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

Article abstract of DOI:10.1002/cbic.201900135

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 buildin

Full text of DOI:10.1002/cbic.201900135

Accepted Article
Title: Probing the substrate promiscuity of isopentenyl phosphate
kinase as a platform for hemiterpene analogue production
Authors: Sean Lund, Taylor Courtney, and Gavin John Williams
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Probing the substrate promiscuity of isopentenyl phosphate
kinase as a platform for hemiterpene analogue production
Sean Lund, [a,b] Taylor Courtney, [a,c] and Gavin J. Williams*[a,d]
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 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.
acidophilum (Figure 1A). By simply providing exogenous
isopentenol (ISO) and dimethylallyl alcohol (DMAA), the ADH
pathway was able to support hemiterpene production sufficient to
drive lycopene production and the prenylation of an aromatic
amino acid via a promiscuous dimethylallyltryptophan synthase
(DMATS).[3] In this way, the short ADH pathway is highly portable
between microbial hosts, it could be easily optimized by enzyme
and metabolic engineering, and also likely reduces the metabolic
burden of the host strain. Furthermore, previous studies have
showed that PhoN displays broad substrate specificity,[7]
suggesting that a variety of non-native and non-natural alcohols
Isoprenoids comprise >80,000 natural products for which
methods to access and diversify their structures are in high
demand given their broad applications as pharmaceuticals,
additives, fragrances, cosmetics, biofuels, and platform chemicals
to produce value-added products.[1] Notably, nature builds all
isoprenoids via the controlled assembly of two hemiterpenes,
isopentenyl
pyrophosphate
(IPP)
and
dimethylallyl
pyrophosphate (DMAPP).[2] Aside from the impressive product
promiscuity and diversity of reactivities catalyzed by terpene
cyclases and P450s, the use of only two building blocks by
isoprenoid metabolism severely restricts the diversity of possible
biosynthetically-derived isoprenoid structures. To address this
need, our group[3] and another[4] have recently demonstrated the
ability of an artificial two-step biosynthetic pathway to produce the
hemiterpenes IPP and DMAPP. Our prototype alcohol dependent
hemiterpene (ADH) pathway uses PhoN, a non-specific acid
phosphatase from Shigella flexneri,[5] in conjunction with IPK,[6] an
isopentenyl phosphate (IP) kinase from Thermoplasma
[a]
Dr. S. Lund, Dr. G.J. Williams
Department of Chemistry
NC State University
2620 Yarbrough Drive, Raleigh, NC 27695 (USA)
E-mail: gjwillia@ncsu.edu
Present address: Amyris, 5885 Hollis St, Suite 100, Emeryville,
California 94608 (USA)
Present address: Department of Chemistry, University of Pittsburgh,
Pittsburgh, PA 15260 (USA)
Comparative Medicine Institute, NC State University, Raleigh, NC
27695 (USA)
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.
[b]
[c]
[d]
Supporting information for this article is given via a link at the end of
the document.
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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.
could be processed by the prototype or engineered ADH pathway
if IPK was also permissive. The substrate specificity of IPK is less
well understood, although natural long chain allylic isoprenoids
are very poor substrates, which has been improved by enzyme
engineering,[8] and two non-natural monophosphates are also
used poorly.[6] Interestingly, the crystal structure of IPK reveals a
hydrophobic pocket for the isopentenyl phosphate alkyl tail that
could also accommodate substrate analogues (Figure 1B).[9]
However, the ability of IPK to utilize a broad array of diverse alkyl
monophosphates has yet to be probed, and the full scope and
utility of the ADH and similar pathways therefore remains
unknown. Accordingly, we set out to construct a panel of diverse
monophosphates to probe the promiscuity of IPK and to begin to
describe and understand its requirements for catalysis.
phosphorylated products (Figure 2A).[10] The panel was designed
to include various alkyl chain lengths (C4-C15), degrees of
branching (e.g. straight chain, a/b-branching), diverse
substituents (e.g. bromo, aryl, and ester functions), and assorted
degrees of unsaturation (alkenes, alkynes, aromatics) in order to
characterize the substrate flexibility of IPK. After isolation of each
alcohol monophosphate and confirmation of the product by high-
resolution mass spectrometry (HRMS) analysis (Supplemental
Table S2), the substrate specificity of the enzyme was initially
assessed in vitro by detecting conversion to the corresponding
pyrophosphate in the presence of ATP by low resolution MS
analysis of the IPK-catalysed reaction mixtures (Figure 2B).
