Origin of Product Selectivity in a Prenyl Transfer Reaction from the Same Intermediate: Exploration of Multiple FtmPT1-Catalyzed Prenyl Transfer Pathways

Abstract

FtmPT1 is a fungal indole prenyltransferase that catalyzes the reaction of tryptophan derivatives with dimethylallyl pyrophosphate to form various biologically active compounds. Herein, we describe detailed studies of FtmPT1 catalysis involving dimethylallyl pyrophosphate and Brevianamide F following the native pathway (yielding Tryprostatin B) and an alternate pathway observed in the Gly115Thr mutant of FtmPT1 yielding a novel cyclized product. Importantly, these two products arise from the same intermediate state, meaning that a step other than the cleavage of the dimethylallyl pyrophosphate (DMAPP; C–O) bond is differentiating between the two product reaction channels. From detailed potential of mean force (PMF) and two-dimensional PMF analyses, we conclude that the rate-limiting step is the cleavage of the C–O bond in DMAPP, while the deprotonation/cyclization step determines the final product distribution. Hence, in the case of FtmPT1, the optimization of the necessary catalytic machinery guides the generation of the final product after formation of the intermediate carbocation.

Thermodynamic and Kinetic Control Determine the Sesquiterpene Reaction Pathways Inside Nanocapsules

“…Sesquiterpene synthases catalyze the formation of a variety of sesquiterpenes, and these enzymes have been studied broadly, using both experimental − and computational tools. − However, it is still unclear how these enzymes produce such a variety of products despite their sequence homology and similar fold. Specifically, how the residues in the active site control the stereoselectivity, despite the inherent reactivity of the carbocations, remains a question. − The reaction cascade is initiated by cleavage of the phosphate group from FPP, leading to the formation of a transoid (2 E ,6 E )-farnesyl cation (farnesyl cation) (Figure ). This conformationally constrained structure can undergo 1,10- or 1,11-cyclization, yielding ( E , E )-germacradienyl and ( E , E )-humulyl cations, respectively.…”

Mechanism of Formation of the Nonstandard Product in the Prenyltransferase Reaction of the G115T Mutant of FtmPT1: A Case of Reaction Dynamics Calling the Shots?

“…Hence, these stationary and rotational states were not discussed in our wild-type paper. 12 Relaxation of the Intermediate State. To complete carbocation formation for the C2−C9 reaction pathway requires a relaxation step along the C5−O6 bond distance (which is restrained in the 2D PMF study) prior to examination of the proton transfer/cyclization step.…”

“…To complete carbocation formation for the C2−C9 reaction pathway requires a relaxation step along the C5−O6 bond distance (which is restrained in the 2D PMF study) prior to examination of the proton transfer/cyclization step. Here again, akin to what was done in the wild-type study, 12 we performed an MD PMF calculation to estimate the free energy released when the C5− O6 bond is relaxed. Figure S3 shows the computed profile.…”

“…16 We recently rationalized the observation of Tryprostatin B formation in native FtmPT1 by showing that the ratedetermining step is the C−O bond cleavage step and that the final proton acceptor for the deprotonation step was Glu102, both of which were consistent with available experimental results. 11,12 The different products formed by native and mutant FtmPT1 follow distinct reaction channels. The formation of the "regular" prenylation product, Tryprostatin B, involves carbon−carbon bond formation between C5 of DMAPP (C5@DMAPP) and C8 of the indole ring (C8@indole) of Brevianamide F. The formation of the "reverse" or novel prenylation product involves the formation of a carbon−carbon bond between C2 of DMAPP (C2@DMAPP) and C9 of the indole ring (C9@indole) (see Figure 1).…”

“…In our previous paper, we studied the regioselectivity of the reactions catalyzed by wild-type FtmPT1. 12 Using potential of mean force simulations, we succeeded in describing the reaction mechanisms for the C5−C8 and C2−C9 reaction pathways. We found that both reaction channels gave essentially identical barrier heights for the prenyl transfer process (e.g., cleavage of the DMAPP C−O bond); for the C2−C9 pathway, the final cyclization/deprotonation step was rate-determining, while carbocation formation was rate-limiting for the regular C5−C8 pathway.…”