Rhombohedral crystals of 2-dehydro-3-deoxygalactarate aldolase from <i>Escherichia coli</i>

Blackwell, Nicholas; Cullis, Paul M.; Cooper, Ronald A.; Izard, Tina

doi:10.1107/s0907444999006502

Cited by 5 publications

(8 citation statements)

References 7 publications

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“…In solution, the enzyme functions as a hexamer (Blackwell et al ., 1999) with point group symmetry 32 (Figure 3A). The crystallographic triad axis coincides with the oligomer's 3‐fold axis.…”

Section: Resultsmentioning

confidence: 99%

“…The enzyme activity is maximal in potassium phosphate buffer (Fish and Blumenthal, 1966). Indeed, crystals were only obtained in the presence of phosphate buffer (Blackwell et al ., 1999). The substrate‐free DDG aldolase crystal structure revealed two phosphate anions bound within the active site pocket (Figure 4A).…”

Section: Resultsmentioning

confidence: 99%

“…Crystals of Se‐Met DDG aldolase were obtained in the same way as the native enzyme (Blackwell et al ., 1999). These crystals also belong to space group R 32 with two polypeptide chains in the asymmetric unit, a solvent content of 0.48 and a volume to protein mass ratio ( V m ) of 1.34 Å 3 /Da.…”

Section: Methodsmentioning

confidence: 99%

“…These crystals also belong to space group R 32 with two polypeptide chains in the asymmetric unit, a solvent content of 0.48 and a volume to protein mass ratio ( V m ) of 1.34 Å 3 /Da. Co‐crystals of DDG aldolase in complex with pyruvate were grown under similar conditions to native DDG aldolase (Blackwell et al ., 1999) with the inclusion of 10 mM ligand to the crystallization drop. Crystals were cryoprotected by including 35% glycerol in the mother liquor.…”

Section: Methodsmentioning

confidence: 99%

“…Aldolase–pyruvate X‐ray data were collected at 100 K on a CCD detector on beam line 9.6 at SRS, Daresbury, with the wavelength set to 0.87 Å and processed with DENZO and SCALEPACK (Otwinowski and Minor, 1997). X‐ray data were collected from a DDG aldolase native crystal at 100 K using a CuKα rotating‐anode source with an R‐AXIS IV imaging plate and processed as described previously (Blackwell et al ., 1999).…”

Section: Methodsmentioning

confidence: 99%

See 4 more Smart Citations

Crystal structures of the metal-dependent 2-dehydro-3-deoxy-galactarate aldolase suggest a novel reaction mechanism

Izard

Blackwell

2000

The EMBO Journal

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Carbon-carbon bond formation is an essential reaction in organic chemistry and the use of aldolase enzymes for the stereochemical control of such reactions is an attractive alternative to conventional chemical methods. Here we describe the crystal structures of a novel class II enzyme, 2-dehydro-3-deoxy-galactarate (DDG) aldolase from Escherichia coli, in the presence and absence of substrate. The crystal structure was determined by locating only four Se sites to obtain phases for 506 protein residues. The protomer displays a modified (alpha/beta)(8) barrel fold, in which the eighth alpha-helix points away from the beta-barrel instead of packing against it. Analysis of the DDG aldolase crystal structures suggests a novel aldolase mechanism in which a phosphate anion accepts the proton from the methyl group of pyruvate.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Crystal structures of the metal-dependent 2-dehydro-3-deoxy-galactarate aldolase suggest a novel reaction mechanism

Izard

Blackwell

2000

The EMBO Journal

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Architecture of intact mammalian pyruvate dehydrogenase complex revealed byin-situstructural analysis

Wang,

Ma,

Chang

et al. 2024

Preprint

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The multi-enzyme pyruvate dehydrogenase complex (PDC) links glycolysis to the citric acid cycle by converting pyruvate into acetyl-coenzyme A and is essential for cellular energy metabolism. Although individual components have been characterized, the structure of intact PDC remains unclear, hampering our understanding of its composition and multi-step catalytic reactions. Here, we report the in-situ architecture of intact PDC within mammalian mitochondria by cryo-electron tomography. The organization of peripheral E1 and E3 multimers varies substantially among PDCs. We discovered 1-46 E1 heterotetramers surrounding each PDC core, and up to 12 E3 dimers locating primarily along the pentagonal openings. Furthermore, we observed dynamic interactions of the substrate translocating lipoyl domains (LDs) with both E1 and E2, implicating the mechanism of substrate channeling. Our findings reveal the intrinsic dynamics of PDC peripheral compositions, suggesting an additional model in which the number of assembled E1 heterotetramer, E3 dimer, and functional LDs of PDC may be regulated to further control its catalytic activity.

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Purification, crystallization and preliminary crystallographic studies on 2-dehydro-3-deoxygalactarate aldolase fromLeptospira interrogans

Huang

Song

et al. 2006

Acta Cryst Sect F

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2-Dehydro-3-deoxygalactarate (DDG) aldolase is a member of the class II aldolase family and plays an important role in the pyruvate-metabolism pathway, catalyzing the reversible aldol cleavage of DDG to pyruvate and tartronic semialdehyde. As it is a potential novel antibiotic target, it is necessary to elucidate the catalytic mechanism of DDG aldolase. To determine the crystal structure, crystals of DDG aldolase from Leptospira interrogans were obtained by the hanging-drop vapour-diffusion method. The crystals diffracted to 2.2 Å resolution using a Cu K rotating-anode X-ray source. The crystal belonged to space group C2, with unit-cell parameters a = 293.5, b = 125.6, c = 87.6 Å , = 100.9 . The V M is calculated to be 2.4 Å 3 Da À1 , assuming there to be 12 protein molecules in the asymmetric unit.

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Rhombohedral crystals of 2-dehydro-3-deoxygalactarate aldolase from Escherichia coli

Cited by 5 publications

References 7 publications

Crystal structures of the metal-dependent 2-dehydro-3-deoxy-galactarate aldolase suggest a novel reaction mechanism

Crystal structures of the metal-dependent 2-dehydro-3-deoxy-galactarate aldolase suggest a novel reaction mechanism

Architecture of intact mammalian pyruvate dehydrogenase complex revealed byin-situstructural analysis

Purification, crystallization and preliminary crystallographic studies on 2-dehydro-3-deoxygalactarate aldolase fromLeptospira interrogans

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