scholarly journals Cloning, expression and characterization of thermostable YdaP from Bacillus licheniformis 9A

2018 ◽  
Vol 65 (1) ◽  
pp. 59-66 ◽  
Author(s):  
Joseph D Wani Lako ◽  
Jada P Yengkopiong ◽  
William HL Stafford ◽  
Marla Tuffin ◽  
Don A Cowan
Keyword(s):  
Bacillus Licheniformis ◽  
Active Site ◽  
Soluble Form ◽  
Significant Activity ◽  
Amino Acid Residues ◽  
Equivalent Site ◽  
Artificial Electron Acceptor ◽  
Triton X ◽  
Sole Product

The Bacillus licheniformis ydaP gene encodes for a pyru­vate oxidase that catalyses the oxidative decarboxyla­tion of pyruvate to acetate and CO2. The YdaP form of this enzyme was purified about 48.6-folds to homoge­neity in three steps. The enzyme was recovered in a soluble form and demonstrated significant activity on pyruvate using 2, 6-dichlorophenolindophenol (DCPIP) as an artificial electron acceptor. HPLC analysis of the YdaP-enzyme catalysed conversion of pyruvate showed acetate as the sole product, confirming the putative identity of pyruvate oxidase. Analysis of the substrate specificity showed that the YdaP enzyme demonstrated preference for short chain oxo acids; however, it was activated by 1% Triton X-100. The YdaP substrate-bind­ing pocket from the YdaP protein differed substantially from the equivalent site in all of the so far character­ized pyruvate oxidases, suggesting that the B. licheni­formis YdaP might accept different substrates. This could allow more accessibility of large substrates into the active site of this enzyme. The thermostability and pH activity of the YdaP enzyme were determined, with optimums at 50ºC and pH 5.8, respectively. The amino acid residues forming the catalytic cavity were identi­fied as Gln460 to Ala480.

scholarly journals Characterization of the Functional Roles of Amino Acid Residues in Acceptor-binding Subsite +1 in the Active Site of the Glucansucrase GTF180 fromLactobacillus reuteri180

2015 ◽  
Vol 290 (50) ◽  
pp. 30131-30141 ◽  
Author(s):  
Xiangfeng Meng ◽  
Tjaard Pijning ◽  
Justyna M. Dobruchowska ◽  
Gerrit J. Gerwig ◽  
Lubbert Dijkhuizen
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Amino Acid Residues ◽  
Functional Roles

scholarly journals Identification of Active-Site Amino Acid Residues in the Chiba Virus 3C-Like Protease

2002 ◽  
Vol 76 (12) ◽  
pp. 5949-5958 ◽  
Author(s):  
Yuichi Someya ◽  
Naokazu Takeda ◽  
Tatsuo Miyamura
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Hepatitis A Virus ◽  
Side Chain ◽  
Significant Activity ◽  
Amino Acid Residues ◽  
Active Site Residues ◽  
3C Protease ◽  
Catalytic Dyad ◽  
Positively Charged

ABSTRACT The 3C-like protease of the Chiba virus, a Norwalk-like virus, is one of the chymotrypsin-like proteases. To identify active-site amino acid residues in this protease, 37 charged amino acid residues and a putative nucleophile, Cys139, within the GDCG sequence were individually replaced with Ala in the 3BC precursor, followed by expression in Escherichia coli, where the active 3C-like protease would cleave 3BC into 3B (VPg) and 3C (protease). Among 38 Ala mutants, 7 mutants (R8A, H30A, K88A, R89A, D138A, C139A, and H157A) completely or nearly completely lost the proteolytic activity. Cys139 was replaceable only with Ser, suggesting that an SH or OH group in the less bulky side chain was required for the side chain of the residue at position 139. His30, Arg89, and Asp138 could not be replaced with any other amino acids. Although Arg8 was also not replaceable for the 3B/3C cleavage and the 3C/3D cleavage, the N-terminal truncated mutant devoid of Arg8 significantly cleaved 3CD into 3C and 3D (polymerase), indicating that Arg8 itself was not directly involved in the proteolytic cleavage. As for position 88, a positively charged residue was required because the Arg mutant showed significant activity. As deduced by the X-ray structure of the hepatitis A virus 3C protease, Arg8, Lys88, and Arg89 are far away from the active site, and the side chain of Asp138 is directed away from the active site. Therefore, these are not catalytic residues. On the other hand, all of the mutants of His157 in the S1 specificity pocket tended to retain very slight activity, suggesting a decreased level of substrate recognition. These results, together with a sequence alignment with the picornavirus 3C proteases, indicate that His30 and Cys139 are active-site residues, forming a catalytic dyad without a carboxylate directly participating in the proteolysis.

Characterization of Acetylcholinesterase in Caco-2 Cells

2002 ◽  
Vol 227 (7) ◽  
pp. 480-486 ◽  
Author(s):  
Lauren R. Plageman ◽  
Giovanni M. Pauletti ◽  
Kenneth A. Skau
Keyword(s):  
Active Site ◽  
Substrate Inhibition ◽  
Extracellular Fluid ◽  
Acetylcholinesterase Activity ◽  
Monomeric Form ◽  
Catalytic Center ◽  
Membrane Bound ◽  
Degree Of Differentiation ◽  
Triton X

Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) was solubilized from cultured Caco-2 cells. It was established that this enzyme activity is acetylcholinesterase by substrate specificity (acetylthiocholine, acetyl-β-methylthiocholine>propionylthiocholine>butyrylthiocholine), substrate inhibition, and specificity of inhibitors (BW284c51>iso-OMPA). The acetylcholinesterase activity increased proportional to the degree of differentiation of the cells. Most of the enzyme was membrane bound, requiring detergent for solubilization, and the active site faced the external fluid. Only one peak of activity, which corresponded to a monomeric form, could be detected on linear sucrose density gradients. The sedimentation of this form of the enzyme was shifted depending on whether Triton X-100 or Brij 96 detergent was used. These results indicate that the epithelial-derived Caco-2 cells produce predominantly an amphiphilic, monomeric form of acetylcholinesterase that is bound to the plasma membrane and whose catalytic center faces the extracellular fluid.

