Identifying and manipulating structural determinates linking catalytic specificities in terpene synthases

Abstract

Terpene synthases are a mechanistically intriguing family of enzymes that catalyze complex, multistep reactions that are capable of generating hundreds of structurally diverse hydrocarbon and oxygenated scaffolds of biological and commercial importance. Interestingly, distantly related terpene synthases from fungi to plants all contain an invariant three-dimensional fold, and molecular comparisons of their active sites indicate that they are enriched with relatively inert amino acid residues that do not react directly with the reaction intermediates. Therefore, catalytic specificity appears to rely on the contour and dynamics of the active site created by the positioning of amino acid backbones and side chains on this catalytic surface and by supporting layers of residues surrounding the synthase active site cavity. Despite the high degree of structural relatedness among terpene synthases, previous studies suggest that no clear relationship between phylogenic organization and catalytic specificities is easily deciphered. We now report on the reciprocal interconversion of catalytic specificities between two distinct yet evolutionarily related terpene synthases based on the systematic identification and mutational replacement of variable residues within and surrounding the active site. Furthermore, we uncover previously undocumented biosynthetic activity during the interconversion, activity that could have been present in a common ancestor of these two highly related synthases. These results provide a simplified means for mapping structural features that are responsible for functional attributes and a strategy for identifying residues that differentiate divergent biosynthetic properties in phylogenetically related terpene synthases.phylogenetic relationships ͉ rational design ͉ sesquiterpene ͉ structure-function T erpenes comprise the most diverse collection of natural products known. Terpene hydrocarbon scaffolds are generated by the action of mono-, sesqui-, and diterpene synthases that catalyze multistep reactions with diphosphorylated substrates of 10 (geranyl diphosphate), 15 [farnesyl diphosphate (FPP)] or 20 (geranylgeranyl diphosphate) carbons (1). The reactions catalyzed by terpene synthases are unparalleled relative to other classes of enzymes because they often consist of a series of stereochemically complex steps. These reactions include ionization of the diphosphate substituent creating an acyclic and reactive carbocation intermediate. In some cases, the isomerization of the all-trans substrate configuration to a cis-trans isomer also occurs before subsequent reactions. Additional steps add increasing complexity and include regio-and stereospecific formation of single or multiple rings, proton eliminations to form double bonds, water quenching of carbocations to create terpene alcohols, and stereospecific hydride, methyl, and methylene migrations. Equally intriguing, the three-dimensional structure of terpene synthases from fungi to plants is highly conserved, including a ''terpene fold'' composed lar...

Conformational analysis of (+)-germacrene A by variable-temperature NMR and NOE spectroscopy

“…As part of our continuing effort to dissect the catalytic features of terpene synthases by molecular comparisons between distinct synthases, 40 we carried out a bioinformatic screen of the lettuce genomic database (http://compositdb.ucdavis.edu/database) and uncovered a putative, but unique terpene synthase gene. While the lettuce gene showed sequence homology to 5-epi-aristolochene synthase, it also showed similarity to several other wellcharacterized terpene synthases including germacrene synthases isolated from chicory (Cichorium intybus), 18 lettuce (Lattuca sativa), 20 and goldenrod (Solidago canadensis).…”

Rational Conversion of Substrate and Product Specificity in a Salvia Monoterpene Synthase: Structural Insights into the Evolution of Terpene Synthase Function

“…Two successful attempts have recently been made to rationally dictate the products formed by a sesquiterpene synthase. Greenhagen et al (2006) applied a concentric contact mapping approach to identify second-tier residues that would enable the conversion of TEAS to HPS. Their work highlighted the importance of outer-tier residues in the structure of the active site and the difficulties involved when applying rational approaches to enzymes whose scaffold might, to some extent, have been affected by evolution.…”

“…Despite high sequence homology (82%), there were >50 residues in the C-terminal region that could have been potential targets for mutagenesis and no strong rationale for eliminating unimportant or essentially conserved amino acids from the selection. A further degree of complexity came from a recent report by Greenhagen et al (2006) on a pair of sesquiterpene synthases. Tobacco (Nicotiana tabacum) 5-epi-aristolochene synthase (TEAS) and Hyoscyamus muticus premnaspirodiene synthase (HPS) are two plant sesquiterpene synthases that share 72% amino acid identity.…”

The Primary Diterpene Synthase Products of Picea abies Levopimaradiene/Abietadiene Synthase (PaLAS) Are Epimers of a Thermally Unstable Diterpenol

“…Our existing understanding of the mechanism of this and other terpene synthases would have predicted that mutations in the active site were very likely to change the proportion of products of multi-product enzymes (11)(12)(13)(14)(15)(16)(17)(18). However, we did notice anecdotally that there was a significant change in the ratio of these PaLAS-like products that depended upon the conditions and temperature of the GC-MS injector and column.…”