Preliminary crystallographic studies of an extremely thermostable KDG aldolase from<i>Sulfolobus solfataricus</i>

Hendry, E; Buchanan, Catriona L.; Russell, Rosemary; Hough, David W.; Reeve, Christopher D.; Danson, Michael J.; Taylor, G.L.

doi:10.1107/s0907444900009859

Cited by 3 publications

(3 citation statements)

References 14 publications

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“…Crystallization of Recombinant KDGA-Several crystal forms of KDGA were previously obtained, the best diffraction data being to 2.15 Å from an orthorhombic crystal form (a ϭ 135.1 Å, b ϭ 135.9 Å, c ϭ 188.7 Å) grown at pH 4.0 (12). Solvent content estimations indicated the presence of two tetramers in the asymmetric unit related by a pseudocentering.…”

Section: Methodsmentioning

confidence: 99%

“…Once the optical density of the cells had reached 0.5 at 600 nm, they were induced with 1 mM isopropyl-␤-D-thiogalactopyranoside and grown for a further 24 h at 250 rpm, 37°C. The purification procedure for the Se-Met derivative was as for the native protein (12) except that all buffers contained 5 mM dithiothreitol. Screening for crystallization conditions had to be repeated as no crystals were obtained under the conditions described for the native protein (8).…”

Section: Methodsmentioning

confidence: 99%

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The Structural Basis for Substrate Promiscuity in 2-Keto-3-deoxygluconate Aldolase from the Entner-Doudoroff Pathway in Sulfolobus solfataricus

Theodossis

Walden

Westwick

et al. 2004

Journal of Biological Chemistry

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The hyperthermophilic Archaea Sulfolobus solfataricus grows optimally above 80°C and metabolizes glucose by a non-phosphorylative variant of the EntnerDoudoroff pathway. In this pathway glucose dehydrogenase and gluconate dehydratase catalyze the oxidation of glucose to gluconate and the subsequent dehydration of gluconate to D-2-keto-3-deoxygluconate (KDG). KDG aldolase (KDGA) then catalyzes the cleavage of KDG to D-glyceraldehyde and pyruvate. It has recently been shown that all the enzymes of this pathway exhibit a catalytic promiscuity that also enables them to be used for the metabolism of galactose. This phenomenon, known as metabolic pathway promiscuity, depends crucially on the ability of KDGA to cleave KDG and D-2-keto-3-deoxygalactonate (KDGal), in both cases producing pyruvate and D-glyceraldehyde. In turn, the aldolase exhibits a remarkable lack of stereoselectivity in the condensation reaction of pyruvate and D-glyceraldehyde, forming a mixture of KDG and KDGal. We now report the structure of KDGA, determined by multiwavelength anomalous diffraction phasing, and confirm that it is a member of the tetrameric N-acetylneuraminate lyase superfamily of Schiff base-forming aldolases. Furthermore, by soaking crystals of the aldolase at more than 80°C below its temperature activity optimum, we have been able to trap Schiff base complexes of the natural substrates pyruvate, KDG, KDGal, and pyruvate plus D-glyceraldehyde, which have allowed rationalization of the structural basis of promiscuous substrate recognition and catalysis. It is proposed that the active site of the enzyme is rigid to keep its thermostability but incorporates extra functionality to be promiscuous.Sulfolobus solfataricus is a hyperthermophilic Archaea that grows optimally at 80 -85°C and pH 2-4, utilizing a wide range of carbon and energy sources (1). Central metabolism in this organism involves a non-phosphorylative variant of the Entner-Doudoroff pathway (2), and similar metabolic routes have been detected in Sulfolobus acidocaldarius (3), Thermoplasma acidophilum (4), Thermoproteus tenax (5), and Aspergillus fungi (6).In S. solfataricus, the enzymes of this non-phosphorylative Entner-Doudoroff pathway have been shown to possess a substrate promiscuity that enables them to catalyze the metabolism of both glucose and galactose (7). The first enzyme in the pathway, glucose dehydrogenase, catalyzes the oxidation of glucose to gluconate and galactose to galactonate. Subsequently, gluconate dehydratase is known to catalyze the dehydration of gluconate to D-2-keto-3-deoxygluconate (KDG), 1 and galactonate to D-2-keto-3-deoxygalactonate (KDGal).2 The third enzyme, KDG aldolase (KDGA), catalyzes the aldolate cleavage of KDG and KDGal, with both substrates yielding pyruvate and D-glyceraldehyde. Moreover, in the aldol condensation reaction, KDGA exhibits no facial selectivity, condensing pyruvate with D-glyceraldehyde to produce a mixture of KDG and KDGal (7). KDGA is thus an extremely unusual aldolase in not exhibiting stereoselectivity with ...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The Structural Basis for Substrate Promiscuity in 2-Keto-3-deoxygluconate Aldolase from the Entner-Doudoroff Pathway in Sulfolobus solfataricus

