Isopentenyl Phosphate Kinases are Ubiquitous and Copy Numbers are Conserved in Plant Genomes

Key message: Isopentenyl phosphate kinase (IPK) is a key enzyme in mevalonate pathway in isoprenoid biosynthesis. We analyzed 37 presumptive IPK sequences from 35 plants. An specific evolution model was found in plant IPKs, which can be used as an new target in studying the plant isoprenoids metabilte. Abstract: Isopentenyl phosphate kinase (IPK) is a recently discovered enzyme played key role in mevalonate pathway in isoprenoid biosynthesis. Here, we showed that IPKs are ubiquitously present in plant genomes. All IPKs previously identified had AAK domain. From 35 plant species with genome assembly data available, we extracted all AAK family members. Using OrthoMCL, we identified a group of 37 sequences in which Arabidopsis IPK was included. Further analysis showed that each peptide sequence in this group has a His residue which is a signature of IPK enzyme, indicating that the genes in this group were IPKs. Not like these in other domains of life which showed spotty distribution over the tree of life, virtually all plant genomes we analyzed here had IPK genes. Further, copy numbers of IPKs were very conserved in that no higher than 2 copies remained in each plant genome. Plant IPKs formed a distinctive clade in phylogenetic tree of plant AAK gene family, and had a phylogenetic topology conformed to that of plant species. The IPKs we identified here would provide new molecular targets for characterization of plant mevalonate pathway, and shed light on biochemistry of plant isoprenoids biosynthesis.

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

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 pathway (ADH) serve to leverage consecutive kinases to convert exogenous alcohols to 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 non-natural pyrophosphates and provide a critical first step in characterizing the rate limiting steps in the artificial ADH pathway.<br>

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

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 pathway (ADH) serve to leverage consecutive kinases to convert exogenous alcohols to 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 non-natural pyrophosphates and provide a critical first step in characterizing the rate limiting steps in the artificial ADH pathway.<br>

Modified mevalonate pathway of the archaeonAeropyrum pernixproceeds viatrans-anhydromevalonate 5-phosphate

The modified mevalonate pathway is believed to be the upstream biosynthetic route for isoprenoids in general archaea. The partially identified pathway has been proposed to explain a mystery surrounding the lack of phosphomevalonate kinase and diphosphomevalonate decarboxylase by the discovery of a conserved enzyme, isopentenyl phosphate kinase. Phosphomevalonate decarboxylase was considered to be the missing link that would fill the vacancy in the pathway between mevalonate 5-phosphate and isopentenyl phosphate. This enzyme was recently discovered from haloarchaea and certain Chroloflexi bacteria, but their enzymes are close homologs of diphosphomevalonate decarboxylase, which are absent in most archaea. In this study, we used comparative genomic analysis to find two enzymes from a hyperthermophilic archaeon,Aeropyrum pernix, that can replace phosphomevalonate decarboxylase. One enzyme, which has been annotated as putative aconitase, catalyzes the dehydration of mevalonate 5-phosphate to form a previously unknown intermediate,trans-anhydromevalonate 5-phosphate. Then, another enzyme belonging to the UbiD-decarboxylase family, which likely requires a UbiX-like partner, converts the intermediate into isopentenyl phosphate. Their activities were confirmed by in vitro assay with recombinant enzymes and were also detected in cell-free extract fromA. pernix. These data distinguish the modified mevalonate pathway ofA. pernixand likely, of the majority of archaea from all known mevalonate pathways, such as the eukaryote-type classical pathway, the haloarchaea-type modified pathway, and another modified pathway recently discovered fromThermoplasma acidophilum.