Figures and data in Discovery of a metabolic alternative to the classical mevalonate pathway

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5 figures and 4 tables

Figures

Figure 1

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The classical and alternative MVA pathways.

Both branches of the MVA pathway begin with acetyl-CoA (and acetoacetyl-CoA) and proceed through a series of enzymatic reactions involving 3-hydroxy-3-methylglutary-CoA Synthase (HMGS), 3-hydroxy-3-methylglutary-CoA Reductase (HMGR, the presumed early rate-limiting step), and mevalonate kinase (MVK) before branching. At the bifurcation, the canonical MVA pathway, highlighted by light blue arrows, guides MVAP (3) through an additional phosphorylation reaction followed by a phosphorylation-dependent decarboxylation carried out by phosphomevalonate kinase (PMK) and diphosphomevalonate decarboxylase (MDD), respectively. The alternative MVA pathway, highlighted with light brown arrows, hypothetically decarboxylates MVAP (3) prior to the phosphorylation reaction carried out by IPK but the former step has not been discovered until now (Grochowski et al., 2006). The enzymes MVK, PMK, MDD, and IPK all consume ATP during catalysis. All enzymes are shown in green type. Statins serve as inhibitors of HMGR as highlighted.

https://doi.org/10.7554/eLife.00672.003

Figure 2

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Phylogenetic distribution of IPK across the three domains of life.

Maximum likelihood tree of selected IPK protein sequences. Eukaryotes are highlighted with blues, selected archaeal clades with grays and a small group of bacteria with purple. The tree is anchored by several bacterial fosfomycin kinases. See Figure 2—source data 1 for an alignment of IPK homologs. See Figure 2—source data 2 for a table of IPK homolog sequences.

https://doi.org/10.7554/eLife.00672.004

Figure 2—source data 1 Alignment of IPKs from the three domains of life.: https://doi.org/10.7554/eLife.00672.005
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Figure 2—source data 2 Additional IPK sequences from an assortment of sequence databases.: https://doi.org/10.7554/eLife.00672.006
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Figure 3

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Fluorescence thermal shift assays of *Roseiflexus castenholzii* MDD-like MPD.

(A) Thermograms for *R. castenholzii* MPD in 100 mM buffer (pH 3.0–3.8 citric acid; pH 4.0–4.8 sodium acetate; pH 5.0–5.8 sodium citrate; pH 6.0–6.8 sodium cacodylate; pH 7.0–7.8 sodium HEPES; pH 8.0–8.8 Tris-HCl; pH 9.0–11.0 CAPSO) colored from red to violet (acidic to alkaline pH depicted in the inset). (B) Negative derivatives of the thermograms (−dF/dT) color-coded as in (A). (C) T_ms for each of the curves show in (A) and (B) plotted as a function of pH. *R. castenholzii* MPD was unfolded from pH 2.2 to 2.8 at 20°C (data now shown).

https://doi.org/10.7554/eLife.00672.014

Figure 4

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In vitro reconstitution and GC-MS analysis of the alternative and classical MVA pathways.

(A) Terminal steps of the MVA pathways shown with enzymes of the alternative (orange) and classical (blue) pathways depicted as arrows. The putative neofunctionalization of MDD to MPD is highlighted by a grey arrow. (B) In vitro assays include either the alternative or the classical enzymes to produce IPP (1) (as shown in panel A) as well as all downstream enzymes, including isopentenyl phosphate isomerase (IPPI) to produce DMAPP (2), farnesyl diphosphate synthase (FPPS) to produce farnesyl diphosphate (FPP, 6) and tobacco 5-*epi*-aristolochene synthase (TEAS) to produce the sesquiterpene product, 5-EA (7). Products are separated by GC and detected by MS ionization and fragmentation. Enzymes used are highlighted in turquoise type. (C) Results of the in vitro reconstitutions of various enzyme combinations. The y-axis of the graph represents combinations of enzymes shown in panel A and panel B and the % conversion to the expected sesquiterpene end product, 5-EA (7) shown as grey bars along the x-axis. Abbreviations of organisms are as follows: Bf = *Branchiostoma floridae* , At = *Arabidopsis thaliana*, Ss = *Sulfolobus solfataricus*, and Rc = *Roseiflexus castenholzii*. Note, RcMPD is colored both orange and blue on the y-axis, depending on whether it is being tested as an MDD (blue) or an MPD (orange).

https://doi.org/10.7554/eLife.00672.015

Figure 5

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Structural comparisons of a bona fide MDD and MPD.

