Isoprenoid biosynthesis constitutes an immensely diverse, highly branched network of pathways that spans both primary and secondary (specialized) metabolism in all organisms. The mevalonate (MVA) pathway or the 1-deoxy-D- xylulose 5-phosphate (DXP) pathway operate in a given organism to produce the two important building blocks for all downstream isoprenoids : isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In Archaea, the biosynthesis of these two vital building blocks remains unclear. The current hypothesis is that Archaea utilize an alternative mevalonate pathway that follows the canonical pathway up until the biosynthesis of phosphomevalonate. At this point, a decarboxylation event followed by a phosphorylation event produces the essential building block, IPP. The latter step is catalyzed by isopentenyl phosphate kinase (IPK). In this work, we solved the structure of IPK from M. jannaschii and successfully used it toward : 1) the design of a deeper active site pocket for binding and catalysis of longer chained isoprenoid monophosphates; 2) the identification and characterization of active IPK homologs in other kingdoms of life. This work contributes towards the design of a synthetic metabolic pathway and reveals new information about the potential existence of a bifurcated mevalonate pathway in all plants and certain other eukaryotic organisms. Farnesyl diphosphate is directly derived from the building blocks IPP and DMAPP and is an essential metabolic intermediate for a variety of downstream primary and secondary metabolic pathways including cholesterol biosynthesis and terpenoid biosynthesis, respectively. Sesquiterpene cyclases (synthases) are part of terpenoid biosynthesis and catalyze the cyclization of farnesyl diphosphate into one or more sesquiterpene products; these chemicals play important biological roles in defense and communication, especially in plants. Here, we explore a variety of mutant and wild type plant sesquiterpene cyclases in attempt to understand several concepts : 1) how these enzymes traverse a defined catalytic landscape to biosynthesize disparate products without compromising their catalytic activities; 2) the structural and functional differences associated with turnover of cis- and trans-FPP by wild type and promiscuous cyclase mutants; 3) how certain sesquiterpene synthases utilize an Arg-Pro motif within the amino terminal domain to interact with the catalytic C-terminal domain and modulate product profile complexity

Unnatural amino acids (Uaas) can be translationally incorporated into proteins in vivo using evolved tRNA/aminoacyl-tRNA synthetase (RS) pairs, affording chemistries inaccessible when restricted to the 20 natural amino acids. To date, most evolved RSs aminoacylate Uaas chemically similar to the native substrate of the wild-type RS; these conservative changes limit the scope of Uaa applications. Here, we adapt Methanosarcina mazei PylRS to charge a noticeably disparate Uaa, O-methyl-l-tyrosine (Ome). In addition, the 1.75 Å X-ray crystal structure of the evolved PylRS complexed with Ome and a non-hydrolyzable ATP analogue reveals the stereochemical determinants for substrate selection. Catalytically synergistic active site mutations remodel the substrate-binding cavity, providing a shortened but wider active site. In particular, mutation of Asn346, a residue critical for specific selection and turnover of the Pyl chemical core, accommodates different side chains while the central role of Asn346 in aminoacylation is rescued through compensatory hydrogen bonding provided by A302T. This multifaceted analysis provides a new starting point for engineering PylRS to aminoacylate a significantly more diverse selection of Uaas than previously anticipated.