Mutagenesis of cycloartenol synthase and lanosterol synthase: Broadening and narrowing product profile
Matsuda, Seiichi P. T.
Doctor of Philosophy
This thesis describes mutagenesis experiments in cycloartenol synthase and lanosterol synthase that allowed the identification of important catalytic residues necessary to broaden and narrow product profiles. These results provided insights into factors critical for redesigning enzyme function. Mutagenesis in Arabidopsis thaliana cycloartenol synthase revealed that His477 is an essential component of the catalytic distinction between cycloartenol synthase and lanosterol synthase. Mutations at position 477 abolish cycloartenol biosynthesis, and subtle structural changes at this position dramatically alter product profile. The His477Asn mutant produces lanosterol as its major product (88%) whereas His477Gln produces primarily parkeol (73%). His477 influences deprotonation more strongly than any of the previously studied catalytic residues, but changes at this position are catalytically irrelevant in the presence of Tyr410Thr and Ile481Val mutations. The His477Asn Tyr410Thr Ile481Val and His477Gln Tyr410Thr Ile481Val triple mutants have the same product profile as the Tyr410Thr Ile481Val double mutant. Homology modeling studies established that His477 is a second-sphere residue that affects catalysis indirectly through its interactions with the active-site residue Tyr410. Changes at His477 strongly affect the location, orientation, and electronics of the Tyr410 side chain. Efforts to modify the catalytic specificity of enzymes consistently show that it is easier to broaden the substrate or product specificity of an accurate enzyme than to restrict the selectivity of one that is promiscuous. Careful examination of the homology model allowed the identification of a combination of mutations necessary to redesign cycloartenol synthase into a highly accurate lanosterol synthase. A double mutant was constructed and characterized and was shown to cyclize oxidosqualene accurately to lanosterol (99%). This catalytic change entailed both relocating polarity with a His477Asn mutation and modifying steric constraints with an Ile481Val mutation, and is among the best examples of redesigning an enzyme to accurately generate a new product. Known lanosterol synthase mutants make monocyclic or tetracyclic byproducts from oxidosqualene. Mutation of Saccharomyces cerevisiae lanosterol synthase at Tyr510 caused partial substrate misfolding and generated a tricyclic byproduct. This novel triterpene, (13alphaH)-isomalabarica-14(27),17 E,21-trien-3beta-ol, is the putative biosynthetic precursor of the isomalabaricane triterpenoids in sponges. The results suggest the facile evolution of tricyclic terpenoids as secondary metabolites in sponges.