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Shikimate kinase (SK) is an enzyme which participates in the fifth step of the shikimate pathway. The functional role shikimate kinase plays in this pathway is to catalyze the ATP-dependent phosphorylation of shikimate into shikimate 3-phosphate (3-phosphoshikimate). This aids in the synthesis of chorismate, which is the precursor to aromatic amino acids and secondary metabolites. Shikimate kinase belongs to the nucleoside monophosphate (NMP) kinase family, which phosphorylates a protein leading to a functional change in the phosphorylated protein | Shikimate kinase (SK) is an enzyme which participates in the fifth step of the shikimate pathway. The functional role shikimate kinase plays in this pathway is to catalyze the ATP-dependent phosphorylation of shikimate into shikimate 3-phosphate (3-phosphoshikimate). This aids in the synthesis of chorismate, which is the precursor to aromatic amino acids and secondary metabolites. Shikimate kinase belongs to the nucleoside monophosphate (NMP) kinase family, which phosphorylates a protein leading to a functional change in the phosphorylated protein. SK is of the transferase class, which acts to transfer a functional group from the donor to acceptor molecule. The protein fold consists of 8 α-helices and 5 β-strands. The length of shikimate kinase is 173 residues and it is found in the cytoplasm. This is a subclass of α/β proteins, meaning an α/β domain exists. SK is composed of Molecule A and B, which together form the asymmetric unit, with SK functioning as a monomer. SK consists of the CORE, LID, and substrate-binding domains. ATP is the co-substrate while magnesium ion is the co-factor. The shikimate pathway is present in bacteria, fungi, higher plants, algae, and apicomplexan. This enzyme has been experimentally observed predominantly in the organism ''Mycobacterium tuberculosis'', but also in ''Helicobacter pylori'', ''Bacteroides thetaiotaomicron'', ''Campylobacter jejuni'', ''Aquifex aeolicus'', ''Coxiella burnetii'', ''Arabidopsis thaliana''. The aroK gene encodes for SK in ''Mycobacterium tuberculosis''. This enzyme is a protein target for rational drug design, which holds great potential due to shikimate kinase being absent in mammals. | ||
==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 | 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 1 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=== | |||
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="skstructure"> 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> | SK is 173 amino acids in length and consists of two molecules: Molecule A from residues 1-112 and 128-173; Molecule B from residues 1-112 and 123-172. However, SK functions as a monomer with Molecules A and B forming the asymmetric unit. A disulfide bridge between Cys162 of Molecules A and B covalently links the two together.<ref name="skstructure"> | ||
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" /> | ||
ADP binds in P-loop, Mg+2 binds nearby and is essential for enzyme activity. Shikimate binding occurs with helices α2, α3, and α4 (N-terminal region).<ref name="skstructure"> | |||
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 via 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 is that of an α/β protein. An α/β/α fold exists consisting of a 5-stranded central parallel β-sheet (strand order 23145) and is flanked by 8 surrounding α-helices (α1 and α8 are to one side while α4, α5, and α7 are on the other side).<ref name="miscinfo" /><ref name="skstructure"> | ||
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 | 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 | ||
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> | ||
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Circular Dichroism | Circular Dichroism | ||
Spectroscopy | |||
Heavy atom derivatisation | |||
Electron density mapping | |||
Chromatography | |||
==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=== | ||