Remarkably, of the 22 monophosphates tested, ions
corresponding to the expected pyrophosphate product were
detected by MS for 17 of them (1-13, 16-19). Only the internal
alkyne 14, ester 15, and the C10/C15 derivatives geranyl
monophosphate (20), neryl monophosphate (21), and farnesyl
monophosphate (22) failed to serve as substrates, as judged by
MS analysis. Monophosphates 1-8, 10, and 12 were the most
robust substrates for IPK (>50% conversion by MS analysis),
9]
A codon optimized IPK gene from T. acidophilum[6,
(Supplemental Table S1) was synthesized and sub-cloned into
pET28a. Following overexpression in E. coli BL21 (DE3), the
enzyme was purified via metal-chelation affinity chromatography.
A panel of diverse monophosphates was synthesized in a single
step from their corresponding alcohols using a previously
reported synthetic procedure that provides
a mixture of
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while 9, 11, 13, 16, 17, 18, and 19 were utilized by IPK more
poorly (<25% conversion by MS analysis).
Table 1. Steady state kinetic parameters of the wild-type IPK-catalysed
Together, this preliminary analysis suggests that the wild-type
IPK displays a broad substrate specificity that is likely limited by
simple steric effects, consistent with a previous study involving
natural isoprenoid monophosphates.[9] In addition, those
monophosphates with structural rigidity (high proportions of sp2
and sp) are also poor substrates for phosphorylation by IPK.
To better understand the substrate specificity of IPK, the
synthesis of the most robust substrates (1-8, 10, 12) was scaled-
up, the monophosphates isolated and the identity of every target
product confirmed by high-resolution mass spectrometry (HRMS),
1H, and 31P NMR spectroscopy (Supporting Information). The
steady state kinetic parameters of IPK were then determined with
each monophosphate. For this purpose, an NADH-coupled
enzyme assay was employed to quantify the consumption of ATP
by IPK-catalysed phosphorylation of each acceptor substrate.[11]
The steady state kinetic parameters kcat and Km were determined
by measuring initial reaction velocities at a fixed concentration of
ATP and variable concentration of the alcohol monophosphate
(Figure 3). The data was fitted to the Michaelis-Menten equation
and the kinetic parameters (kcat, Km, kcat/Km) extracted (Table 1
and Supplemental Figure S1).
phosphorylation of selected alcohol monophosphates.[a]
Substrate [b]
kcat [s-1]
44 ± 1
32 ± 2
12 ± 1
187 ± 4
226 ± 14
92 ± 4
112 ± 6
18 ± 2
34 ± 4
44 ± 4
Km [µM]
28 ± 4
kcat/Km [M-1 s-1]
1.6 x 106
2.3 x 105
2.4 x 105
4.0 x 105
9.8 x 105
1.7 x 105
1.6 x 105
5.5 x 104
5.0 x 104
9.9 x 104
1
2
140 ± 36
52 ± 23
3
4
470 ± 32
231 ± 32
543 ± 73
718 ± 99
322 ± 136
670 ± 218
445 ± 116
5
6
7
8
10
12
[a] Steady-state kinetic parameters were measured by using an NADH-
coupled enzyme assay for product formation, as described in the
Experimental Section. Kinetic parameters (± standard error) were
determined by fitting the data to the Michaelis–Menten equation by using
Prism; [b] See Figure 2B for structures.
consistent with a previous study,[8] some correlation is observed
between overall length of the substrate and catalytic efficiency.
