scholarly journals Efficient independent activity of a monomeric, monofunctional dehydroquinate synthase derived from the N-terminus of the pentafunctional AROM protein of Aspergillus nidulans

1994 ◽  
Vol 301 (1) ◽  
pp. 297-304 ◽  
Author(s):  
J D Moore ◽  
J R Coggins ◽  
R Virden ◽  
A R Hawkins
Keyword(s):  
Escherichia Coli ◽  
Catalytic Activity ◽  
Aspergillus Nidulans ◽  
Chelating Agents ◽  
Size Exclusion ◽  
Functional Domain ◽  
N Terminus ◽  
Exclusion Chromatography ◽  
Km Value ◽  
Dehydroquinate Synthase

The dehydroquinate synthase (DHQ synthase) functional domain from the pentafunctional AROM protein of Aspergillus nidulans has previously been overproduced in Escherichia coli [van den Hombergh, Moore, Charles and Hawkins (1992) Biochem J. 284, 861-867]. We now report the purification of this domain to homogeneity and subsequent characterization. The monofunctional DHQ synthase was found to retain efficient catalytic activity when compared with the intact pentafunctional AROM protein of Neurospora crassa [Lambert, Boocock and Coggins (1985) Biochem J. 226, 817-829]. The apparent kcat. was estimated to be 8 s-1, and the apparent Km values for NAD+ and 3-deoxy-D-arabino-heptulosonate phosphate (DAHP) were 3 microM and 2.2 microM respectively. These values are similar to those reported for the intact N. crassa enzyme, except that the apparent Km for NAD+ reported here is 15-fold higher. The monofunctional DHQ synthase domain is inactivated by treatment with chelating agents in the absence of substrates and is re-activated by the addition of metal ions; among those tested, Zn2+ gave the highest kcat./Km value. The enzyme is inactivated by diethyl pyrocarbonate; both the substrate, DAHP, and the product phosphate protected against inactivation. Size-exclusion chromatography suggested an M(r) of 43,000 for the monofunctional domain, indicating that it is monomeric and compactly folded. The c.d. spectrum confirmed that the domain has a compact globular conformation; the near-u.v. c.d. of zinc- and cobalt-reactivated domains were superimposable.

scholarly journals Effect of Posttranslational Modifications on the Structure and Activity of FTO Demethylase

2021 ◽  
Vol 22 (9) ◽  
pp. 4512
Author(s):  
Michał Marcinkowski ◽  
Tomaš Pilžys ◽  
Damian Garbicz ◽  
Jan Piwowarski ◽  
Damian Mielecki ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Size Exclusion Chromatography ◽  
Posttranslational Modifications ◽  
Size Exclusion ◽  
Saxs Data ◽  
Wide Range ◽  
Exclusion Chromatography ◽  
Demethylase Activity ◽  
Structure And Activity

The FTO protein is involved in a wide range of physiological processes, including adipogenesis and osteogenesis. This two-domain protein belongs to the AlkB family of 2-oxoglutarate (2-OG)- and Fe(II)-dependent dioxygenases, displaying N6-methyladenosine (N6-meA) demethylase activity. The aim of the study was to characterize the relationships between the structure and activity of FTO. The effect of cofactors (Fe2+/Mn2+ and 2-OG), Ca2+ that do not bind at the catalytic site, and protein concentration on FTO properties expressed in either E. coli (ECFTO) or baculovirus (BESFTO) system were determined using biophysical methods (DSF, MST, SAXS) and biochemical techniques (size-exclusion chromatography, enzymatic assay). We found that BESFTO carries three phosphoserines (S184, S256, S260), while there were no such modifications in ECFTO. The S256D mutation mimicking the S256 phosphorylation moderately decreased FTO catalytic activity. In the presence of Ca2+, a slight stabilization of the FTO structure was observed, accompanied by a decrease in catalytic activity. Size exclusion chromatography and MST data confirmed the ability of FTO from both expression systems to form homodimers. The MST-determined dissociation constant of the FTO homodimer was consistent with their in vivo formation in human cells. Finally, a low-resolution structure of the FTO homodimer was built based on SAXS data.

scholarly journals Effect of mutations in the transmethylase and dehydrogenase/chelatase domains of sirohaem synthase (CysG) on sirohaem and cobalamin biosynthesis

1998 ◽  
Vol 330 (1) ◽  
pp. 121-129 ◽  
Author(s):  
C. Sarah WOODCOCK ◽  
Evelyne RAUX ◽  
Florence LEVILLAYER ◽  
Claude THERMES ◽  
Alain RAMBACH ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Catalytic Activity ◽  
Catalytic Domain ◽  
Conserved Residues ◽  
Pseudomonas Denitrificans ◽  
E Coli ◽  
Cobalamin Biosynthesis ◽  
N Terminus ◽  
Low Levels ◽  
Cobyric Acid

The Escherichia coli CysG protein (sirohaem synthase) catalyses four separate reactions that are required for the transformation of uroporphyrinogen III into sirohaem, initially two S-adenosyl-L-methionine-dependent transmethylations at positions 2 and 7, mediated through the C-terminal, or CysGA, catalytic domain of the protein, and subsequently a ferrochelation and dehydrogenation, mediated through the N-terminal, or CysGB, catalytic domain of the enzyme. This report describes how the deletion of the NAD+-binding site of CysG, located within the first 35 residues of the N-terminus, is detrimental to the activity of CysGB but does not affect the catalytic activity of CysGA, whereas the mutation of a number of phylogenetically conserved residues within CysGA is detrimental to the transmethylation reaction but does not affect the activity of CysGB. Further studies have shown that CysGB is not essential for cobalamin biosynthesis because the presence of the Salmonella typhimurium CobI operon with either cysGA or the Pseudomonas denitrificans cobA are sufficient for the synthesis of cobyric acid in an E. coli cysG deletion strain. Evidence is also presented to suggest that a gene within the S. typhimurium CobI operon might act as a chelatase that, at low levels of cobalt, is able to aid in the synthesis of sirohaem.