Interactions between the terminal phosphate of 6F-MVAPP and the surrounding amino acid residues from the crystal structure of *S. epidermidis* MDD (PDB ID 3QT7(Barta et al., 2011)) and the 3D model of MPD from *R. castenholzii*. (A) *S. epidermidis* MDD has multiple interactions with the diphosphate of MVAPP (5). Atoms are colored by type with carbon gold. (B) The active site model of *R. castenholzii* MPD lacks many of the key interactions shown by *S. epidermidis* MDD in panel A. Atoms are colored by type with carbon green. (C) Interactions between the monophosphate of modeled 6F-MVAP and the surrounding amino acids in a superposition of the modeled *R. castenholzii* MPD, backbone atoms and carbon colored green, on the crystal structure of *S. epidermidis* MDD, backbone atoms and carbon colored gold. In *R. castenholzii* MPD, two divergent side chains, Arg83 and Lys161, putatively provide additional electrostatic interactions with the single phosphate group of MVAP (3). These amino acid side chains would clash with the second phosphate of MVAPP (5). These models suggest that the predicted active site topology of *R. castenholzii* MPD facilitates substrate recognition of MVAP (3) through complementary charged and polarized hydrogen bonds and excludes MVAPP (5) through steric incompatibility with its second phosphate.

https://doi.org/10.7554/eLife.00672.016

Tables

Table 1

Phylogenetic distribution of IPK in Eukarya

https://doi.org/10.7554/eLife.00672.007

Classification	IPK-bearing species	Species with no IPK
Mammals		35 mammalian genomes
Birds		Gallus gallus
		Taeniopygia guttata
		Meleagris gallopavo
Reptiles	Anolis carolensis
Reptiles	Philodryas olfersii (EST)*
Amphibians	Notophthalmus viridescens (EST)	Xenopus tropicalis
Fish	Callorhinchus milii (partial IPK from draft genome)	Danio rerio
		Oryzias latipes
		Takifugu rubripes
		Tetraodon nigrovidris
		Gasterosteus aculeatus
Invertebrate chordates	Branchiostoma floridae	Ciona intestinalis
Invertebrate chordates	Saccoglossus kowalevskii	Ciona savignyi
Echinoderms (Deuterostomes)	Paracentrotus lividus (EST) (almost complete assembly)
Echinoderms (Deuterostomes)	Strongylocentrotus purpuratus (almost complete prediction)
Arthropods	Homarus americanus (EST)	Drosophila sp. (Cassera et al., 2004)
		Apis mellifera
		Aedes aegypti
		Anopheles gambiae
		Culex quinquefasciatus
		Pediculus humanus
		Ixodes scapularis
		Daphnia pulex
Other bilaterians	Hirudo medicinalis (EST)	Capitella teleta
	Eisenia fetida (EST)	Helobdella robusta
	Lottia gigantea	Schistosoma mansoni
	Aplysia kurodai (EST)	Schistosoma japonicum
	Aplysia californica (partial prediction)	Brugia malayi
		Meloidogyne incognita
		Pristionchus pacificus
Cnidarians	Acropora palmate (EST)	Hydra magnapilliata
	Montastraea faveolata (EST)
	Nematostella vectensis (predicted)
Other early metazoans	Trichoplax adherens	Amphimedon queenslandica
Pre-metazoans (within Holozoa)		Monosiga brevicollis
		Salpingoeca rosetta
		Capsaspora owczarzaki
Fungi	Spizellomyces punctatus	34 fungal genomes
	Rhizopus oryzae
	Mucor circinelloides
	Epichloe festucae (EST)‡
Green plants	all plants, including:
	Selaginella moellendorffii
	Physcomitrella patens
	Ostreococcus tauri
	Ostreococcus lucimarinus
	Micromonas pusilla
	Chlamydomonas reinhardtii
	Volvox carteri
Amoebozoa	Dictyostelium discoideum
	Polysphondylium pallidum
	Entamoeba hislolytica§
	Entamoeba invadens§
	Entamoeba dispar§
Alveolata	Perkinsus marinus	Plasmodium sp. (Lynen, 1967)
		Cryptosporidium sp. (Hedden and Kamiya, 1997)
		Tetrahymena thermophila
		Paramecium tetraurelia
		Ichthyophthirius multifilis
Diatoms	Thalassiosira pseudonana
Diatoms	Phaeodactylum tricornutum
Kinetoplastida		Trypanosoma sp. (Chew and Bryant, 2007)
Kinetoplastida		Leishmania sp. (Chew and Bryant, 2007)
Excavates		Giardia lamblia
Excavates		Trichomonas vaginalis
Others	Malawimonas jakobiformis (EST)	Naegleria gruberi
Others	Alexandrium tamarense	Naegleria gruberi

*

EST stands for expressed sequence tag. All other IPKs were found in draft or complete genomes.
‡

Since IPK was not found in related fungal genomes, this may be a case of horizontal transfer or even EST library contamination.
§

Greater similarity to IPK from a clade of archaea than to eukaryotes.