Overall, and consistent with previous reports, longer substrates
are poor substrates. For instance, the kcat/Km values for the series
of C4-C6 olefins decreases in the order 4 > 6 > 8. The C1-
branched olefin is a notable exception¾the conversion of 9 was
too low to be kinetically characterized. Interestingly, a 3-fold
decrease in catalytic efficiency is observed when the p-bond
geometry of 7 is switched from Z to E in substrate 8.
Interestingly, the poor Km of IPK with each non-natural
substrate tested here suggests that any IPK promiscuity towards
monophosphates that are native to E. coli would not manifest in
off-target phosphorylation of crucial metabolic intermediates. This
observation indicates that IPK is a viable candidate for processing
non-natural alcohol monophosphates to the corresponding
pyrophosphate in vivo. The crystal structure of IPK has been used
to rationally expand the specificity of the enzyme towards the
longer chain isoprenoid monophosphates geranyl- and farnesyl
monophosphate.[8, 12] This suggests that the substrate specificity
of IPK can be further manipulated to accept poorly utilized
monophosphates. Furthermore, IPK and various homologues
have been shown to synthesize IPP via two sequential
phosphorylations of isopentenol,[13] albeit poorly, and this activity
has been targeted by enzyme engineering.[13a] Therefore we
predict that wild-type or mutant IPK can also access a variety of
non-natural monophosphates directly from the corresponding
alcohol.
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.
Gratifyingly, the kcat/Km determined for IP (1) was within 10%
of previously reported kinetic constants for the T. acidophilum
IPK.[6] Notably, the kcat of the enzyme with several of the
substrates was higher than that with the natural substrate, 1. For
example, the kcat with 4 and 5 were 4.25-fold and 5.1-fold higher
than that with 1 (Table 1). However, any increase in kcat with a
non-natural substrate is offset by higher Km values for each
substrate. Subsequently, IPK displays a poorer catalytic efficiency
(kcat/Km) with every non-natural substrate tested, as compared to
the natural substrate 1. For example, the best non-natural
substrate 5 is only ~1.6-fold less efficient than 1, as judged by the
kcat/Km, while the worst substrate 10 is ~32-fold less efficient, as
judged by the kcat/Km. Thus, the catalytic efficiencies of IPK with
the non-natural substrates span 20-fold. In addition, and
The broad substrate specificity of IPK revealed herein for the
first time, coupled with the previously established promiscuity of
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the buffer was exchanged with protein storage buffer (50 mM Tris-HCl, 100
mM NaCl, and 20% (v/v) glycerol at pH 8.0). Protein aliquots were flash
frozen with a dry ice isopropanol bath before storage at -80 °C. Protein
purity was confirmed by SDS-PAGE and concentration was determined by
absorbance using a Pierce Bradford Protein Assay kit.
PhoN, enables the production of non-natural isoprenoid-
pyrophosphates via the ADH pathway[3] in E. coli to be
investigated. The kinetic studies described here are a crucial first
step in identifying the kinetic bottlenecks in the ADH pathway. We
anticipate that the ability to access diverse derivatives of IPP and
DMAPP could be coupled to the known promiscuity of
downstream isoprenoid biosynthetic machinery, including
prenyltransferases[14] and terpene cyclases,[15] to expand the
structural diversity of the terpenoids.
General procedure for synthesis of isoprenoid monophosphates:[10]
400 μmol of the neat alcohol substrate was added to a 15 mL falcon tube.
