was replaced by a C–O bond, was found to be turned over as efficiently as diuron, suggesting that the substrate specificity is predominantly determined by steric factors. The discovery of PuhA and PuhB on separate continents, and the absence of any other close homologues in the available sequence databases, poses a challenging question regarding the evolutionary origins of these enzymes.

scholarly journals Purification and biochemical characterization of recombinant Persicaria minor β-sesquiphellandrene synthase

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e2961 ◽  
Author(s):  
De-Sheng Ker ◽  
Sze Lei Pang ◽  
Noor Farhan Othman ◽  
Sekar Kumaran ◽  
Ee Fun Tan ◽  
...  
Keyword(s):  
Active Site ◽  
Metal Binding ◽  
Binding Motif ◽  
Multiple Sequence ◽  
Sesquiterpene Synthase ◽  
Catalytic Active Site ◽  
Soluble Aggregate ◽  
Persicaria Minor ◽  
Metal Binding Motif

Background Sesquiterpenes are 15-carbon terpenes synthesized by sesquiterpene synthases using farnesyl diphosphate (FPP) as a substrate. Recently, a sesquiterpene synthase gene that encodes a 65 kDa protein was isolated from the aromatic plant Persicaria minor. Here, we report the expression, purification and characterization of recombinant P. minor sesquiterpene synthase protein (PmSTS). Insights into the catalytic active site were further provided by structural analysis guided by multiple sequence alignment. Methods The enzyme was purified in two steps using affinity and size exclusion chromatography. Enzyme assays were performed using the malachite green assay and enzymatic product was identified using gas chromatography-mass spectrometry (GC-MS) analysis. Sequence analysis of PmSTS was performed using multiple sequence alignment (MSA) against plant sesquiterpene synthase sequences. The homology model of PmSTS was generated using I-TASSER server. Results Our findings suggest that the recombinant PmSTS is mainly expressed as inclusion bodies and soluble aggregate in the E. coli protein expression system. However, addition of 15% (v/v) glycerol to the protein purification buffer and removal of N-terminal 24 amino acids of PmSTS helped to produce homogenous recombinant protein. Enzyme assay showed that recombinant PmSTS is active and specific to the C15 substrate FPP. The optimal temperature and pH for the recombinant PmSTS are 30 °C and pH 8.0, respectively. The GC-MS analysis further showed that PmSTS produces β-sesquiphellandrene as a major product and β-farnesene as a minor product. MSA analysis revealed that PmSTS adopts a modified conserved metal binding motif (NSE/DTE motif). Structural analysis suggests that PmSTS may binds to its substrate similarly to other plant sesquiterpene synthases. Discussion The study has revealed that homogenous PmSTS protein can be obtained with the addition of glycerol in the protein buffer. The N-terminal truncation dramatically improved the homogeneity of PmSTS during protein purification, suggesting that the disordered N-terminal region may have caused the formation of soluble aggregate. We further show that the removal of the N-terminus disordered region of PmSTS does not affect the product specificity. The optimal temperature, optimal pH, Km and kcat values of PmSTS suggests that PmSTS shares similar enzyme characteristics with other plant sesquiterpene synthases. The discovery of an altered conserved metal binding motif in PmSTS through MSA analysis shows that the NSE/DTE motif commonly found in terpene synthases is able to accommodate certain level of plasticity to accept variant amino acids. Finally, the homology structure of PmSTS that allows good fitting of substrate analog into the catalytic active site suggests that PmSTS may adopt a sesquiterpene biosynthesis mechanism similar to other plant sesquiterpene synthases.

Structures of c-di-GMP/cGAMP degrading phosphodiesterase VcEAL: identification of a novel conformational switch and its implication

2019 ◽  
Vol 476 (21) ◽  
pp. 3333-3353 ◽  
Author(s):  
Malti Yadav ◽  
Kamalendu Pal ◽  
Udayaditya Sen
Keyword(s):  
Active Site ◽  
Metal Binding ◽  
Metal Ion ◽  
Triosephosphate Isomerase ◽  
Catalytic Mechanism ◽  
Degradation Mechanism ◽  
Structural Basis ◽  
Conserved Residues ◽  
Phosphodiester Bond ◽  
Cyclic Dinucleotides

