The characterisation of the shikimate pathway enzyme dehydroquinase from<i>Pisum sativum</i>

Deka, Ranjit K.; Anton, Ian A.; Dunbar, Bryan; Coggins, John R.

doi:10.1016/0014-5793(94)00710-1

Cited by 32 publications

(20 citation statements)

References 41 publications

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“…Salmonella typhi [8,9], Enterococcus faecalis [10] and Bacillus subtilis [11] all possess monofunctional DHQases. In plants such as Physcomitrella patens [12], Nicotiana tabacum [13] and Pisum sati um [14], DHQase is part of a bifunctional polypeptide that also carries shikimate dehydrogenase activity, the next enzyme in the pathway. The DHQase in the shikimate pathway of fungi and yeast occurs as part of a pentafunctional polypeptide chain carrying the activities of the five consecutive steps leading to the production of 5-enoylpyruvylshikimate-3-phosphate [15,16].…”

Section: Introductionmentioning

confidence: 99%

Cloning, sequencing, expression, purification and preliminary characterization of a type II dehydroquinase from Helicobacter pylori

Bottomley

Clayton²,

Chalk³

et al. 1996

Biochemical Journal

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A heat-stable dehydroquinase was purified to near homogeneity from a plate-grown suspension of the Gram-negative stomach pathogen Helicobacter pylori, and shown from both its subunit and native molecular masses to be a member of the type II family of dehydroquinases. This was confirmed by N-terminal amino acid sequence data. The gene encoding this activity was isolated following initial identification, by random sequencing of the H. pylori genome, of a 96 bp fragment, the translated sequence of which showed strong identity to a C-terminal region of other type II enzymes. Southern blot analysis of a cosmid library identified several potential clones, one of which complemented an Escherichia coliaroD point mutant strain deficient in host dehydroquinase. The gene encoding the H. pylori type II dehydroquinase (designated aroQ) was sequenced. The translated sequence was identical to the N-terminal sequence obtained directly from the purified protein, and showed strong identity to other members of the type II family of dehydroquinases. The enzyme was readily expressed in E. coli from a plasmid construct from which several milligrams of protein could be isolated, and the molecular mass of the protein was confirmed by electrospray MS. The aroQ gene in H. pylori may function in the central biosynthetic shikimate pathway of this bacterium, thus opening the way for the construction of attenuated strains as potential vaccines as well as offering a new target for selective enzyme inhibition.

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Section: Introductionmentioning

confidence: 99%

Cloning, sequencing, expression, purification and preliminary characterization of a type II dehydroquinase from Helicobacter pylori

Bottomley

Clayton²,

Chalk³

et al. 1996

Biochemical Journal

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“…In higher organisms this activity is part of a multifunctional enzyme. In plants shikimate dehydrogenase is associated with type I dehydroquinase to form a bifunctional enzyme (14), whereas in fungi, such as Neurospora crassa, this enzyme forms the fifth domain of the pentafunctional AROM polypeptide, which catalyzes five of seven steps of the shikimate pathway (15). However, the molecular basis of 3-dehydroshikimate recognition and enzymatic reduction is not known.…”

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confidence: 99%

“…Most of the residues absolutely conserved in the SDH family are located in this pocket, i.e. Ser 14 (67), a serine or a threonine is also always observed in the SDH family (Fig. 3).…”

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Structures of Shikimate Dehydrogenase AroE and Its Paralog YdiB

Michel

Roszak

Sauvé

et al. 2003

Journal of Biological Chemistry

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106

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Shikimate dehydrogenase catalyzes the fourth step of the shikimate pathway, the essential route for the biosynthesis of aromatic compounds in plants and microorganisms. Absent in metazoans, this pathway is an attractive target for nontoxic herbicides and drugs. Escherichia coli expresses two shikimate dehydrogenase paralogs, the NADP-specific AroE and a putative enzyme YdiB. Here we characterize YdiB as a dual specificity quinate/shikimate dehydrogenase that utilizes either NAD or NADP as a cofactor. Structures of AroE and YdiB with bound cofactors were determined at 1.5 and 2.5 Å resolution, respectively. Both enzymes display a similar architecture with two ␣/␤ domains separated by a wide cleft. Comparison of their dinucleotide-binding domains reveals the molecular basis for cofactor specificity. Independent molecules display conformational flexibility suggesting that a switch between open and closed conformations occurs upon substrate binding. Sequence analysis and structural comparison led us to propose the catalytic machinery and a model for 3-dehydroshikimate recognition. Furthermore, we discuss the evolutionary and metabolic implications of the presence of two shikimate dehydrogenases in E. coli and other organisms.The shikimate pathway, which links metabolism of carbohydrates to biosynthesis of aromatic compounds, is essential to plants, bacteria, and fungi (1) as well as apicomplexan parasites (2). This seven-step metabolic route leads from phosphoenolpyruvate and erythrose 4-phosphate to chorismate, the common precursor for the synthesis of folic acid, ubiquinone, vitamins E and K, and aromatic amino acids (1). This pathway is absent in metazoans, which must obtain the essential amino acids phenylalanine and tryptophan from their diet. Therefore, enzymes of this pathway are important targets for the development of nontoxic herbicides (3), as well as antimicrobial (4) and antiparasite (2) agents. The sixth step in the pathway, catalyzed by 5-enolpyruvylshikimate-3-phosphate synthase, has already been successfully targeted, with the development of glyphosate, a broad spectrum herbicide (5). However, after 20 years of extensive use, glyphosate-resistant weeds have recently emerged (6), emphasizing the importance of maintaining target diversity. In order to design new inhibitors, crystal structures of several enzymes of the shikimate pathway have been elucidated recently: 3-dehydroquinate synthase (7), type I and II dehydroquinases (8), type I and II shikimate kinases (9, 10), and 5-enolpyruvylshikimate-3-phosphate synthase (11), catalyzing the second, third, fifth, and sixth steps of the pathway, respectively.Shikimate dehydrogenase (EC 1.1.1.25) catalyzes the fourth reaction in the shikimate pathway, the NADP-dependent reduction of 3-dehydroshikimate to shikimate (Fig. 1A). Whereas dehydrogenases usually form oligomers, shikimate dehydrogenase, coded by the gene aroE in Escherichia coli, is present as a monomer in most bacteria (12, 13). In higher organisms this activity is part of a multifunctional enzym...

