Selectivity of Fungal Sesquiterpene Synthases: Role of the Active Site's H-1α Loop in Catalysis

Abstract

Sesquiterpene synthases are responsible for the cyclization of farnesyl pyrophosphate into a myriad of structurally diverse compounds with various biological activities. We examine here the role of the conserved active site H-␣1 loop in catalysis in three previously characterized fungal sesquiterpene synthases. The H-␣1 loops of Cop3, Cop4, and Cop6 from Coprinus cinereus were altered by site-directed mutagenesis and the resultant product profiles were analyzed by gas chromatography-mass spectrometry and compared to the wild-type enzymes. In addition, we examine the effect of swapping the H-␣1 loop from the promiscuous enzyme Cop4 with the more selective Cop6 and the effect of acidic or basic conditions on loop mutations in Cop4. Directed mutations of the H-␣1 loop had a marked effect on the product profile of Cop3 and Cop4, while little to no change was shown in Cop6. Swapping of the Cop4 and Cop6 loops with one another was again shown to influence the product profile of Cop4, while the product profile of Cop6 remained identical to the wild-type enzyme. The loop mutations in Cop4 also implicate specific residues responsible for the pH sensitivity of the enzyme. These results affirm the role of the H-␣1 loop in catalysis and provide a potential target to increase the product diversity of terpene synthases.Sesquiterpene synthases catalyze the cyclization of farnesyl pyrophosphate (FPP) to structurally diverse C 15 -hydrocarbons. These enzymes belong to the large group of terpene synthases that convert isoprene pyrophosphate substrates into hundreds of described terpenoid compounds by employing some of the most complex carbon-carbon forming reactions known (8). Many terpenoids are biologically active and are produced by plants, bacteria, and fungi such as, for example, antibiotics, toxins, and pheromones (5, 9).Catalysis in this class of enzymes is dependent on the presence of three Mg 2ϩ ions coordinated by two conserved motifs, an aspartate-rich DDXXD/E and an NSE/DTE motif, flanking the entrance of the active site. This Mg 2ϩ cluster binds the pyrophosphate (PP i ) group of FPP and positions the isoprenyl chain in the hydrophobic substrate binding pocket of the enzyme (10). Substrate binding triggers a conformational change that results in the closure of the active site and concurrent PP i cleavage to generate an initial transoid, allylic carbocation (6,7,35). This carbocation is then transferred along the isoprenyl chain and eventually quenched either by a water molecule or through proton abstraction. The binding pocket determines folding of the isoprenyl chain and chaperones the reactive carbocation intermediates until the final quenching step (20), thereby defining the product profile of a particular sesquiterpene synthase.Crystal structures have been solved for several microbial and plant sesquiterpene synthases (1,7,14,30). All enzymes share the same ␣-helical fold characteristic for ionization-dependent terpene synthases. Plant enzymes possess an additional, catalytically inactive N-terminal domain that h...

Biosynthesis of Terpenoid Natural Products in Fungi

“…3. Biochemical studies as well as structural modeling and mutagenesis provided new insights into their cyclization mechanism as well as their product specificities [21,22]. Cuprenene-synthase Cop6 proved to be a highly product-specific enzyme, while all the other Cop enzymes were less selective; in the case of Cop4, product selectivity could be influenced by reaction conditions as well as site-directed mutagenesis [21,22].…”

Improved selectivity of an engineered multi-product terpene synthase

“…9 These synthases include the α-cuprenene synthase Cop6, and the multi-product terpene synthases Cop3 and Cop4. Though mutation of residues in the Cop6 H-α1 loop caused little change in product selectivity, both Cop4 and Cop3 showed significant changes in product compositions upon mutation.…”

“…For example, mutation to the H-α1 loop of sesquiterpene synthases Cop4 and Cop6 severely impeded the kinetic behavior of these enzymes. 9 In another example, thermostabilization of the sesquiterpene tobacco epi-aristolochene synthase caused lowered activity in the thermostabilized mutant. 8 It is a general problem in terpene synthase engineering that mutations in these enzymes often lead to decreased activity.…”

“…Several groups have recently taken on the challenge of altering and controlling the course of terpene synthase reactions using mutagenesis. [4][5][6][7][8][9][10][11][12] These studies have largely focused on mutations either surrounding the active site or at locations identified in close homologues. This limitation exists because there has been a lack of an efficient system with which to screen the large number of mutants generated via random mutagenesis, many of which may be misfolded.…”

Structure-based virtual screening of hypothetical inhibitors of the enzyme longiborneol synthase—a potential target to reduce Fusarium head blight disease

“…Nevertheless, this difference in the Mg ions binding groups would be critical for the precise mechanism of longiborneol production. A large loop called H-1α has been shown to be important, participating in the lead covering the active site after ligand and Mg 2+ ions binding [62]. In our model, this loop looks disordered, as in the unligated enzyme.…”