Cyclic dinucleotides (CDNs) have emerged as the central molecules that aid bacteria to adapt and thrive in changing environmental conditions. Therefore, tight regulation of intracellular CDN concentration by counteracting the action of dinucleotide cyclases and phosphodiesterases (PDEs) is critical. Here, we demonstrate that a putative stand-alone EAL domain PDE from Vibrio cholerae (VcEAL) is capable to degrade both the second messenger c-di-GMP and hybrid 3′3′-cyclic GMP–AMP (cGAMP). To unveil their degradation mechanism, we have determined high-resolution crystal structures of VcEAL with Ca2+, c-di-GMP-Ca2+, 5′-pGpG-Ca2+ and cGAMP-Ca2+, the latter provides the first structural basis of cGAMP hydrolysis. Structural studies reveal a typical triosephosphate isomerase barrel-fold with substrate c-di-GMP/cGAMP bound in an extended conformation. Highly conserved residues specifically bind the guanine base of c-di-GMP/cGAMP in the G2 site while the semi-conserved nature of residues at the G1 site could act as a specificity determinant. Two metal ions, co-ordinated with six stubbornly conserved residues and two non-bridging scissile phosphate oxygens of c-di-GMP/cGAMP, activate a water molecule for an in-line attack on the phosphodiester bond, supporting two-metal ion-based catalytic mechanism. PDE activity and biofilm assays of several prudently designed mutants collectively demonstrate that VcEAL active site is charge and size optimized. Intriguingly, in VcEAL-5′-pGpG-Ca2+ structure, β5–α5 loop adopts a novel conformation that along with conserved E131 creates a new metal-binding site. This novel conformation along with several subtle changes in the active site designate VcEAL-5′-pGpG-Ca2+ structure quite different from other 5′-pGpG bound structures reported earlier.

Characterization of Impressed Current Technique to Model Corrosion of Reinforcement in Concrete

2020 ◽  
Vol 11 (1) ◽  
pp. 93-99
Author(s):  
Abu Zakir Morshed ◽  
Sheikh Shakib ◽  
Tanzim Jahin
Keyword(s):  
Immersion Time ◽  
Chloride Content ◽  
Engineering Science ◽  
Coastal Regions ◽  
Faraday’S Law ◽  
Current Technique ◽  
Corrosion Cell ◽  
Impressed Current

Corrosion of reinforcement is an important durability concern for the structures exposed to coastal regions. Since corrosion of reinforcement involves long periods of time, impressed current technique is usually used to accelerate the corrosion of reinforcement in laboratories. Characterization of impressed current technique was the main focus of this research,which involved determination of optimum chloride content and minimum immersion time of specimens for which the application of Faraday’s law could be efficient. To obtain optimum chloride content, the electrolytes in the corrosion cell were prepared similar to that of concrete pore solutions. Concrete prisms of 200 mm by 200 mm by 300 mm were used to determine the minimum immersion time for saturation. It was found that the optimum chloride content was 35 gm/L and the minimum immersion time for saturation was 140 hours. Accounting the results, a modified expression based on Faraday’s law was proposed to calculate weight loss due to corrosion. Journal of Engineering Science 11(1), 2020, 93-99

The Determination of Sense via Deleuze and Blanchot: Paradoxes of the Habitual, the Immemorial, and the Eternal Return

2008 ◽  
Vol 2 (2) ◽  
pp. 155-177 ◽  
Author(s):  
Eugene Brently Young
Keyword(s):  
Eternal Return

Eternal return is the paradox that accounts for the interplay between difference and repetition, a dynamic at the heart of Deleuze's philosophy, and Blanchot's approach to this paradox, even and especially through what it elides, further illuminates it. Deleuze draws on Blanchot's characterisations of difference, forgetting, and the unlivable to depict the ‘sense’ produced via eternal return, which, for Blanchot, is where repetition implicates or ‘carries’ pure difference. However, for Deleuze, difference and the unlivable are also developed by the living repetition or ‘contraction’ of habit, which results in his distinctive characterization of ‘force’, ‘levity’, and sense in eternal return.

SEM/EDS Characterization of Uncommon Metal Whiskers and Determination of Underlying Growth Mechanisms

2018 ◽  
Author(s):  
D. Basak ◽  
L. H. Ponce
Keyword(s):  
Reliability Analysis ◽  
Case Studies ◽  
Whisker Growth ◽  
Innovation Systems ◽  
Growth Mechanisms ◽  
Crystal Oscillator ◽  
Analysis Laboratory ◽  
Investigative Techniques

Abstract Two case-studies on uncommon metals whiskers, performed at the Reliability Analysis Laboratory (RAL) of Northrop Grumman Innovation Systems, are presented. The components analyzed are an Oven Controlled Crystal Oscillator (OCXO) and an Electromechanical Relay. Investigative techniques were used to determine the chemical and physical makeup of the metal whiskers and develop an understanding of the underlying effects and mechanisms that caused the conditions conducive to whisker growth.

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