Sandbox Reserved 763: Difference between revisions
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==Structure== | ==Structure== | ||
The crystal structure of [[2iyq]] from the Protein Data Bank <ref name="PDB">Protein Data Bank http://www.rcsb.org/pdb/explore.do?structureId=2IYQ</ref> showing shikimate kinase from ''Mycobacterium tuberculosis'' complexed with ADP and shikimate is shown to the right as the <scene name='56/564039/Defaultscene/1'>default scene</scene>. In addition, the 2D model of [[2gij]], showing the space filling details of the asymmetric unit of MtSK, is shown to the left. | The crystal structure of [[2iyq]] from the Protein Data Bank <ref name="PDB">Protein Data Bank http://www.rcsb.org/pdb/explore.do?structureId=2IYQ</ref> showing shikimate kinase from ''Mycobacterium tuberculosis'' complexed with ADP and shikimate is shown to the right as the <scene name='56/564039/Defaultscene/1'>default scene</scene>. In addition, the 2D model of [[2gij]], showing the space filling details of the asymmetric unit of MtSK, is shown to the left. | ||
===3D Structures in Different Organisms=== | ===3D Structures in Different Organisms=== | ||
''Mycobacterium tuberculosis'' | ''Mycobacterium tuberculosis'' | ||
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===Secondary Structural Elements=== | ===Secondary Structural Elements=== | ||
[[Image:Secondary2iyq.PNG|300px|left|thumb| Secondary Structural Elements<ref name="PDB" />]] | [[Image:Secondary2iyq.PNG|300px|left|thumb| Secondary Structural Elements<ref name="PDB" />]] | ||
Using [[2iyq]] as an example, the secondary structure of shikimate kinase can be observed in terms of strands and helices shown <scene name='56/564039/Helixsheet/1'>here</scene>. There are 5 strands, shown in yellow, and 9 helices (8 α-helices and | Using [[2iyq]] as an example, the secondary structure of shikimate kinase can be observed in terms of strands and helices shown <scene name='56/564039/Helixsheet/1'>here</scene>. There are 5 strands, shown in yellow, and 9 helices (8 α-helices and a single 3<sub>10</sub> helix) which are shown in pink. Shown to the left is a 2D depiction of the secondary structural content of shikimate kinase (strands are yellow, helices are pink) and corresponding residues. | ||
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===Isoforms=== | ===Isoforms=== | ||
In '' Escherichia coli'' the shikimate kinase reaction is catalyzed by two different isoforms: SKI and SKII (Km= 20 μM Km= 200 μM, respectively) with 30% sequence identify. This is very unusual for two isoforms to occur in the middle of a biosynthetic pathway. Therefore, what may be occurring is that shikimate is at a branch point for two pathways. It is believed that SKII is the isoform participating in chorismate biosynthesis, while the role of the SKI isoform is unclear.<ref name=" | In '' Escherichia coli'' the shikimate kinase reaction is catalyzed by two different isoforms: SKI and SKII (Km= 20 μM Km= 200 μM, respectively) with 30% sequence identify. This is very unusual for two isoforms to occur in the middle of a biosynthetic pathway. Therefore, what may be occurring is that shikimate is at a branch point for two pathways. It is believed that SKII is the isoform participating in chorismate biosynthesis, while the role of the SKI isoform is unclear.<ref name="3dstructure">The three-dimensional structure of shikimate kinase. Journal of Molecular Biology http://www.sciencedirect.com.prox.lib.ncsu.edu/science/article/pii/S0022283698917557</ref> | ||
===Oligomeric State=== | ===Oligomeric State=== | ||
Through experiments involving size exclusion liquid chromatography and gel filtration chromatography, the oligomeric state of homogeneous MtSK was found. The molecular mass was found to be 20.7 kDa and when compared to the suggested value of 18.5 kDa, it was found that MtSK is a monomer in solution.<ref>The Mode of Action of Recombinant Mycobacterium tuberculosis Shikimate Kinase: Kinetics and Thermodynamics Analyses http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061918</ref> | |||
Through experiments involving size exclusion liquid chromatography and gel filtration chromatography, the oligomeric state of homogeneous MtSK was found. The molecular mass was found to be 20.7 kDa and when compared to the suggested value of 18.5 kDa, it was found that MtSK is a monomer in solution.<ref>The Mode of Action of Recombinant Mycobacterium tuberculosis Shikimate Kinase: Kinetics and Thermodynamics Analyses http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061918</ref> | |||
