Taming the Reactivity of Monoterpene Synthases To Guide Regioselective Product Hydroxylation

Monoterpenoids are industrially important natural products with applications in the flavours, fragrances, fuels and pharmaceutical industries. Most monoterpenoids are produced by plants, but recently two bacterial monoterpene synthases have been identified, including a cineole synthase (bCinS). Unlike plant cineole synthases, bCinS is capable of producing nearly pure cineole from geranyl diphosphate in a complex cyclisation cascade that is tightly controlled. Here we have used a multidisciplinary approach to show that Asn305 controls water attack on the α-terpinyl cation and subsequent cyclisation and deprotonation of the α-terpineol intermediate, key steps in the cyclisation cascade which direct product formation towards cineole. Mutation of Asn305 results in variants that no longer produce α-terpineol or cineole. Molecular dynamics simulations revealed that water coordination is disrupted in all variants tested. Quantum mechanics calculations indicate that Asn305 is most likely a (transient) proton acceptor for the final deprotonation step. Our synergistic approach gives unique insight into how a single residue, Asn305, tames the promiscuous chemistry of monoterpene synthase cyclisation cascades. It does this by tightly controlling the final steps in cineole formation catalysed by bCinS to form a single hydroxylated monoterpene product.     

Protein fold comparison by the alignment of topological strings

Using the definitions of protein folds encoded in a text string,a dynamic programming algorithm was devised to compare theseand identify their largest common substructure and calculatethe distance (in terms of the number of edit operations) thatthis lay from each structure. This provided a metric on whichthe folds were clustered into a ‘phylogenetic’ tree.This construction differs from previous automatic structureclustering algorithms as it has explicit representation of thestructures at ‘ancestral’ branching nodes, evenwhen these have no corresponding known structure. The resultingtree was compared with that compiled by an ‘expert’in the field and while there was broad agreement, differenceswere found that resulted from differing degrees of emphasisbeing placed on the types of operations that can be used totransform structures. Some concluding speculations on the relationshipof such trees to the evolutionary history and folding of theproteins are advanced. Received June 19, 2003; revised September 13, 2003; accepted October 21, 2003