Overproduction in Escherichia coli of the dehydroquinate synthase domain of the Aspergillus nidulans pentafunctional AROM protein

1992 ◽  
Vol 284 (3) ◽  
pp. 861-867 ◽  
Author(s):  
J P T W van den Hombergh ◽  
J D Moore ◽  
I G Charles ◽  
A R Hawkins
Keyword(s):  
Escherichia Coli ◽  
Aspergillus Nidulans ◽  
Dna Sequences ◽  
Specific Activity ◽  
Stop Codon ◽  
Cell Protein ◽  
Shikimate Dehydrogenase ◽  
E Coli ◽  
Prokaryotic Expression Vector ◽  
Dehydroquinate Synthase

The pentafunctional AROM protein of Aspergillus nidulans is encoded by the complex aromA locus and catalyses steps 2-6 in the synthesis of chorismate, the common precursor for the aromatic amino acids and p-aminobenzoic acid. DNA sequences encoding the 3-dehydroquinate synthase (DHQ synthase) and 3-dehydroquinase domains of the AROM protein have been amplified with the inclusion of a translational stop codon at the C-terminus by PCR technology. These amplified fragments of DNA have been subcloned into the prokaryotic expression vector pKK233-2 and expressed in Escherichia coli. As a result, the DHQ synthase domain is overproduced in E. coli, forming 30% of total cell protein, and can be purified to greater than 80% homogeneity by a simple two-step protocol. The 3-dehydroquinase domain is produced at a specific activity 8-fold greater than the corresponding activity encoded by the aromA gene in A. nidulans. The qutB gene of A. nidulans encoding quinate dehydrogenase has similarly been subjected to PCR amplification and expression in E. coli. The quinate dehydrogenase is not overproduced, but is active in E. coli as a shikimate dehydrogenase, as the presence of the qutB gene allows the growth of an E. coli mutant strain lacking shikimate dehydrogenase on minimal medium lacking aromatic-amino-acid supplementation.

The N-terminal 1–55 residues domain of pyruvate dehydrogenase from Escherichia coli assembles as a dimer in solution

2019 ◽  
Vol 32 (6) ◽  
pp. 271-276
Author(s):  
Yuanyuan Wang ◽  
Zemao Gong ◽  
Han Fang ◽  
Dongming Zhi ◽  
Hu Tao
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Critical Role ◽  
Size Exclusion ◽  
Titration Calorimetry ◽  
Stable Complex ◽  
Binding Assays ◽  
Exclusion Chromatography ◽  
Multiple Copies

Abstract The pyruvate dehydrogenase complex (PDHc) from Escherichia coli is a large protein complex consisting of multiple copies of the pyruvate dehydrogenase (E1ec), dihydrolipoamide acetyltransferase (E2ec) and dihydrolipoamide dehydrogenase (E3ec). The N-terminal domain (NTD, residues 1–55) of E1ec plays a critical role in the interaction between E1ec and E2ec and the whole PDHc activity. Using circular dichroism, size-exclusion chromatography and dynamic light scattering spectroscopy, we show that the NTD of E1ec presents dimeric assembly under physiological condition. Pull-down and isothermal titration calorimetry binding assays revealed that the E2ec peripheral subunit-binding domain (PSBD) forms a very stable complex with the NTD, indicating the isolated NTD functionally interacts with PSBD and the truncated E1ec (E1ec∆NTD) does not interact with PSBD. These findings are important to understand the mechanism of PDHc and other thiamine-based multi-component enzymes.

scholarly journals Cyclodextrin-grafted polymers functionalized with phosphanes: a new tool for aqueous organometallic catalysis

2014 ◽  
Vol 10 ◽  
pp. 2642-2648 ◽  
Author(s):  
Jonathan Potier ◽  
Stéphane Menuel ◽  
David Mathiron ◽  
Véronique Bonnet ◽  
Frédéric Hapiot ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Nmr Spectroscopy ◽  
Size Exclusion Chromatography ◽  
Water Soluble ◽  
Size Exclusion ◽  
Grafted Polymers ◽  
Organometallic Catalysis ◽  
Exclusion Chromatography

New cyclodextrin (CD)-grafted polymers functionalized with water-soluble phosphanes were synthesized in three steps starting from polyNAS. Once characterized by NMR spectroscopy and size-exclusion chromatography, they were used as additives in Rh-catalyzed hydroformylation of 1-hexadecene. The combined supramolecular and coordinating properties of these polymers allowed increasing the catalytic activity of the reaction without affecting the selectivities.

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