Functional and biochemical characterization of a recombinant Arabidopsis thaliana 3-deoxy-D-manno-octulosonate 8-phosphate synthase

An open reading frame, encoding for KDOPS (3-deoxy-D-manno-octulosonate 8-phosphate synthase), from Arabidopsis thaliana was cloned into a T7-driven expression vector. The protein was overexpressed in Escherichia coli and purified to homogeneity. Recombinant A. thaliana KDOPS, in solution, displays an apparent molecular mass of 76 kDa and a subunit molecular mass of 31.519 kDa. Unlike previously studied bacterial KDOPSs, which are tetrameric, A. thaliana KDOPS appears to be a dimer in solution. The optimum temperature of the enzyme is 65 °C and the optimum pH is 7.5, with a broad peak between pH 6.5 and 9.5 showing 90% of maximum activity. The enzyme cannot be inactivated by EDTA or dipicolinic acid treatment, nor it can be activated by a series of bivalent metal ions, suggesting that it is a non-metallo-enzyme, as opposed to the initial prediction that it would be a metallo-enzyme. Kinetic studies showed that the enzyme follows a sequential mechanism with Km=3.6 μM for phosphoenolpyruvate and 3.8 μM for D-arabinose 5-phosphate and kcat=5.9 s−1 at 37 °C. On the basis of the characterization of A. thaliana KDOPS and phylogenetic analysis, plant KDOPSs may represent a new, distinct class of KDOPSs.

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Biochemical Characterization of Bovine Brain Myristoyl-CoA:Protein N-Myristoyltransferase Type 2

Journal of Biomedicine and Biotechnology ◽

10.1155/2009/907614 ◽

2009 ◽

Vol 2009 ◽

pp. 1-9 ◽

Cited By ~ 1

Author(s):

Ponniah Selvakumar ◽

Ashakumary Lakshmikuttyamma ◽

Rajendra K. Sharma

Keyword(s):

Metal Ion ◽

Biochemical Characterization ◽

Kinetic Studies ◽

Bovine Brain ◽

Covalent Attachment ◽

Reading Frame ◽

Gene Coding ◽

Acid Protein

Protein N-myristoylation is a lipidic modification which refers to the covalent attachment of myristate, a 14-carbon saturated fatty acid, to the N-terminal glycine residue of a number of mammalian, viral, and fungal proteins. In this paper, we have cloned the gene coding for myristoyl-CoA:protein N-myristoyltransferase (NMT) fromBos tarusbrain. The open reading frame codes for a 410-amino-acid protein and overexpressed inEscherichia coli. Kinetic studies suggested that bovine brain NMT2 and human NMT1 show significant differences in their peptide substrate specificities. The metal ionCa2+had stimulatory effects on NMT2 activity whileMn2+andZn2+inhibited the enzyme activity. In addition, NMT2 activity was inhibited by various organic solvents and other detergents while NMT1 had a stimulatory effect. Biochemical characterization suggested that both forms of NMT have unique characteristics. Further analysis towards functional role NMT2 will lead the development of therapeutic target for the progression of various diseases such as cancer, cardiovascular diseases, and neurodegenerative diseases.

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Biochemical characterization of a glycoside hydrolase family 43 β-D-galactofuranosidase from the fungus Aspergillus niger

10.1101/2021.10.27.466152 ◽

2021 ◽

Author(s):

Gregory S Bulmer ◽

Fang Wei Yuen ◽

Naimah Begum ◽

Bethan S Jones ◽

Sabine S Flitsch ◽

...

