Evidence that the active site in type II dehydroquinase from <i>Streptomyces coelicolor</i> is near the single tryptophan

Boam, Deborah J.; Price, Nicholas C.; Kelly, Sharon M.; Krel, Tino; Coggins, John R.

doi:10.1042/bst025093s

Cited by 2 publications

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“…Moreover, the relatively low value of the Stern-Volmer constant (K sv ) for quenching by succinimide (0.74 M −" ) is consistent with an internal location for the side chain of the introduced tryptophan (Figure 8). To place these results in context, the K sv (for succinimide quenching) of various proteins containing a single tryptophan residue range from 0.26 M −" for type I dehydroquinase from Salmonella typhi (buried tryptophan [29]), to 4.8 and 3.2 M −" for shikimate kinase of Erwinia chrystanthemi and type II dehydroquinase from Streptomyces coelicolor respectively (exposed tryptophans [30,31]). Unfolding of the F67W protein in 6 M guanidinium chloride led to a red shift in the emission peak to 351 nm and resulted in a K sv for succinimide quenching of 2.6 M −" (Figure 8), which is of a similar order to that observed for other single-tryptophan-containing proteins when unfolded [30][31][32].…”

Section: Site-directed Mutagenesismentioning

confidence: 99%

“…To place these results in context, the K sv (for succinimide quenching) of various proteins containing a single tryptophan residue range from 0.26 M −" for type I dehydroquinase from Salmonella typhi (buried tryptophan [29]), to 4.8 and 3.2 M −" for shikimate kinase of Erwinia chrystanthemi and type II dehydroquinase from Streptomyces coelicolor respectively (exposed tryptophans [30,31]). Unfolding of the F67W protein in 6 M guanidinium chloride led to a red shift in the emission peak to 351 nm and resulted in a K sv for succinimide quenching of 2.6 M −" (Figure 8), which is of a similar order to that observed for other single-tryptophan-containing proteins when unfolded [30][31][32]. In some lipid binding proteins the fluorescence emission spectrum of tryptophan residues within or near the binding site can be altered by ligand binding [33,34], but in F67W no such changes were detectable on the addition of oleic or linoleic acid (results not shown).…”

Section: Site-directed Mutagenesismentioning

confidence: 99%

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A surface-associated retinol- and fatty acid-binding protein (Gp-FAR-1) from the potato cyst nematode Globodera pallida: lipid binding activities, structural analysis and expression pattern

Prior¹,

Jones²,

Blok³

et al. 2001

Biochem. J.

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Parasitic nematodes produce at least two structurally novel classes of small helix-rich retinol- and fatty-acid-binding proteins that have no counterparts in their plant or animal hosts and thus represent potential targets for new nematicides. Here we describe a protein (Gp-FAR-1) from the plant-parasitic nematode Globodera pallida, which is a member of the nematode-specific fatty-acid- and retinol-binding (FAR) family of proteins but localizes to the surface of this species, placing it in a strategic position for interaction with the host. Recombinant Gp-FAR-1 was found to bind retinol, cis-parinaric acid and the fluorophore-tagged lipids 11-(dansylamino)undecanoic acid and dansyl-D,L-alpha-amino-octanoic acid. The fluorescence emission characteristics of the dansylated analogues indicated that the entire ligand enters the binding cavity. Fluorescence competition experiments showed that Gp-FAR-1 binds fatty acids in the range C(11) to C(24), with optimal binding at C(15). Intrinsic fluorescence analysis of a mutant protein into which a tryptophan residue had been inserted supported computer-based predictions of the position of this residue at the protein's interior and possibly also at the binding site. Of direct relevance to plant defence systems was the observation that Gp-FAR-1 binds two lipids (linolenic and linoleic acids) that are precursors of plant defence compounds and the jasmonic acid signalling pathway. Moreover, Gp-FAR-1 was found to inhibit the lipoxygenase-mediated modification of these substrates in vitro. Thus not only does Gp-FAR-1 function as a broad-spectrum retinol- and fatty-acid-binding protein, the results are consistent with the idea that Gp-FAR-1 is involved in the evasion of primary host plant defence systems.

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Section: Site-directed Mutagenesismentioning

confidence: 99%

Section: Site-directed Mutagenesismentioning

confidence: 99%

A surface-associated retinol- and fatty acid-binding protein (Gp-FAR-1) from the potato cyst nematode Globodera pallida: lipid binding activities, structural analysis and expression pattern

Prior¹,

Jones²,

Blok³

et al. 2001

Biochem. J.

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The Shikimate Pathway

Herrmann

Weaver

1999

Annu. Rev. Plant. Physiol. Plant. Mol. Biol.

1,055

778

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The shikimate pathway links metabolism of carbohydrates to biosynthesis of aromatic compounds. In a sequence of seven metabolic steps, phosphoenolpyruvate and erythrose 4-phosphate are converted to chorismate, the precursor of the aromatic amino acids and many aromatic secondary metabolites. All pathway intermediates can also be considered branch point compounds that may serve as substrates for other metabolic pathways. The shikimate pathway is found only in microorganisms and plants, never in animals. All enzymes of this pathway have been obtained in pure form from prokaryotic and eukaryotic sources and their respective DNAs have been characterized from several organisms. The cDNAs of higher plants encode proteins with amino terminal signal sequences for plastid import, suggesting that plastids are the exclusive locale for chorismate biosynthesis. In microorganisms, the shikimate pathway is regulated by feedback inhibition and by repression of the first enzyme. In higher plants, no physiological feedback inhibitor has been identified, suggesting that pathway regulation may occur exclusively at the genetic level. This difference between microorganisms and plants is reflected in the unusually large variation in the primary structures of the respective first enzymes. Several of the pathway enzymes occur in isoenzymic forms whose expression varies with changing environmental conditions and, within the plant, from organ to organ. The penultimate enzyme of the pathway is the sole target for the herbicide glyphosate. Glyphosate-tolerant transgenic plants are at the core of novel weed control systems for several crop plants.

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Evidence that the active site in type II dehydroquinase from Streptomyces coelicolor is near the single tryptophan

Cited by 2 publications

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A surface-associated retinol- and fatty acid-binding protein (Gp-FAR-1) from the potato cyst nematode Globodera pallida: lipid binding activities, structural analysis and expression pattern

A surface-associated retinol- and fatty acid-binding protein (Gp-FAR-1) from the potato cyst nematode Globodera pallida: lipid binding activities, structural analysis and expression pattern

The Shikimate Pathway

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