Trichloroacetonitrile (1 mL, 10 mmol) was then added and the mixture was
allowed to incubate at room temperature for 5 min. Bis-triethylammonium
phosphate (TEAP) solution was prepared by slowly adding solution A (25
mL phosphoric acid, 94 mL acetonitrile) to solution B (110 mL triethylamine,
100 mL acetonitrile) to generate a solution that was 38% solution A and
62% solution B. To the mixture of alcohol and trichloroacetonitrile was
added 1 mL of TEAP solution. The mixture was then incubated in a 37 °C
water bath for 5 min before another addition of TEAP was added. A total
of three additions of TEAP solution were added and incubated. The
mixture was then separated by column chromatography using 6:2.5:0.5
iPrOH: conc. NH4OH:H2O with silica as the stationary phase. Prior to
loading the column, the reaction mixture was diluted 20% (v/v) with
chromatography buffer and the resulting precipitate was pelleted by
centrifugation. Generally, each column was eluted with a total of 400 mL
of eluent with a total silica load of 50 mL pre-equilibrated stationary phase
slurry. Fractions of 10 mL (around 24 total) were collected after the yellow
colour of the solvent front disappeared and were analysed using a
Shimadzu single quadrupole LCMS-2020. Those containing the
monophosphorylated compound, [M-H]-, free of di- or tri-phosphorylated
were used to assay IPK activity (see below) and pooled. The pooled
fractions were then concentrated in vacuo to remove isopropanol and
acetonitrile. The concentrated mixture was then filtered using a 0.2 μm
cellulose filter and frozen at -80 °C. After being frozen overnight, the
sample was lyophilized yielding a salt. The diammonium salt was then
characterized and stored frozen as 250 μL 25 mM aliquots.
Experimental Section
General Methods: All plasmids were verified by DNA sequencing.
Purifications of all DNA were performed with kits from BioBasic. Synthetic
oligonucleotides were purchased from IDT (Coralville, IA, USA). All plate
reader assays were performed using a BioTek Hybrid Synergy 4 plate
reader (Winooski, VT, USA). Restriction enzymes were purchased from
New England Biolabs (Ipswich, MA, USA). Polymerase chain reactions
were conducted using Phire Hot Start II DNA Polymerase from
ThermoFisher Scientific (Waltham, MA, USA). Chemicals were purchased
from Sigma Aldrich (St. Louis, MO, USA) and Alfa Aesar (Haverhill, MA,
USA).
Gene cloning: Isopentenyl monophosphate kinase (IPK) from
9]
Thermoplasma acidophilum DSM 1728[6,
was codon-optimized and
synthesized by Genewiz, Inc (see Supplemental Table S1). The ipk gene
was PCR amplified from the synthesized template and sub-cloned into
pET28a using NdeI and XhoI restriction sites to generate an N-terminally
hexa-histidine tagged fusion. Briefly, PCR was performed using Phire Hot
Start II polymerase (ThermoFisher) according to the supplier’s protocol,
purified by agarose gel electrophoresis, digested with NdeI and XhoI, and
purified again. The digested PCR product and similarly treated pET28a
were ligated at room temperature with T4 ligase (New England BioLabs)
according to the supplier’s protocol. The ligation mixture was then
transformed into E. coli DH5α and plated onto LB agar plates containing
50 μg/mL kanamycin. Individual colonies were picked, grown in the
presence of kanamycin, plasmids purified and the ipk gene sequence
verified by DNA sequencing (Genewiz).
Mass spectrometry assay for initial assessment of IPK activity:
Fractions from the synthesis of the monophosphates were concentrated in
vacuo and resuspended in water at 5 mg/mL. Enzymatic reaction mixtures
contained 50 mM Tris (pH 8.0), 2.5 mM MgCl2, 0.05 mM DTT, 1 mM ATP,
40 µL of substrate (1 mg/mL final), and 4.2 µg of enzyme in a total volume
of 200 µL. For each monophosphate, a reaction mixture without enzyme
was setup as a control. Reactions were incubated overnight at 37 °C and
5 µL analysed initially by low-resolution LC-MS for pyrophosphate product
formation. In every case, the pyrophosphate products were
indistinguishable from the chemically synthesized diphosphates (by-
products of the monophosphate syntheses) with respect to retention time
and m/z ([M-H]-). LC-MS experiments were conducted using a Shimadzu
LC-MS 2020 single quadrupole instrument with a Phenomenex Kinetex
UPLC C18 column (2.1 X 50 mm, 2.6 μm particle, 100 Å pores) column.