Keyword(s):

Aspergillus Niger ◽

Glycoside Hydrolase ◽

Biochemical Characterization ◽

Maximum Activity ◽

Glycoside Hydrolase Family ◽

Enzyme Specificity ◽

Fungal Enzymes ◽

Glycoside Hydrolase Family 43 ◽

Hydrolase Family

β-D-Galactofuranose (Galf) and its polysaccharides are found in bacteria, fungi and protozoa but do not occur in mammalian tissues, and thus represent a specific target for anti-pathogenic drugs. Understanding the enzymatic degradation of these polysaccharides is therefore of great interest, but the identity of fungal enzymes with exclusively galactofuranosidase activity has so far remained elusive. Here we describe the identification and characterization of a galactofuranosidase from the industrially important fungus Aspergillus niger. Phylogenetic analysis of glycoside hydrolase family 43 subfamily 34 (GH43_34) members revealed the occurrence of three distinct clusters and, by comparison with specificities of characterized bacterial members, suggested a basis for prediction of enzyme specificity. Using this rationale, in tandem with molecular docking, we identified a putative β-D-galactofuranosidase from A. niger which was recombinantly expressed in Escherichia coli. The Galf-specific hydrolase, encoded by xynD demonstrates maximum activity at pH 5, 25 °C towards 4-Nitrophenyl-β-galactofuranoside (pNP-βGalf), with a Km of 17.9 ± 1.9 mM and Vmax of 70.6 ± 5.3 μmol min-1. The characterization of this first fungal GH43 galactofuranosidase offers further molecular insight into the degradation of Galf-containing structures and may inform clinical treatments against fungal pathogens.

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Molecular and Biochemical Characterization of the xlnD-Encoded 3-Hydroxybenzoate 6-Hydroxylase Involved in the Degradation of 2,5-Xylenol via the Gentisate Pathway in Pseudomonas alcaligenes NCIMB 9867

Journal of Bacteriology ◽

10.1128/jb.187.22.7696-7702.2005 ◽

2005 ◽

Vol 187 (22) ◽

pp. 7696-7702 ◽

Cited By ~ 28

Author(s):

Xiaoli Gao ◽

Chew Ling Tan ◽

Chew Chieng Yeo ◽

Chit Laa Poh

Keyword(s):

Escherichia Coli ◽

Fusion Protein ◽

Molecular Mass ◽

Electron Donor ◽

Biochemical Characterization ◽

Aromatic Substrates ◽

Gentisate Pathway ◽

A Subunit ◽

Pseudomonas Alcaligenes

ABSTRACT The xlnD gene from Pseudomonas alcaligenes NCIMB 9867 (strain P25X) was shown to encode 3-hydroxybenzoate 6-hydroxylase I, the enzyme that catalyzes the NADH-dependent conversion of 3-hydroxybenzoate to gentisate. Active recombinant XlnD was purified as a hexahistidine fusion protein from Escherichia coli, had an estimated molecular mass of 130 kDa, and is probably a trimeric protein with a subunit mass of 43 kDa. This is in contrast to the monomeric nature of the few 3-hydroxybenzoate 6-hydroxylases that have been characterized thus far. Like other 3-hydroxybenzoate 6-hydroxylases, XlnD could utilize either NADH or NADPH as the electron donor. P25X harbors a second 3-hydroxybenzoate 6-hydroxylase II that was strictly inducible by specific aromatic substrates. However, the degradation of 2,5-xylenol and 3,5-xylenol in strain P25X was found to be dependent on the xlnD-encoded 6-hydroxylase I and not the second, strictly inducible 6-hydroxylase II.

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Genetic Context and Biochemical Characterization of the IMP-18 Metallo-β-Lactamase Identified in aPseudomonas aeruginosaIsolate from the United States

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00858-10 ◽

2010 ◽

Vol 55 (1) ◽

pp. 140-145 ◽

Cited By ~ 15

Author(s):

Luisa Borgianni ◽

Silvia Prandi ◽

Laurie Salden ◽

Gisela Santella ◽

Nancy D. Hanson ◽

...

