Structural studies of triacylglycerol lipase from Candida cylindracea, 5-aminolaevulinic acid dehydratase from Escherichia coli and succinyl-CoA transferase from pig heart
Structural studies of triacylglycerol lipase from Candida cylindracea, 5-aminolaevulinic acid dehydratase from Escherichia coli and succinyl-CoA transferase from pig heart
The enzymes triacylglycerol lipase from Candida cylindracea, 5-aminolaevulinic acid dehydratase from Escherichia coli and succinyl-CoA transferase from pig heart were purified to electrophoretic homogeneity.
The lipase was crystallised and data were collected to 3.5Å resolution on a rotating anode source. Crystals were in the space group P21 with cell dimensions a = 94.3Å, b = 117.0Å, c = 114.2Å, and β = 109.2°. The structure was solved using molecular replacement with the triacylglycerol lipase from Geotrichum candidum. Four molecules were present per asymmetric unit.
5-Aminolaevulinic acid dehydratase as its Schiff's base adduct with the substrate analogue, laevulinic acid, was crystallised in the space group I422 with cell dimensions a = b = 130.7Å and c = 142.4Å. Data were collected both at room temperature and 100K at the SRS, Daresbury to 2.0Å resolution. Cell dimensions decreased on freezing to a = b = 126.4Å and c = 140.5Å. After searching for heavy atom derivatives, the structure was finally solved by molecular replacement with 5-aminolaevulinic acid dehydratase from Saccharmoyces cerevisiae. One monomer was present in the asymmetric unit. Initial electron density maps show the structure to be dominated by a TIM barrel fold. Density at the active site lysine 246 indicates the presence of laevulinic acid. The enzyme was also crystallised as its sodium borohydride-reduced adduct with both the substrate, 5-aminolaevulinic acid, and the inhibitor, laevulinic acid, irreversibly bound in the active site. This was confirmed with matrix assisted laser desorption time of flight mass spectrometry. Crystals grown of these forms await data collection.
Succinyl-CoA transferase was crystallised in the space group P21 with two main cell forms: a = 75.3.3Å, b = 134.3Å, c = 104.9Å, β = 100.3° and a = 74.9Å, b = 134.5Å, c = 102.3Å, β = 104.5°. Data were collected at 100K at the ESRF, Grenoble to 2.2Å resolution. Calculations indicated two dimers per asymmetric unit. A search for heavy atom derivatives is ongoing. The first mass spectrometric analysis of intact protein was performed. This showed the Mr of the protein to be 52189.9±4.1, close to the predicted Mr of 52197, indicating the absence of previously reported glycosylation.
University of Southampton
1997
Lewis, Gareth Raymond
(1997)
Structural studies of triacylglycerol lipase from Candida cylindracea, 5-aminolaevulinic acid dehydratase from Escherichia coli and succinyl-CoA transferase from pig heart.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
The enzymes triacylglycerol lipase from Candida cylindracea, 5-aminolaevulinic acid dehydratase from Escherichia coli and succinyl-CoA transferase from pig heart were purified to electrophoretic homogeneity.
The lipase was crystallised and data were collected to 3.5Å resolution on a rotating anode source. Crystals were in the space group P21 with cell dimensions a = 94.3Å, b = 117.0Å, c = 114.2Å, and β = 109.2°. The structure was solved using molecular replacement with the triacylglycerol lipase from Geotrichum candidum. Four molecules were present per asymmetric unit.
5-Aminolaevulinic acid dehydratase as its Schiff's base adduct with the substrate analogue, laevulinic acid, was crystallised in the space group I422 with cell dimensions a = b = 130.7Å and c = 142.4Å. Data were collected both at room temperature and 100K at the SRS, Daresbury to 2.0Å resolution. Cell dimensions decreased on freezing to a = b = 126.4Å and c = 140.5Å. After searching for heavy atom derivatives, the structure was finally solved by molecular replacement with 5-aminolaevulinic acid dehydratase from Saccharmoyces cerevisiae. One monomer was present in the asymmetric unit. Initial electron density maps show the structure to be dominated by a TIM barrel fold. Density at the active site lysine 246 indicates the presence of laevulinic acid. The enzyme was also crystallised as its sodium borohydride-reduced adduct with both the substrate, 5-aminolaevulinic acid, and the inhibitor, laevulinic acid, irreversibly bound in the active site. This was confirmed with matrix assisted laser desorption time of flight mass spectrometry. Crystals grown of these forms await data collection.
Succinyl-CoA transferase was crystallised in the space group P21 with two main cell forms: a = 75.3.3Å, b = 134.3Å, c = 104.9Å, β = 100.3° and a = 74.9Å, b = 134.5Å, c = 102.3Å, β = 104.5°. Data were collected at 100K at the ESRF, Grenoble to 2.2Å resolution. Calculations indicated two dimers per asymmetric unit. A search for heavy atom derivatives is ongoing. The first mass spectrometric analysis of intact protein was performed. This showed the Mr of the protein to be 52189.9±4.1, close to the predicted Mr of 52197, indicating the absence of previously reported glycosylation.
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Published date: 1997
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Local EPrints ID: 463100
URI: http://eprints.soton.ac.uk/id/eprint/463100
PURE UUID: 79f1d051-3170-4b40-b13f-bc056313ebc3
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Date deposited: 04 Jul 2022 20:44
Last modified: 04 Jul 2022 20:44
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Author:
Gareth Raymond Lewis
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