===Active Residues=== | ===Active Residues=== | ||
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===Ligands=== | ===Ligands=== | ||
The liganded state of SK includes binary complexes with SO4 or MgADP ([[1l4y]]) and ternary complexes with shikimate as the first ligand and SO4 ([[2g1k]]), ADP ([[1u8a]]), MgADP, or AMPPCP (an ATP analogue) ([[1zyu]]) as the second ligand.<ref name="miscinfo" /> | The liganded state of SK includes binary complexes with SO4 or MgADP ([[1l4y]]) and ternary complexes with shikimate as the first ligand and SO4 ([[2g1k]]), ADP ([[1u8a]]), MgADP, or AMPPCP (an ATP analogue) ([[1zyu]]) as the second ligand.<ref name="miscinfo" /> | ||
Magnesium ion hold influence over the position of shikimate hydroxy groups. This ion has a role in the transfer of the γ-phosphate of ATP to the 3-hydroxy group on shikimate. Chloride ions increase enzyme affinity for ADP and ATP and help to bind the nucleotide substrate in correct orientation.<ref name="mgcl"> Effects of the magnesium and chloride ions and shikimate on the structure of shikimate kinase from Mycobacterium tuberculosis Structural Biology and Crystallization Communications | Magnesium ion hold influence over the position of shikimate hydroxy groups. This ion has a role in the transfer of the γ-phosphate of ATP to the 3-hydroxy group on shikimate. Chloride ions increase enzyme affinity for ADP and ATP and help to bind the nucleotide substrate in correct orientation.<ref name="mgcl"> Effects of the magnesium and chloride ions and shikimate on the structure of shikimate kinase from Mycobacterium tuberculosis Structural Biology and Crystallization Communications | ||
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MgADP | MgADP | ||
Shikimate interacts | Shikimate interacts viah intermolecular hydrogen bonds with Asp34, Arg58, Gly80, and Arg136<ref name="miscinfo" /><ref name="inhibitors"> Identification of new potential Mycobacterium tuberculosis shikimate kinase inhibitors through molecular docking simulations. Journal of Molecular Modeling http://link.springer.com.prox.lib.ncsu.edu/article/10.1007%2Fs00894-011-1113-5 | ||
</ref> | </ref> | ||
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===Protein Fold=== | ===Protein Fold=== | ||
Protein folding from a random coil to native state with correct 3D structure is essential for proper protein function. The primary sequence of amino acids determines the folded structure. The topology of this enzyme | Protein folding from a random coil to native state with correct 3D structure is essential for proper protein function. The primary sequence of amino acids determines the folded structure. The topology of this enzyme shows an alpha/beta/alpha fold consisting of a 5-stranded central parallel beta sheet and 8 surrounding alpha-helices.<ref name="miscinfo" /> | ||
Shikimate kinase is a convenient protein to use in protein folding studies. This is because it is one of the smallest kinases and is a monomeric enzyme. It has been shown that the proposed refolding model includes a rapid hydrophobic collapse and then a slower secondary structure formation.<ref name="fold">The refolding of type II shikimate kinase from Erwinia chrysanthemi | Shikimate kinase is a convenient protein to use in protein folding studies. This is because it is one of the smallest kinases and is a monomeric enzyme lacking disulfide bonds. It has been shown that the proposed refolding model includes a rapid hydrophobic collapse and then a slower secondary structure formation.<ref name="fold">The refolding of type II shikimate kinase from Erwinia chrysanthemi | ||
after denaturation in urea. European Journal of Biochemistry http://onlinelibrary.wiley.com/doi/10.1046/j.1432-1033.2002.ejb.02862.x/pdf</ref> | after denaturation in urea. European Journal of Biochemistry http://onlinelibrary.wiley.com/doi/10.1046/j.1432-1033.2002.ejb.02862.x/pdf</ref> | ||
Look into how the protein fold could be disrupted (ie pH, temperature studies etc) | |||
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Circular Dichroism | Circular Dichroism | ||
==Mechanism of Action== | ==Mechanism of Action== | ||
[[Image:Shikimate_kinase_reaction.png|400px|left|thumb| Shikimate kinase catalyzed reaction]] | [[Image:Shikimate_kinase_reaction.png|400px|left|thumb| Shikimate kinase catalyzed reaction]] | ||
===Reaction Pathway=== | ===Reaction Pathway=== | ||