Keyword(s):

United States ◽

Biochemical Characterization ◽

Gene Cassette ◽

The United States ◽

Turnover Rates ◽

Reading Frame ◽

Class 1 ◽

Original Array ◽

Genetic Context

ABSTRACTThe production of metallo-β-lactamase (MBL) is an important mechanism of resistance to β-lactam antibiotics, including carbapenems. Despite the discovery and emergence of many acquired metallo-β-lactamases, IMP-type determinants (now counting at least 27 variants) remain the most prevalent in some geographical areas. In Asian countries, and notably Japan, IMP-1 and its closely related variants are most widespread. Some other variants have been detected in other countries and show either an endemic (e.g., IMP-13 in Italy) or sporadic (e.g., IMP-12 in Italy or IMP-18 in the United States) occurrence. The IMP-18-producingPseudomonas aeruginosastrain PS 297 from the southwestern United States carried at least two class 1 integrons. One was identical to In51, while the other, named In133and carrying theblaIMP-18gene cassette in the third position, showed an original array of five gene cassettes, includingaacA7,qacF,aadA1, and an unknown open reading frame (ORF). Interestingly. In133differed significantly from In96, theblaIMP-18-carrying integron identified in aP. aeruginosaisolate from Mexico. The meropenem and ertapenem MIC values were much lower forEscherichia colistrains producing IMP-18 (0.06 and 0.12 μg/ml, respectively) than for strains producing IMP-1 (2 μg/ml for each). Kinetic data obtained with the purified enzyme revealed lower turnover rates of IMP-18 than of other IMP-type enzymes with most substrates.

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Molecular Characterization of Saccharomyces cerevisiae TFIID

Molecular and Cellular Biology ◽

10.1128/mcb.22.16.6000-6013.2002 ◽

2002 ◽

Vol 22 (16) ◽

pp. 6000-6013 ◽

Cited By ~ 78

Author(s):

Steven L. Sanders ◽

Krassimira A. Garbett ◽

P. Anthony Weil

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcription Factor ◽

Molecular Mass ◽

Molecular Characterization ◽

Biochemical Characterization ◽

Gel Filtration ◽

Calculated Molecular Mass ◽

General Transcription Factor

ABSTRACT We previously defined Saccharomyces cerevisiae TFIID as a 15-subunit complex comprised of the TATA binding protein (TBP) and 14 distinct TBP-associated factors (TAFs). In this report we give a detailed biochemical characterization of this general transcription factor. We have shown that yeast TFIID efficiently mediates both basal and activator-dependent transcription in vitro and displays TATA box binding activity that is functionally distinct from that of TBP. Analyses of the stoichiometry of TFIID subunits indicated that several TAFs are present at more than 1 copy per TFIID complex. This conclusion was further supported by coimmunoprecipitation experiments with a systematic family of (pseudo)diploid yeast strains that expressed epitope-tagged and untagged alleles of the genes encoding TFIID subunits. Based on these data, we calculated a native molecular mass for monomeric TFIID. Purified TFIID behaved in a fashion consistent with this calculated molecular mass in both gel filtration and rate-zonal sedimentation experiments. Quite surprisingly, although the TAF subunits of TFIID cofractionated as a single complex, TBP did not comigrate with the TAFs during either gel filtration chromatography or rate-zonal sedimentation, suggesting that TBP has the ability to dynamically associate with the TFIID TAFs. The results of direct biochemical exchange experiments confirmed this hypothesis. Together, our results represent a concise molecular characterization of the general transcription factor TFIID from S. cerevisiae.

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Biochemical characterization of the novel endo -β-mannanase At Man5-2 from Arabidopsis thaliana

Plant Science ◽

10.1016/j.plantsci.2015.10.002 ◽

2015 ◽

Vol 241 ◽

pp. 151-163 ◽

Cited By ~ 3

Author(s):

Yang Wang ◽

Shoaib Azhar ◽

Rosaria Gandini ◽

Christina Divne ◽

Ines Ezcurra ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Biochemical Characterization ◽

The Novel

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Biochemical Characterization of a New β-Agarase from Cellulophaga Algicola

