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. 2019 Sep 2;20(17):2217-2221.
doi: 10.1002/cbic.201900135. Epub 2019 Jul 22.

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

Sean Lund  1   2 , Taylor Courtney  1   3 , Gavin J Williams  1   4
Affiliations

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

Sean Lund et al. Chembiochem. .

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.

Keywords: hemiterpenes; kinases; phosphorylation; synthetic biology; terpenoids.

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Figures

Figure 1.
Figure 1.
The two-enzyme ‘alcohol dependent hemiterpene’ pathway. (A) Scheme illustrating the role of PhoN and IPK in converting ISO and DMAA to IPP and DMAPP. (B) Active site of wild-type IPK (PDB: 3LKK). Residues surrounding the isopentenyl phosphate ligand (cyan sticks) are shown as green sticks.
Figure 2.
Figure 2.
Substrate promiscuity of wild-type IPK. (A) General synthetic procedure describing the preparation of a panel of alcohol monophosphates (B) Substrate scope of wild-type IPK determined by MS analysis; Blue (✱✱), >50% conversion by MS analysis; Red (✱), <25% conversion; Black, no conversion detected
Figure 3.
Figure 3.
Kinetic characterization of the wild-type IPK-catalysed phosphorylation. (A) Scheme illustrating the coupled enzyme assay. PK, pyruvate kinase; LDH, lactate dehydrogenase; PEP, phospho(enol)pyruvate. (B) Michaelis-Menten curve of wild-type IPK with 4 as the variable substrate.
See this image and copyright information in PMC

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