Theodossis

Walden

Westwick

et al. 2004

Journal of Biological Chemistry

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“…acidocaldarius, and Tpt. tenax have been reported (295)(296)(297)(298). They belong to the protein family of Schiff base-forming class I aldolases, in which the archaeal KD(P)G aldolases appear to belong to a distinct subgroup of enzymes (SCOP database).…”

Section: -Keto-3-deoxy-(6-phospho)gluconate Aldolase [Kd(p)ga]mentioning

confidence: 99%

Carbohydrate Metabolism in Archaea: Current Insights into Unusual Enzymes and Pathways and Their Regulation

Bräsen

Esser

Rauch

et al. 2014

Microbiol Mol Biol Rev

280

294

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SUMMARY The metabolism of Archaea , the third domain of life, resembles in its complexity those of Bacteria and lower Eukarya . However, this metabolic complexity in Archaea is accompanied by the absence of many “classical” pathways, particularly in central carbohydrate metabolism. Instead, Archaea are characterized by the presence of unique, modified variants of classical pathways such as the Embden-Meyerhof-Parnas (EMP) pathway and the Entner-Doudoroff (ED) pathway. The pentose phosphate pathway is only partly present (if at all), and pentose degradation also significantly differs from that known for bacterial model organisms. These modifications are accompanied by the invention of “new,” unusual enzymes which cause fundamental consequences for the underlying regulatory principles, and classical allosteric regulation sites well established in Bacteria and Eukarya are lost. The aim of this review is to present the current understanding of central carbohydrate metabolic pathways and their regulation in Archaea . In order to give an overview of their complexity, pathway modifications are discussed with respect to unusual archaeal biocatalysts, their structural and mechanistic characteristics, and their regulatory properties in comparison to their classic counterparts from Bacteria and Eukarya . Furthermore, an overview focusing on hexose metabolic, i.e., glycolytic as well as gluconeogenic, pathways identified in archaeal model organisms is given. Their energy gain is discussed, and new insights into different levels of regulation that have been observed so far, including the transcript and protein levels (e.g., gene regulation, known transcription regulators, and posttranslational modification via reversible protein phosphorylation), are presented.

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Use of a novel microtitration protocol to obtain diffraction-quality crystals of 4-hydroxy-2-oxoglutarate aldolase fromBos taurus

2014

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The enzyme 4-hydroxy-2-oxoglutarate aldolase (HOGA) catalyses the retroaldol degradation of 4-hydroxy-2-oxoglutarate to pyruvate and glyoxylate as part of the hydroxyproline catabolic pathway in mammals. Mutations in the coding region of the human HOGA gene are associated with primary hyperoxaluria type 3, a disease characterized by excessive oxalate production and ultimately stone deposition. Native HOGA was purified from bovine kidney using an improved and streamlined purification protocol from which two crystal forms were obtained using two different approaches. Vapour diffusion using PEG 3350 as a precipitant produced monoclinic crystals that belonged to space group C2 and diffracted to 3.5 Å resolution. By comparison, orthorhombic crystals belonging to space group I222 or I2 1 2 1 2 1 and diffracting to beyond 2.25 Å resolution were obtained using a novel microtitration protocol with ammonium sulfate. The latter crystal form displayed superior diffraction quality and was suitable for structural determination by X-ray crystallography.

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Preliminary crystallographic studies of an extremely thermostable KDG aldolase fromSulfolobus solfataricus

Cited by 3 publications

References 14 publications

The Structural Basis for Substrate Promiscuity in 2-Keto-3-deoxygluconate Aldolase from the Entner-Doudoroff Pathway in Sulfolobus solfataricus

The Structural Basis for Substrate Promiscuity in 2-Keto-3-deoxygluconate Aldolase from the Entner-Doudoroff Pathway in Sulfolobus solfataricus

Carbohydrate Metabolism in Archaea: Current Insights into Unusual Enzymes and Pathways and Their Regulation

Use of a novel microtitration protocol to obtain diffraction-quality crystals of 4-hydroxy-2-oxoglutarate aldolase fromBos taurus

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