International Journal of Molecular Sciences ◽

10.3390/ijms20092143 ◽

2019 ◽

Vol 20 (9) ◽

pp. 2143 ◽

Cited By ~ 5

Author(s):

Han ◽

Zhang ◽

Yang

Keyword(s):

Structural Model ◽

Biochemical Characterization ◽

Maximum Activity ◽

Optimal Temperature ◽

Salt Tolerant ◽

Wide Ph Range ◽

Specific Activities ◽

Ph Range ◽

Layer Chromatography

Cellulophaga algicola DSM 14237, isolated from the Eastern Antarctic coastal zone, was found to be able to hydrolyze several types of polysaccharide materials. In this study, a predicted β-agarase (CaAga1) from C. algicola was heterologously expressed in Escherichia coli. The purified recombinant CaAga1 showed specific activities of 29.39, 20.20, 14.12, and 8.99 U/mg toward agarose, pure agar, and crude agars from Gracilaria lemaneiformis and Porphyra haitanensis, respectively. CaAga1 exhibited an optimal temperature and pH of 40 oC and 7, respectively. CaAga1 was stable over a wide pH range from 4 to 11. The recombinant enzyme showed an unusual thermostability, that is, it was stable at temperature below or equal to 40oC and around 70 oC, but was thermolabile at about 50 oC. With the agarose as the substrate, the Km and Vmax values for CaAga1 were 1.19 mg/mL and 36.21 U/mg, respectively. The reducing reagent (dithiothreitol) enhanced the activity of CaAga1 by more than one fold. In addition, CaAga1 was salt-tolerant given that it retained approximately 70% of the maximum activity in the presence of 2 M NaCl. The thin layer chromatography results indicated that CaAga1 is an endo-type β-agarase and efficiently hydrolyzed agarose into neoagarotetraose (NA4) and neoagarohexaose (NA6). A structural model of CaAga1 in complex with neoagarooctaose (NA8) was built by homology modeling and explained the hydrolysis pattern of CaAga1.

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Characterization of Streptococcus pneumoniae TrmD, a tRNA Methyltransferase Essential for Growth

Journal of Bacteriology ◽

10.1128/jb.186.8.2346-2354.2004 ◽

2004 ◽

Vol 186 (8) ◽

pp. 2346-2354 ◽

Cited By ~ 23

Author(s):

Karen O'Dwyer ◽

Joseph M. Watts ◽

Sanjoy Biswas ◽

Jennifer Ambrad ◽

Michael Barber ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Kinetic Studies ◽

Mobility Shift ◽

Regulation Of Expression ◽

E Coli ◽

Trna Species ◽

Sequential Mechanism ◽

Trna Methyltransferase ◽

Gel Mobility Shift

ABSTRACT Down-regulation of expression of trmD, encoding the enzyme tRNA (guanosine-1)-methyltransferase, has shown that this gene is essential for growth of Streptococcus pneumoniae. The S. pneumoniae trmD gene has been isolated and expressed in Escherichia coli by using a His-tagged T7 expression vector. Recombinant protein has been purified, and its catalytic and physical properties have been characterized. The native enzyme displays a molecular mass of approximately 65,000 Da, suggesting that streptococcal TrmD is a dimer of two identical subunits. In fact, this characteristic can be extended to several other TrmD orthologs, including E. coli TrmD. Kinetic studies show that the streptococcal enzyme utilizes a sequential mechanism. Binding of tRNA by gel mobility shift assays gives a dissociation constant of 22 nM for one of its substrates, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathit{tRNA}_{\mathit{CAG}}^{\mathit{Leu}}\) \end{document} . Other heterologous nonsubstrate tRNA species, like \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathit{tRNA}_{\mathit{GGT}}^{\mathit{Thr}}\) \end{document} , tRNAPhe, and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathit{tRNA}_{\mathit{TGC}}^{\mathit{Ala}}\) \end{document} , bind the enzyme with similar affinities, suggesting that tRNA specificity is achieved via a postbinding event(s).

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