Serine Palmitoyltransferase
Created by Tamana Ghani
Serine palmitoyltransferase (pdb id=2xbn) is a PLP-dependent enzyme that exists in all organisms, both eukaryotic and prokaryotic, and has been most extensively studied in the bacterium Sphingomonas paucimobilis. The first and rate-determining step of the sphingolipid biosynthetic pathway is catalyzed by SPT, producing 3-ketodihydrosphingosine from the condensation of serine and palmitoyl-CoA.
The physiological form of SPT in S. paucimobilis is a symmetrical homodimer found in the cytoplasm with two identical monomeric subunits, each of which contains three domains. The monomeric tertiary structure contains an N-terminal domain about 80 residues in length comprised of an alpha helix and three-stranded antiparallel beta-sheet, followed by a central catalytic domain of about 200 residues and a seven-stranded parallel beta-sheet, and then the C-terminal domain of about 100 residues that interacts with the N-terminal domain of the other subunit (1). The SPT monomer is a single chain consisting predominately of beta sheets and alpha helices, although turns and random coils are also present to a lesser degree.
Each monomer of SPT contains a ligand binding site where the PLP cofactor, PMP, is covalently bonded to the K-265 residue as an internal aldimine/Schiff base; hence the dimer oligomeric state of the enzyme contains two active sites. This internal aldimine is the holoenzyme form of SPT, however the binding of the serine substrate to the PLP cofactor changes the conformation to an external aldimine where PLP is no longer covalently linked to Lys265 in the active site (2). PLP is transferred to the substrate serine and the PLP-serine complex is held in the active site where binding of the substrate palmitoyl-CoA to PLP induces a further conformational change that causes the alpha-proton of Serine to become perpendicular to the plane of the PLP-Ser external aldimine (3). Once the product is released, the internal aldimine form of the enzyme is regenerated. This conformational change only occurs during the catalytic cycle, however, and SPT does not exist in different conformations naturally.
The PLP cofactor is essential to the function of SPT and the residues involved in binding the cofactor in the active site are: His-234, His-159, Ala-160, Ser-161, Lys-265, and Asp-231. Hydrogen bonds are formed with this ligand by the side chains of residues Asn-138, Asp-231, His-234 and Thr-262 and also with the main chains of Gly-134 and Tyr-135. His-159 and Arg-378 play multiple roles in the recognition and binding of the Serine substrate and subsequent stabilization of the external aldimine conformation (4). A water molecule is also found in the active site hydrogen bonded to the hydroxyl group of PLP and is displaced during catalysis by binding of serine. Magnesium, which plays a role in regulating the activity of SPT by preventing upregulation, has a binding site on the enzyme through an ionic interaction with the residue Phe80 (5).
SPT is a ubiquitous enzyme among all organisms because sphingolipids are essential for proper cellular function. Inhibition of the function of SPT has been observed with the cyclic amino acid mimic, cycloserine (6). Cycloserine exists as a synthetically made L-enantiomer and a naturally occurring D-enantiomer that is commonly used in drugs to treat tuberculosis and certain neurological disorders; however the usefulness of both is compromised due to their toxicity in inhibiting sphingolipid biosynthesis. The essential PLP cofactor is disabled by cycloserine because the amino acid mimic becomes bonded to the prosthetic group and forms a stable 3-hydroxyisoxazole/pyridixal 5'-phosphate (PMP) adduct at the site where serine should bind. The covalent bond to Lys-265 is broken by binding of cycloserine, and this inhibition of the cofactor is irreversible as long as cycloserine is present at certain concentrations near the enzyme.
The structure of SPT is strikingly similar to that of 8-amino-7-oxonanoate synthase ( AONS), another enzyme in the alpha-oxoamine synthase family. Both enzymes are symmetrical homodimers in their oligomeric state with similar active site organization and relative orientation of domains (7). The residues His-159, Ala-160 and Ser-161 are conserved in the ligand binding sites of both SPT and AONS, and a salt bridge between the pyridinium nitrogen atom of the cofactor and oxygen atom of Asp-231 is present in both enzymes to stabilize the intermediate product during catalysis (8). Furthermore, cycloserine also inhibits AONS through bond formation with its PLP cofactor. The structural similarities of SPT and AONS are indicative of their similar functions as PLP dependent enzymes, emphasizing the relationship between protein structure and function.