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“…Besides being mechanistically distinct, the two classes of DHQase have very different biophysical properties and are apparently unrelated at the level of primary structure (10,11). The type I enzymes are dimers with a molecular mass of about 46,000 Da; they are heat-labile and use a mechanism that involves the formation of a Schiff-base intermediate followed by the abstraction of a proton by a general base (12)(13)(14)(15). In the case of the type I Escherichia coli enzyme the lysine residue has been located (Lys-170) by trapping the Schiff-base intermediate by borohydride reduction (12,13), and this residue is conserved in all type I sequences (12) as is His-143 (15,16) which is the general base.…”

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Localization of the Active Site of Type II Dehydroquinases

Krell

Horsburgh

Cooper

et al. 1996

Journal of Biological Chemistry

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A novel method based on electrospray mass spectrometry (Krell, T., Pitt, A. R., and Coggins, J. R. (1995) FEBS Lett. 360, 93-96) has been used to localize active site residues in the type I and type II dehydroquinases. Both enzymes have essential hyper-reactive arginine residues, and the type II enzymes have an essential tyrosine residue. The essential hyper-reactive Arg-23 of the Streptomyces coelicolor type II enzyme has been replaced by lysine, glutamine, and alanine residues. The mutant enzymes were purified and shown by CD spectroscopy to be structurally similar to the wild-type enzyme. All three mutant enzymes were much less active, for example the k cat of the R23A mutant was 30,000-fold reduced. The mutants all had reduced K m values, indicating stronger substrate binding, which was confirmed by isothermal titration calorimetry experiments. A role for Arg-23 in the stabilization of a carbanion intermediate is proposed. Comparison of the amino acid sequence around the hyper-reactive arginine residues of the two classes of enzymes indicates that there is a conserved structural motif that might reflect a common substrate binding fold at the active center of these two classes of enzyme.It is generally believed that enzymes have evolved to catalyze reactions by optimal mechanisms. With the exception of the four classes of proteinases (1) and the two classes of aldolases (2), examples of mechanistically different pairs of enzymes that catalyze the same reaction are very rare. This unusual situation is found with the two classes of dehydroquinase that catalyze the dehydration of 3-dehydroquinate to form 3-dehydroshikimate (3). The reaction is common to two metabolic pathways: the biosynthetic shikimate pathway that is used for the synthesis of aromatic compounds in plants and micro-organisms (4) and the catabolic quinate pathway that enables fungi and some other micro-organism to use quinate as a carbon and energy source (5). The type I dehydroquinases (DHQase) 1 catalyze a cis elimination and are only involved in the biosynthesis of shikimate (6), whereas the type II enzymes, which catalyze a trans elimination (1), have been found to have either a biosynthetic (7) or a catabolic role (8) and in at least one species a dual role (9). Besides being mechanistically distinct, the two classes of DHQase have very different biophysical properties and are apparently unrelated at the level of primary structure (10, 11). The type I enzymes are dimers with a molecular mass of about 46,000 Da; they are heat-labile and use a mechanism that involves the formation of a Schiff-base intermediate followed by the abstraction of a proton by a general base (12-15). In the case of the type I Escherichia coli enzyme the lysine residue has been located (Lys-170) by trapping the Schiff-base intermediate by borohydride reduction (12, 13), and this residue is conserved in all type I sequences (12) as is 16) which is the general base. Both have also been identified as active site residues although their role in substrate binding or catal...

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The characterisation of the shikimate pathway enzyme dehydroquinase fromPisum sativum

Cited by 32 publications

References 41 publications

Cloning, sequencing, expression, purification and preliminary characterization of a type II dehydroquinase from Helicobacter pylori

Cloning, sequencing, expression, purification and preliminary characterization of a type II dehydroquinase from Helicobacter pylori

Structures of Shikimate Dehydrogenase AroE and Its Paralog YdiB

Localization of the Active Site of Type II Dehydroquinases

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