Catalytic Role of Conserved Asparagine, Glutamine, Serine, and Tyrosine Residues in Isoprenoid Biosynthesis Enzymes

We report the results of an investigation into the catalytic role of highly conserved amide-containing (asparagine, glutamine) and OH-containing (serine, tyrosine) residues in several prenyltransferases. We first obtained the X-ray structure of cyclolavandulyl diphosphate synthase containing two molecules of the substrate analogue dimethylallyl (S)-thiolodiphosphate (DMASPP). The two molecules have diphosphate group orientations similar to those seen in other ζ -fold (cis head-to-tail and head-to-middle) prenyltransferases, with one diphosphate moiety forming a bidentate chelate with Mg2+ in the so-called S1 site (which is typically the allylic binding site in ζ -fold proteins) and the second diphosphate binding to Mg2+ in the so-called S2 site (which is typically the homoallylic binding site in ζ -fold proteins) via a single P1O1 oxygen. The latter interaction can facilitate direct phosphate-mediated proton abstraction via P1O2 or, more likely, by an indirect mechanism in which P1O2 stabilizes a basic asparagine species that removes H+, which is then eliminated via an Asn-Ser shuttle. The universal occurrence of Asn-Ser pairs in ζ -fold proteins leads to the idea that the highly conserved amide-containing (Asn, Gln) and OH-containing (Tyr) residues seen in many "head-to-head" prenyltransferases such as squalene synthase and dehydrosqualene synthase might play similar roles in H+ elimination. Structural, bioinformatics, and mutagenesis investigations indeed indicate an important role of these residues in catalysis, with the results of density functional theory calculations showing that Asn bound to Mg2+ can act as a general (imine-like) base while Gln, Tyr, and H2O form a proton channel that is adjacent to the conventional (Asp-rich) "active site". Taken together, our results lead to mechanisms of proton elimination from carbocations in numerous prenyltransferases in which neutral species (Asn, Gln, Ser, Tyr, and H2O) act as proton shuttles, complementing the more familiar roles of acidic groups (in Asp and Glu), which bind to Mg2+, and basic groups (primarily Arg), which bind to diphosphates, in isoprenoid biosynthesis. © 2018 American Chemical Society.