The selective addition of water

Resch, Verena; Hanefeld, Ulf

doi:10.1039/c4cy00692e

Cited by 83 publications

(86 citation statements)

References 122 publications

(137 reference statements)

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“…Binding of the (His) 6 -tagged PfFH∆40/EcFumA to Ni-NTA agarose was continued with gentle shaking for 3 h. The tube was transferred back to the chamber and the beads were washed with 50 ml of lysis buffer followed by washes with 10 mM and 20 mM imidazole containing lysis buffer (10 ml each) and the protein eluted directly with 500 mM imidazole in lysis buffer. An equal volume of 100 % glycerol was added to the eluate such that the final concentration of glycerol is 50 %.…”

Section: Generation Of Plasmid Constructs-mentioning

confidence: 99%

“…The fragment was cloned into modified pET21b (Novagen, Merck, USA) using restriction enzyme sites BamHI and SalI, to obtain the construct pET-PfFH∆40 that encodes the protein with an N-terminal (His) 6 -tag. For functional complementation in the fh null strain of E. coli, pQE30 plasmid (Qiagen, Germany) containing full length (PfFHFL) and two different N-terminus deleted constructs (PfFH∆40, PfFH∆120) of the P. falciparum fh gene were used.…”

Section: Generation Of Plasmid Constructs-mentioning

confidence: 99%

“…This shows that the PfFH can functionally complement the deficiency of fumarate hydratase activity in ∆fumACB strain and validates that the P. falciparum enzyme is indeed fumarate hydratase. The slow growth of PfFH∆120 expressing ∆fumACB E. coli strain indicates that residues 40-120 play a role in the structure and/or function of PfFH despite these residues being conserved only in Plasmodial fumarate hydratase sequences and not in others (S1 Fig). Activity of PfFH∆40-PfFH∆40 was expressed with an N-terminal (His) 6 -tag in Codon plus BL21 (DE3) RIL, purified using Ni-NTA affinity chromatography (Fig 3a) and reconstituted in vitro with Fe-S cluster. The UV-visible spectrum with absorption maxima at 360 and 405 nm indicates the presence of 4Fe-4S cluster in the enzyme and 78 % reconstitution efficiency using an Ɛ value of 16,000 M -1 cm -1 at 410 nm (30).…”

Section: Pffh Complements Fumarase Deficiency In E Coli-mentioning

confidence: 99%

“…The stereospecific reaction involves the anti-addition of a water molecule across the carbon-carbon double bond of fumarate resulting in the formation of S-malate (L-malate). The reverse reaction proceeds with the elimination of a molecule of water from malate in an antifashion (4)(5)(6). FH is found in two biochemically distinct forms; class I FH a thermolabile, oxygen sensitive, 4Fe-4S cluster containing enzyme and, class II FH, a stable, oxygen insensitive and ironindependent enzyme (7).…”

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confidence: 99%

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Biochemical characterization and essentiality of fumarate hydratase

Jayaraman¹,

Suryavanshi²,

Kalale

et al. 2018

Journal of Biological Chemistry

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Plasmodium falciparum (Pf), the causative agent of malaria, has an iron-sulfur cluster-containing class I fumarate hydratase (FH) that catalyzes the interconversion of fumarate to malate, a wellknown reaction in the tricarboxylic acid cycle. In humans, the same reaction is catalyzed by class II FH that has no sequence or structural homology with the class I enzyme from Plasmodium. Fumarate is generated in large quantities in the parasite as a byproduct of AMP synthesis and is converted to malate by FH and then used in the generation of the key metabolites oxaloacetate, aspartate, and pyruvate. Previous studies have identified the FH reaction as being essential to P. falciparum, but biochemical characterizations of PfFH that may provide leads for the development of specific inhibitors are lacking. Here, we report on the kinetic characterization of purified recombinant PfFH, functional complementation of fh deficiency in Escherichia coli, and mitochondrial localization in the parasite. We found that the substrate analog mercaptosuccinic acid is a potent PfFH inhibitor, with a K i value in the nanomolar range. The fh gene could not be knocked out in Plasmodium berghei when transfectants were introduced into BALB/c mice; however, fh knockout was successful when C57BL/6 mice were used as host, suggesting that the essentiality of the fh gene to the parasite was mouse strain dependent.Plasmodium falciparum (Pf), the causative agent of the most lethal form of malaria, during its intra-erythrocytic asexual stages, derives ATP primarily from glycolysis with low contribution from mitochondrial pathways (1, 2). The bulk of pyruvate formed is converted to lactic acid with a minor amount entering the tricarboxylic acid (TCA) cycle, the flux through which is upregulated in sexual stages (2). Key intermediates that anaplerotically feed into the TCA cycle are α-ketoglutarate derived from glutamate, oxaloacetate (OAA) from phosphoenolpyruvate, and fumarate from adenosine 5΄-monophosphate (AMP) synthesis. Synthesis of AMP in the parasite is solely from inosine-5΄-monophosphate (IMP) through a pathway involving the enzymes adenylosuccinate synthetase (ADSS) and adenylosuccinate lyase (ASL). The net reaction of ADSS and ASL involves consumption of GTP and aspartate and, generation of GDP, P i and fumarate. In the rapidly dividing parasite with an AT-rich genome and high energy requirements, leading to a high demand for adenine pools, one would expect a high flux of fumarate generation. The parasite does not secrete fumarate but instead, the carbon derived from this metabolite can be traced in malate, OAA, aspartate, pyruvate (through PEP) and lactate (3). The metabolic significance of this fumarate anaplerosis is still obscure. In this context, fumarate hydratase (FH, fumarase) the key enzyme to metabolize fumarate becomes an Fumarate hydratase (fumarase, E.C. 4.2.1.2) catalyzes the reversible conversion of fumarate to malate. The stereospecific reaction involves the anti-addition of a water molecule across the carbon-carbon...

