From Protein Structure to Function with Bioinformatics.pdf

284 J.D. Watson and J.M. Thorntonand tense (T) states (from Staphylococcus aureus and Chlorobium tepidum,respectively). The two enzymes show low sequence identity (27.3%) but thesame overall architecture and domain organization: both enzymes are tetrameric(forming dimers of dimers – see Fig. 11.3 for images of the individual dimers)and are made up of a catalytic domain (PDT domain) and a regulatory domain(ACT domain). Based on these two PDT structures the authors proposed that thePDT active site is located in the cleft between two PDT subdomains (Fig. 11.4).This structure-based prediction was supported by sequence analysis andmutagenesis data. The mapping of conserved residues from multiple sequencealignments onto the structure located the most highly conserved residues at thebottom of the cleft between subdomains. Residues, shown to be critical for PDTactivity in E. coli by site-directed mutagenesis, were shown to be equivalent toresidues associated with the cleft between the two PDT subdomains (Zhanget al. 2000). Additional mutagenesis data for Corynebacterium glutamicum PDTconfirmed equivalent residues to be involved in substrate binding and/or catalyticactivity (Hsu et al. 2004). All in all, the data suggest that the cleft and conservedresidues within it form the active site of prephenate dehydratase, withT168 being the most likely key catalytic residue.The identification of the likely active site was followed by identification of theallosteric control site. The location of this l-Phe binding site in PDT is similar tothe effector binding sites observed in several other ACT domain containingenzymes involved in binding amino acids or other small ligands. The presence ofbound l-Phe in the structure allowed the visualisation of how its interaction withthe ACT domains in the Chlorobium tepidum structure. The examination of thebinding residues shows that most interactions are likely to be non-specific, whichexplains why other amino acids such as methionine can also bind inside the pocketand regulate catalytic activity (Liberles et al. 2005).Fig. 11.3 PDT structures deposited by the MCSG illustrating the overall similarities and structuraldifferences: (a) The R-state structure from Staphylococcus aureus (PDB entry 2qmw) (b)The T-state structure from Chlorobium tepidum (PDB entry 2qmx). The two enzymes share only27.3% sequence identity