Terpene Cyclases And Prenyltransferases: Structures And Mechanisms Of Action

Terpenes and isoprenoids are the most diverse small organic molecules on Earth. Here, we obtained the structures of sesquisabinene and santalene synthases from Santalum album L. in the presence or absence of substrate, substrate-like, or product-like molecules for a total of eight structures. All proteins crystallized in an "open", noncatalytic conformation and, in the case of the FPP structure, three Mg2+ (MgABC) were bound in the active site region. We found that one of these Mg2+ (MgB), involved in pocket closure to the catalytically active conformation, interacted with a single inner-sphere amino acid, a bidentate diphosphate, and a facial arrangement of three water molecules, H2O-a,b,c: fac-[Mg(H2O)(3)XPP(Asn/Asp)]. Paradoxically, the same arrangement of ligands is found in closed-form, catalytically active "head-to-tail" prenyltransferases, such as farnesyl diphosphate synthase (FPPS), leading to the proposal that in these proteins, pocket closure is due to interaction with outer-sphere ligands (typically, two Asps) with H2O-a,b. We also discovered an extensive H-bond network that is present in related cyclases and prenyltransferases: the D-motif. This motif is centered around the totally conserved outer-sphere Asp that is bonded via H2O-c to MgB and comprises five amino acids, including a very highly conserved residue in the cyclases, an Arg, and a highly conserved residue in the alpha HT proteins, a Gln, and we propose that this motif may be involved in catalysis per se. Finally, using bioinformatics and mutagenesis, we show that many of the most highly conserved residues in these and other cyclases are outside the active site region and are in the noncatalytic beta-domain, reflecting their evolutionary origin from beta gamma domain proteins, while a highly conserved alpha-domain Arg forms a salt bridge with a Glu that serves to stabilize the catalytically active alpha beta-domain structure.