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Section: Generation Of Plasmid Constructs-mentioning

confidence: 99%

Section: Generation Of Plasmid Constructs-mentioning

confidence: 99%

Section: Pffh Complements Fumarase Deficiency In E Coli-mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Biochemical characterization and essentiality of fumarate hydratase

Jayaraman¹,

Suryavanshi²,

Kalale

et al. 2018

Journal of Biological Chemistry

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“…[1][2][3][4][5] Da sie so den Zugang zu chiralen sekundären und tertiären Alkoholen ermçglichen, sind FAHYs vielversprechende Hilfsmittel in chemischen Industrien fürd ie Herstellung einer Reihe wichtiger Chemikalien wie etwa Aromastoffe, [6][7][8] Te nside, Schmiermittel und Ausgangsstoffe fürd ie Polymersynthese. [14][15][16][17][18][19][20][21] Unter Berücksichtigung all dieser Faktoren ermçglichen FAHYs den Zugang zu Reaktionen, die mit organisch-chemischenM ethoden nicht durchführbar sind, wodurch sich klare Vorteile fürd en Einsatz dieser Enzymgruppe in der organischen Synthese ergeben. [14][15][16][17][18][19][20][21] Unter Berücksichtigung all dieser Faktoren ermçglichen FAHYs den Zugang zu Reaktionen, die mit organisch-chemischenM ethoden nicht durchführbar sind, wodurch sich klare Vorteile fürd en Einsatz dieser Enzymgruppe in der organischen Synthese ergeben.…”

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Weiterentwicklung der Substrattoleranz von Elizabethkingia meningoseptica Oleathydratase zur regio‐ und stereoselektiven Hydratisierung von Ölsäurederivaten

et al. 2019

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Die Addition von Wasser an nichtaktivierte Kohlenstoff-Kohlenstoff-Doppelbindungen von Fettsäuren wird durch Fettsäurehydratasen (FAHYs) katalysiert, und ermçglichtd ie hochselektive Oxyfunktionalisierung von erneuerbaren, çlhaltigen Rohstoffen. Da fürdie Erkennung und Bindung von Substraten die freie Carboxylatgruppe bislang als unerlässlich galt, ist der Einsatz von FAHYs gegenwärtig auf die Hydratisierung freier Fettsäuren limitiert. Diese Arbeit zeigt erstmals die Hydratisierung von Ölsäurederivaten ohne freier Carboxylatfunktion am Beispiel der Elizabethkingia meningoseptica Oleathydratase (OhyA). Im Zuge bioinformati-scherB indestudien von Substraten an die 3D-Struktur der OhyA sowie durch Aminosäuresequenzvergleiche konnten konservierte Aminosäurereste in der Substratbindetasche des Enzyms identifiziert werden. Der rationale Austausch dieser Reste führte zu Enzymvarianten mit bis zu 18-fachv erbessertem Umsatz bestimmter Ölsäurederivate ohne Beeinträchtigung der exzellenten Regio-und Stereoselektivitätd er Olefinhydratisierung.

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Biocatalysis

Torrelo

Hanefeld

Hollmann

2015

Kirk-Othmer Encyclopedia of Chemical Technology

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Biocatalysis, the use of enzymes—either as isolated enzymes or in whole cells—to accelerate chemical transformations, is one of the approaches available to the organic chemist. Key advantages of biocatalysis are its high selectivity and the high rate of chemical transformations at relatively mild reaction conditions, and as a result, the simplified downstream processing and more cost‐effective synthesis. Therefore, it is not surprising that interest in industrial biocatalysis has been on the rise. The aim of this chapter is to provide the process chemist with a general overview of the available enzymes and the chemistries they enable and to discuss concepts in biocatalysis highlighted by selected examples.

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The selective addition of water

Abstract: Water is omnipresent and unreactive. How to speed up water addition and even make it selective are highlighted in this perspective.

Cited by 83 publications

References 122 publications

Biochemical characterization and essentiality of fumarate hydratase

Biochemical characterization and essentiality of fumarate hydratase

Weiterentwicklung der Substrattoleranz von Elizabethkingia meningoseptica Oleathydratase zur regio‐ und stereoselektiven Hydratisierung von Ölsäurederivaten

Biocatalysis

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