1ur5: Difference between revisions
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==Overview== | ==Overview== | ||
Malate dehydrogenase (MDH) from the moderately thermophilic bacterium, Chloroflexus aurantiacus (CaMDH) is a tetrameric enzyme, while MDHs from, mesophilic organisms usually are dimers. To investigate the potential, contribution of the extra dimer-dimer interface in CaMDH with respect to, thermal stability, we have engineered an intersubunit disulfide bridge, designed to strengthen dimer-dimer interactions. The resulting mutant, (T187C, containing two 187-187 disulfide bridges in the tetramer) showed a, 200-fold increase in half-life at 75 degrees C and an increase of 15 deg., C in apparent melting temperature compared to the wild-type. The crystal, structure of the mutant (solved at 1.75 A resolution) was essentially, identical with that of the wild-type, with the exception of the added, ... | Malate dehydrogenase (MDH) from the moderately thermophilic bacterium, Chloroflexus aurantiacus (CaMDH) is a tetrameric enzyme, while MDHs from, mesophilic organisms usually are dimers. To investigate the potential, contribution of the extra dimer-dimer interface in CaMDH with respect to, thermal stability, we have engineered an intersubunit disulfide bridge, designed to strengthen dimer-dimer interactions. The resulting mutant, (T187C, containing two 187-187 disulfide bridges in the tetramer) showed a, 200-fold increase in half-life at 75 degrees C and an increase of 15 deg., C in apparent melting temperature compared to the wild-type. The crystal, structure of the mutant (solved at 1.75 A resolution) was essentially, identical with that of the wild-type, with the exception of the added, inter-dimer disulfide bridge and the loss of an aromatic intra-dimer, contact. Remarkably, the mutant and the wild-type had similar temperature, optima and activities at their temperature optima, thus providing a clear, case of uncoupling of thermal stability and thermoactivity. The results, show that tetramerization may contribute to MDH stability to an extent, that depends strongly on the number of stabilizing interactions in the, dimer-dimer interface. | ||
==About this Structure== | ==About this Structure== | ||
1UR5 is a | 1UR5 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Chloroflexus_aurantiacus Chloroflexus aurantiacus] with CD, CL, NA and NAD as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Malate_dehydrogenase Malate dehydrogenase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.1.1.37 1.1.1.37] Structure known Active Site: AC1. Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1UR5 OCA]. | ||
==Reference== | ==Reference== | ||
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[[Category: tricarboxylic acid cycle]] | [[Category: tricarboxylic acid cycle]] | ||
''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on | ''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Mon Nov 5 14:41:40 2007'' | ||
Revision as of 15:36, 5 November 2007
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STABILIZATION OF A TETRAMERIC MALATE DEHYDROGENASE BY INTRODUCTION OF A DISULFIDE BRIDGE AT THE DIMER/DIMER INTERFACE
OverviewOverview
Malate dehydrogenase (MDH) from the moderately thermophilic bacterium, Chloroflexus aurantiacus (CaMDH) is a tetrameric enzyme, while MDHs from, mesophilic organisms usually are dimers. To investigate the potential, contribution of the extra dimer-dimer interface in CaMDH with respect to, thermal stability, we have engineered an intersubunit disulfide bridge, designed to strengthen dimer-dimer interactions. The resulting mutant, (T187C, containing two 187-187 disulfide bridges in the tetramer) showed a, 200-fold increase in half-life at 75 degrees C and an increase of 15 deg., C in apparent melting temperature compared to the wild-type. The crystal, structure of the mutant (solved at 1.75 A resolution) was essentially, identical with that of the wild-type, with the exception of the added, inter-dimer disulfide bridge and the loss of an aromatic intra-dimer, contact. Remarkably, the mutant and the wild-type had similar temperature, optima and activities at their temperature optima, thus providing a clear, case of uncoupling of thermal stability and thermoactivity. The results, show that tetramerization may contribute to MDH stability to an extent, that depends strongly on the number of stabilizing interactions in the, dimer-dimer interface.
About this StructureAbout this Structure
1UR5 is a Single protein structure of sequence from Chloroflexus aurantiacus with CD, CL, NA and NAD as ligands. Active as Malate dehydrogenase, with EC number 1.1.1.37 Structure known Active Site: AC1. Full crystallographic information is available from OCA.
ReferenceReference
Stabilization of a tetrameric malate dehydrogenase by introduction of a disulfide bridge at the dimer-dimer interface., Bjork A, Dalhus B, Mantzilas D, Eijsink VG, Sirevag R, J Mol Biol. 2003 Dec 5;334(4):811-21. PMID:14636605
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