Carbocation intermediates can be stabilized in the active site of a terpenoid cyclase by weakly polar interactions involving charge–charge, charge–dipole, and charge–quadrupole interactions with suitably oriented amino acid side chains. (26−28) Additionally, these studies show that many carbocation intermediates in terpenoid cyclization cascades are subject to electron delocalization (e.g., through hyperconjugation and other through-bond coupling) that influences and directs the cyclization trajectory. Quantum mechanical studies of terpenoid cyclase reaction mechanisms indicate that tertiary carbocation intermediates are much more common than secondary carbocation intermediates. Since some terpenoid cyclases generate hydroxylated products, reflecting quenching of the final carbocation intermediate by a water molecule, it is logical that these cyclases have evolved with suitable water management strategies to prevent premature quenching of carbocation intermediates. (23−25) Although such potential nucleophiles could rapidly quench a carbocation intermediate, they will not do so as long as they are fixed in positions where they are not properly oriented to react. However, this does not preclude the presence of occasional polar groups or solvent molecules in the terpenoid synthase active site (e.g., as observed in bornyl diphosphate synthase from Salvia officinalis and aristolochene synthase from Aspergillus terreus). Such highly reactive intermediates could potentially alkylate and inactivate the enzyme, so the terpenoid synthase typically contains a nonpolar active site pocket that enables effective management and manipulation of these intermediates. Figure 1Ī hallmark of most reactions catalyzed by a terpenoid synthase is the cascade of multiple carbocation intermediates that define the reaction coordinate of catalysis. Moreover, since cyclic terpenoids typically cannot be generated from linear precursors in the absence of an enzyme, the catalytic rate enhancement of a terpenoid cyclase over the uncatalyzed rate is immeasurably large. Terpenoid cyclization reactions are the most complex reactions found in nature, in that on average more than half of the substrate carbon atoms undergo changes in bonding, hybridization, and stereochemistry during the course of a multistep cyclization cascade. (5−19) These reactions are catalyzed by enzymes known as terpenoid synthases terpenoid synthases that catalyze cyclization reactions are also known as terpenoid cyclases. (1,2) The structural complexity of this vast chemical library belies relatively simple biosynthetic roots: head-to-tail coupling reactions of 5-carbon precursors yield linear, achiral C 5 n isoprenoid diphosphates ( n = 1, 2, 3, etc.) ( Figure 1), (3,4) which in turn undergo cyclization reactions to yield a myriad of products typically containing multiple fused rings and stereocenters (some examples are shown in Figure 2). Currently numbering more than 80000 members in a greater family that also includes steroids and carotenoids, the terpenome accounts for nearly one-third of all compounds currently characterized in the Dictionary of Natural Products ( ). Terpenes, also known as terpenoids or isoprenoids, comprise the most chemically and structurally diverse family of natural products. Here, I review key advances in terpenoid cyclase structural and chemical biology, focusing mainly on terpenoid cyclases and related prenyltransferases for which X-ray crystal structures have informed and advanced our understanding of enzyme structure and function. The role of the terpenoid cyclase as a template for catalysis is paramount to its function, and protein engineering can be used to reprogram the cyclization cascade to generate alternative and commercially important products. The past two decades have witnessed structural, functional, and computational studies illuminating the modes of substrate activation that initiate the cyclization cascade, the management and manipulation of high-energy carbocation intermediates that propagate the cyclization cascade, and the chemical strategies that terminate the cyclization cascade. Terpenoid cyclases catalyze the most complex chemical reactions in biology, in that more than half of the substrate carbon atoms undergo changes in bonding and hybridization during a single enzyme-catalyzed cyclization reaction. The year 2017 marks the twentieth anniversary of terpenoid cyclase structural biology: a trio of terpenoid cyclase structures reported together in 1997 were the first to set the foundation for understanding the enzymes largely responsible for the exquisite chemodiversity of more than 80000 terpenoid natural products.
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