Probing the Substrate Promiscuity of Isopentenyl Phosphate Kinase as a Platform for Hemiterpene Analogue Production

Abstract

Isoprenoids are a large class of natural products with wide‐ranging applications. Synthetic biology approaches to the manufacture of isoprenoids and their new‐to‐nature derivatives are limited due to the provision in nature of just two hemiterpene building blocks for isoprenoid biosynthesis. To address this limitation, artificial chemo‐enzymatic pathways such as the alcohol‐dependent hemiterpene (ADH) pathway serve to leverage consecutive kinases to convert exogenous alcohols into pyrophosphates that could be coupled to downstream isoprenoid biosynthesis. To be successful, each kinase in this pathway should be permissive of a broad range of substrates. For the first time, we have probed the promiscuity of the second enzyme in the ADH pathway—isopentenyl phosphate kinase from Thermoplasma acidophilum—towards a broad range of acceptor monophosphates. Subsequently, we evaluate the suitability of this enzyme to provide unnatural pyrophosphates and provide a critical first step in characterizing the rate‐limiting steps in the artificial ADH pathway.

Modular Chemoenzymatic Synthesis of Terpenes and their Analogues

“…Those substrate affinities that could be measured were all comparable, suggesting that Ec THIM's promiscuity arises from weak non‐specific contacts with its substrates. All products from reactions with Ec THIM were accepted as substrates by Mj IPK; the resulting diphosphates were characterised by 1 H and 31 P NMR and LC‐MS (Supporting Information, Figures S6 and S7 and Table S2) …”

“…All products from reactions with EcTHIM were accepted as substrates by MjIPK; the resulting diphosphates were characterised by 1 H and 31 P NMR and LC-MS (Supporting Information, Figures S6 and S7 and Table S2). [42] Synthesis of (2E,6E)-FDP ( 7) was achieved using a 1:2 ratio of 3 to 4, EcTHIM, MjIPK, and the (2E,6E)-FDP synthase from Geobacillus stearothermophilus (GsFDPS, Scheme 1 a). In the presence of magnesium (5 mm), a necessary cofactor for the enzymes (Supporting Information, Figures S9 and S10), 7 precipitated from solution.…”

Molecular Basis for the Substrate Promiscuity of Isopentenyl Phosphate Kinase from Candidatus methanomethylophilus alvus

“…IPKs themselves are Mg 2+ -dependent enzymes known to be highly efficient catalysts with their natural substrates (k cat /K M ∼ 10 6 M −1 s −1 ). 8,12,31−35 A handful of studies also document the enzymes' substrate specificity toward non-natural alkylmonophosphates (alkyl-Ps), 31,35,36 which is further augmented by engineering studies producing IPK variants capable of diphosphorylating the long-chain isoprene units geranyl and farnesyl monophosphate (GP and FP, respectively). 33,37 These studies confirm that the generalized enzyme class is highly promiscuous toward monophosphorylated substrates, including those bearing chemoselective functionalities (alkynes, azides, and terminal alkenes).…”

Extension of the Terpene Chemical Space: the Very First Biosynthetic Steps

“…All compounds were substrates of the two enzymes with comparable kinetic parameters except GP, found to be a poor substrate. In the context of the newly developed TMP ( vide supra ), Williams′ group [23] extended the repertoire of unnatural substrates of the Thermoplasma acidophilum IPK by testing a library of 17 monophosphates as potential substrates in addition to IP, DMAP, GP, neryl phosphate and farnesyl phosphate (FP). Five monophosphates were not substrates, seven gave less than 25 % conversion and eight gave more than 50 % conversion in addition to IP and DMAP (Scheme 3B).…”