Prokaryotic expression and characterization of a keratinolytic protease from Aspergillus niger

Biologia ◽  
2015 ◽  
Vol 70 (2) ◽  
Author(s):  
Xiaoling Chen ◽  
Bo Zhou ◽  
Meng Xu ◽  
Gang Jia ◽  
Zhiqing Huang ◽  
...  
Keyword(s):  
Aspergillus Niger ◽  
Ethylenediaminetetraacetic Acid ◽  
Signal Sequence ◽  
Sodium Dodecyl ◽  
Recombinant Enzyme ◽  
Protease Gene ◽  
Amino Acid Residues ◽  
Triton X ◽  
Keratinolytic Protease

AbstractIn this study, a keratinolytic protease gene (named as kerD) from Aspergillus niger was cloned. The full-length coding sequence of kerD consists of 1,251 bp and encodes a protein with 416 amino acid residues with a molecular mass of 43,831 Da. A DNA fragment encoding mature kerD without its signal sequence was inserted into the expression vector pET30a(+) and successfully expressed in Escherichia coli. The recombinant protein was purified to approximately 100% purity using Ni-IDA affinity chromatography, and identified by Western blot. The recombinant enzyme had an optimal pH of 8.0 and was stable at pH 7.0-9.0. It exhibited an optimal temperature for activity of 70 °C and was stable at 30-50 °C. It was highly inhibited by 1,10-phenanthroline, ethylenediaminetetraacetic acid and sodium dodecyl sulphate, but activated by phenylmethanesulfonyl fluoride, Mg2+, Fe2+, Mn2+, Cu2+, Zn2+, Ca2+, dithiothreitol, Triton X-100, dimethyl sulfoxide and isopropyl alcohol. The recombinant enzyme could hydrolyse a broad range of protein substrates.

scholarly journals Characterization of a New Member of the Flavoprotein Disulfide Reductase Family of Enzymes fromMycobacterium tuberculosis

2004 ◽  
Vol 279 (50) ◽  
pp. 52694-52702 ◽  
Author(s):  
Argyrides Argyrou ◽  
Matthew W. Vetting ◽  
John S. Blanchard
Keyword(s):  
Active Site ◽  
Isotope Effects ◽  
High Specificity ◽  
Anomalous Scattering ◽  
Amino Acid Residues ◽  
Kinetic Isotope ◽  
Isomorphous Replacement ◽  
Potent Inhibition ◽  
Lipoamide Dehydrogenase

ThelpdA(Rv3303c) gene fromMycobacterium tuberculosisencoding a new member of the flavoprotein disulfide reductases was expressed inEscherichia coli, and the recombinant LpdA protein was purified to homogeneity. LpdA is a homotetramer and co-purifies with one molecule of tightly but noncovalently bound FAD and NADP+per monomer. Although annotated as a probable lipoamide dehydrogenase inM. tuberculosis, LpdA cannot catalyze reduction of lipoyl substrates, because it lacks one of two cysteine residues involved in dithiol-disulfide interchange with lipoyl substrates and a His-Glu pair involved in general acid catalysis. The crystal structure of LpdA was solved by multiple isomorphous replacement with anomalous scattering, which confirmed the absence of these catalytic residues from the active site. Although LpdA cannot catalyze reduction of disulfide-bonded substrates, it catalyzes the NAD(P)H-dependent reduction of alternative electron acceptors such as 2,6-dimethyl-1,4-benzoquinone and 5-hydroxy-1,4-naphthaquinone. Significant primary deuterium kinetic isotope effects were observed with [4S-2H]NADH establishing that the enzyme promotes transfer of the C4-proShydride of NADH. The absence of an isotope effect with [4S-2H]NADPH, the lowKmvalue of 0.5 μmfor NADPH, and the potent inhibition of the NADH-dependent reduction of 2,6-dimethyl-1,4-benzoquinone by NADP+(Ki∼ 6 nm) and 2′-phospho-ADP-ribose (Ki∼ 800 nm), demonstrate the high affinity of LpdA for 2′-phosphorylated nucleotides and that the physiological substrate/product pair is NADPH/NADP+rather than NADH/NAD+. Modeling of NADP+in the active site revealed that LpdA achieves the high specificity for NADP+through interactions involving the 2′-phosphate of NADP+and amino acid residues that are different from those in glutathione reductase.

scholarly journals Leishmania major possesses a unique HemG-type protoporphyrinogen IX oxidase

2014 ◽  
Vol 34 (4) ◽  
Author(s):  
Dagmar Zwerschke ◽  
Simone Karrie ◽  
Dieter Jahn ◽  
Martina Jahn
Keyword(s):  
Amino Acid ◽  
Electron Acceptor ◽  
Active Site ◽  
Leishmania Major ◽  
Amino Acid Residues

Characterization of the first eukaryotic HemG-type protoporphyrinogen IX oxidase from Leishmania major. The cofactor, the electron acceptor and essential active-site amino acid residues were determined for the enzyme. A model for haemoprotein formation for macrophage associated L. major was deduced.

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