From Proteopedia

Revision as of 01:16, 4 December 2013

Shikimate Kinase

Shikimate kinase 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 is a protein kinase, an enzyme which phosphorylates a protein leading to a functional change in the phosphorylated protein, of the transferase class. A transferase acts to transfer a functional group from the donor to acceptor molecule. The protein fold consists of 9 helices and 5 strands. The length of shikimate kinase ranges from 161-214 residues and it is found in the cytoplasm. This is a subclass of alpha/beta proteins, meaning an alpha/beta domain exists. The subunit is that of a monomer. This enzyme consists of the CORE, LED, and substrate-binding domains. ATP is the cosubstrate while magnesium ion is the cofactor. Though the shikimate pathway is present in microorganisms and plants, shikimate kinase is found only in the bacteria taxon. This enzyme is found predominantly in the organism Mycobacterium tuberculosis, but also in Helicobacter pylori, Bacteroides thetaiotaomicron, Campylobacter jejuni, Aquifex aeolicus, Coxiella burnetii, Arabidopsis thaliana. This enzyme is a protein target for rational drug design against tuberculosis, which holds great potential due to shikimate kinase being absent in mammals.

Structure

The crystal structure of 2iyq from the Protein Data Bank ^[1] showing shikimate kinase from Mycobacterium tuberculosis complexed with ADP and shikimate is shown to the right as the . 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

Mycobacterium tuberculosis

2g1j - MtSK

2iyt - MtSK (unliganded, open lid)

1iyw - MtSK (open lid) + ATP

2g1k, 2iyr, 2iyx - MtSK + shikimate

2iys - MtSK (open lid) + shikimate

2iyy - MtSK + shikimate-3-phosphate

2iys - MtSK (open lid) + shikimate

2iyu, 2iyv - MtSK (open lid) + ADP

1l4u, 1l4y, 1u8a, 2dft - MtSK + ADP

2iyw - MtSK (open lid) + ATP

3baf - MtSK + AMP-PNP

2dfn, 1we2, 2iyq, 1u8a - MtSK + ADP + shikimate

2iyz - MtSK + ADP + shikimate-3-phosphate

4bqs - MtSK + ADP + shikimic acid derivative

1zyu - MtSK + AMPPCP + shikimate

Helicobacter pylori

1zuh - HpSK

3mrs - HpSK (mutant)

1zui - HpSK + shikimate

3n2e - HpSK (mutant) + inhibitor

3muf - HpSK + ADP + shikimate-3-phosphate

Other Organisms

2pt5 - SK - Aquifex Aeolicus

3trf - SK - Coxiella burnetii

3vaa - SK - Bacteroides thetaiotaomicron

1via - SK - Campylobacter jejuni

3nwj - SK - Arabidopsis thaliana

Secondary Structural Elements

Secondary Structural Elements^[1]

Using 2iyq as an example, the secondary structure of shikimate kinase can be observed in terms of strands and helices shown . There are 5 strands, shown in yellow, and 9 helices, 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.

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.^[2]

Active Residues

Using 2iyq as an example, the active residues were binding in shikimate kinase occurs is shown to the left. There are three domains present in Shikimate kinase: the CORE domain, and substrate-binding domain, and LID domain. Each domain plays a role in binding and is related to the active residues. The binding site for nucleotides is in the CORE, shikimate binds in the substrate-binding domain, and once ATP or shikimate binds, the LID domain closes over the active site.^[3]

Ligands

Ligands associated with shikimate kinase include ADP, CL, SKM, and TRS.

Adenosine-5'-sisphosphate (ADP)

Chloride ion (CL)

(3R,4S,5R)-3,4,5-trihydroxycyclohex-1-ene-1-caroxylic acid (SKM)

2-amino-2-hydroxymethyl-propane-1,3-diol (TRS)

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.

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.^[4]

Look into how the protein fold could be disrupted (ie pH, temperature studies etc)

Methods Used to Solve

Mechanism of Action

Shikimate kinase catalyzed reaction

Describe how protein functions. Structures of Ligands/inhibitors/important steps in reaction pathway.

The substrates involved with shikimate kinase are shikimate and ATP. The products involved are shikimate 3-phosphate and ADP.

Implications or Possible Application

Medical importance - related disease, if used as drug target.

If protein is found in other organisms.

Other uses (ie antibiotics etc)

References