The class I terpenoid cyclase epi-isozizaene synthase (EIZS) utilizes the universal achiral isoprenoid substrate, farnesyl diphosphate, to generate epi-isozizaene as the predominant sesquiterpene cyclization product and at least five minor sesquiterpene products, making EIZS an ideal platform for the exploration of fidelity and promiscuity in a terpenoid cyclization reaction. The hydrophobic active site contour of EIZS serves as a template that enforces a single substrate conformation, and chaperones subsequently formed carbocation intermediates through a well-defined mechanistic sequence. Here, we have used the crystal structure of EIZS as a guide to systematically remold the hydrophobic active site contour in a library of 26 site-specific mutants. Remolded cyclization templates reprogram the reaction cascade not only by reproportioning products generated by the wild-type enzyme but also by generating completely new products of diverse structure. Specifically, we have tripled the overall number of characterized products generated by EIZS. Moreover, we have converted EIZS into six different sesquiterpene synthases: F96A EIZS is an (E)-β-farnesene synthase, F96W EIZS is a zizaene synthase, F95H EIZS is a β-curcumene synthase, F95M EIZS is a β-acoradiene synthase, F198L EIZS is a β-cedrene synthase, and F96V EIZS and W203F EIZS are (Z)-γ-bisabolene synthases. Active site aromatic residues appear to be hot spots for reprogramming the cyclization cascade by manipulating the stability and conformation of critical carbocation intermediates. A majority of mutant enzymes exhibit only relatively modest 2–100-fold losses of catalytic activity, suggesting that residues responsible for triggering substrate ionization readily tolerate mutations deeper in the active site cavity.

Caspases are fundamental to many essential biological processes, including apoptosis, differentiation, and inflammation. Unregulated caspase activity is also implicated in the development and progression of several diseases, such as cancer, neurodegenerative disorders, and sepsis. Unfortunately, it is difficult to determine exactly which caspase(s) of the 11 isoforms that humans express is responsible for specific biological functions. This lack of resolution is primarily due to highly homologous active sites and overlapping substrates. Currently available peptide-based inhibitors and probes are based on specificity garnered from peptide substrate libraries. For example, the canonical tetrapeptide LETD was discovered as the canonical sequence that is optimally recognized by caspase-8; however, LETD-based inhibitors and substrates promiscuously bind to other isoforms with equal affinity, including caspases-3, -6, and -9. In order to mitigate this problem, we report the identification of a new series of compounds that are >100-fold selective for inhibiting the initiator caspases-8 and -9 over the executioner caspases-3, -6, and -7.C aspases are a family of cysteine-dependent aspartatedirected proteases with 11 human isoforms that are traditionally known for their indispensible roles in the initiation (caspases-2, -8, -9, -10) and execution (caspases-3, -6, -7) of apoptosis. 1 Other family members, including caspases-1, -4, and -5, are important regulators of inflammation and induce pyroptotic cell death as a result of microbial infection. 2 Intriguingly, recent reports implicate caspase activity as being critical for a variety of other essential biological processes, such as DNA repair signaling and tumor suppression, 3 skeletal muscle differentiation, 4 B-cell proliferation, 5 dendritic pruning and neuronal plasticity, 6 embryonic stem cell differentiation through Nanog cleavage, 7 nuclear factor kappa-light-chainenhancer of activated B cells (NF-κB) activation, 8 lymphocyte and monocyte differentiation and development, 9 and keratinocyte differentiation and skin barrier formation. 10 The utility of caspase-dependent proteolysis in an array of cellular functions is continually expanding, and additional caspase roles will likely be discovered with the development of highly potent and specific chemical inhibitors and probes.Active caspases have many significant irreversible consequences and as such are stored as inactive proenzymes, or procaspases, inside of the cell. 11 Intriguingly, several small molecules have been identified in high-throughput screens that promote executioner procaspase maturation using in vitro and in vivo models. 12−14 Aberrant caspase activity is implicated in the development and progression of several diseases, such as neurodegenerative disorders, 15 cancer, 16 cardiovascular disease, 17 and sepsis. 18 Moreover, caspases represent potential drug discovery targets for the treatment of these diseases, and it is therefore imperative to elucidate the exact biological roles of each i...

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