Separation was achieved using a series of linear gradients developed from
0.1% formic acid in H2O (A) to 0.1% formic acid in acetonitrile (B) at 0.2
mL/min using the following protocol: 0-2.2 min, 95-1% A; 2.21-2.6 min, 1%
A; 2.61-2.62 min, 1-95% A; 2.63-3.5 min, 95% A. Each IPK reaction was
also subjected to HRMS analysis in negative-mode on a Thermo Fisher
Scientific Exactive Plus operating with a heated ESI source connected to
a UV detector with a Phenomenex Kinetex UPLC C18 column (2.1 X 50
mm, 2.6 µm particle, 100 Å pores). 1 μL was injected onto and separated
using a series of linear gradients developed from 20 mM NH4HCO3 in H2O
(A) to 4:1 acetonitrile: H2O (B) at 0.2 mL/min using the following protocol:
0-2 min, 100-80% A; 2-6 min, 80-0% A; 6-7 min, 0% A; 7-7.1 min, 0-100%
A; 7.1-12 min, 100% A.
Expression and purification of IPK: The sequence verified pET28a-IPK
plasmid was transformed into E. coli BL21 (DE3) for protein expression. A
single colony was used to inoculate a 3 mL culture in LB media
supplemented with 50 μg/mL kanamycin. A 1 L culture containing 50
μg/mL kanamycin in LB media was then inoculated with 1 mL of the
overnight culture and grown to an OD600 of ~0.6 at 37 °C with shaking at
300 rpm at which point protein expression was induced by the addition of
1 mM isopropyl β-D-1-thiogalactopyranoside. The temperature of the
incubator-shaker was reduced to 30 °C and the culture incubated for
approximately 18 h. The culture was pelleted at 4,000 rpm for 10 min, the
supernatant was decanted, the cell pellet resuspended in 15 mL of lysis
buffer (100 mM Tris-HCl, 300 mM NaCl, 10% glycerol, pH 8.0) and lysed
by sonication. The lysate was then pelleted at 4,500 rpm for 10 min,
decanted, and the soluble protein was spun down at 15,000 rpm for 1 h.
The resulting soluble fraction was then purified by fast protein liquid
chromatography (FPLC) using nickel-bead column chromatography for the
extraction of His6-tagged proteins. The column was first equilibrated with
wash buffer (50 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 8.0)
prior to loading of the soluble fraction. The soluble fraction was then eluted
with elution buffer (50 mM Tris-HCl, 500 mM NaCl, 200 mM imidazole, pH
8.0) using a gradient of 0% elution buffer 0-7.5 min, 0-50% 7.5-18 min, 50-
100% 18-22 min, 100% 22-27.5 min, and equilibrated with 0% elution
buffer 27.5-35 min. Fractions containing the desired protein were identified
by SDS-PAGE and pooled. The pooled protein was then concentrated
using a 10 kDa molecular weight cut-off filter (Millipore Amicon-Ultra) and
Michaelis-Menten kinetics: Initial reaction velocities were determined by
coupling the consumption of ATP to the depletion of NADH and monitoring
the change in absorbance at 340 nm in a 96-well plate at 30 °C using a
Biotek Synergy 4 plate reader (Winooski, VT).[11] Each 200 µL assay
mixture contained 50 mM Tris-HCl (pH 8.0), 25 mM KCl, 2.5 mM MgCl2,
0.05 mM DTT, 1 mM ATP, 320 µM NADH, 400 µM phosphoenolpyruvate,
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Entry for the Table of Contents
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Sean Lund, Taylor Courtney, & Gavin J.
Williams*
Page No. – Page No.
Probing the substrate promiscuity of
isopentenyl phosphate kinase as a
platform for hemiterpene analogue
production
Isopentenyl phosphate kinase is a key component of an artificial pathway for
hemiterpene production. The specificity of the kinase was probed with a panel of
non-natural analogues, revealing promiscuity towards a variety of alkenyl, alkynyl,
and aromatic monophosphates. This broad substrate specificity sets the stage for
probing the ability of artificial hemiterpene pathways to provide precursors for
isoprenoid biosynthesis.
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