Table 2

Conserved residues in IPK

https://doi.org/10.7554/eLife.00672.008

Highly-conserved residues*	Partially conserved residues
K6, G8, G9, K15, G54, H60, P140, G144, D213, T215, G216	K221†, G253‡, T254‡

*

Numbering is in accordance with IPK from M. jannaschii.
†

Conserved in nearly all species except one possibly due to a gene prediction error.
‡

May be invariant, but mis-predicted in several sequences.

Table 3

Steady-state kinetic constants for enzymes of the MVA pathway*

https://doi.org/10.7554/eLife.00672.009

Organism	gi number	Enzyme	Substrate	K_M (μM)	k_cat (s⁻¹)	K_i (μM)	k_cat/K_M (s⁻¹μM⁻¹)	R² (†)
M. jannaschii	15668214	IPK	IP	4.3 (± 0.6)‡	1.46 (± 0.03)	–§	0.34 (± 0.05)	0.90
M. maripaludis	132664414	IPK	IP	21.4 (± 4.3)	15.2 (± 1.4)	877 (± 550)	0.71 (± 0.16)	0.99
S. solfataricus	15897030	IPK	IP	23.6 (± 4.8)	0.91 (± 0.05)	–	0.04 (± 0.01)	0.92
	15899698	PMK	MVAP	23.1 (± 3.8)	1.98 (± 0.10)	5500 (± 2200)	0.09 (± 0.01)	0.98
R. castenholzii	156743980	IPK	IP	4.3 (± 0.7)	1.70 (± 0.04)	–	0.40 (± 0.06)	0.96
	156740939	MPD#	MVAP	152 (± 38)	1.7 (± 0.1)	–	0.011 (± 0.003)	0.98
	156740939	MDD	MVAPP	ND#	ND	ND	ND	ND
B. floridae	NA¶	IPK	IP	13.3 (± 2.0)	27.2 (± 1.2)	2820 (± 1700)	2.05 (± 0.32)	0.98
	260829481	PMK	MVAP	38.0 (± 7.3)	12.6 (± 0.4)	–	0.33 (± 0.06)	0.93
	260794527	MDD	MVAPP	15.1 (± 3.7)	1.36 (± 0.09)	–	0.09 (± 0.02)	0.89
A. thaliana	22329798	IPK	IP	0.79 (± 0.35)	1.9 (± 0.2)	522 (± 381)	2.4 (± 1.1)	0.95
	15222502	PMK	MVAP	11.8 (± 2.0)	20.9 (± 0.7)	–	1.77 (± 0.31)	0.97
	15224931	MDD	MVAPP	15.7 (± 5.0)	2.02 (± 0.13)	–	0.13 (± 0.04)	0.89
T. adhaerens	195996013	IPK	IP	3.1 (± 1.6)	2.4 (± 0.2)	–	0.77 (0.40)	0.79

*

See Table 3—source data 1, Table 3—source data 2, Table 3—source data 3, and Table 3—source data 4 for Michaelis-Menten fitted kinetic curves for each enzyme.
†

R2 represents goodness of fit for the kinetic curve to the experimental data. R2 = 1.0 − (SS_reg/SS_tot), where SS_reg = sum of squares, SS_tot = sum of squares of the distances between each point and a horizontal line passing through the average of all y values.
‡

Values in parentheses represent standard error (or propagation of error) for each calculated kinetic constant.
§

K_i constant was not calculated or was not applicable.
#

Not detected.
¶

B. floridae IPK sequence was predicted from scaffold_167 of the genome assembly (Putnam et al., 2008) using genewise and manual annotation.

Table 3—source data 1 Steady-state kinetic plots for IPKs listed above each curve.: https://doi.org/10.7554/eLife.00672.010
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Table 3—source data 2 Steady-state kinetic plots for PMKs listed above each curve.: https://doi.org/10.7554/eLife.00672.011
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Table 3—source data 3 Steady-state kinetic plots for MDDs listed above each curve.: https://doi.org/10.7554/eLife.00672.012
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Table 3—source data 4 Stead-state kinetic plots for MPD from R. castenholzii.: https://doi.org/10.7554/eLife.00672.013
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Table 4

Active site phosphate-binding residues identified in MDDs across Archaea, Bacteria and Eukarya*

https://doi.org/10.7554/eLife.00672.017

MDD-like active site residues
S. epidermidis	Tyr18	Lys21	Ile27	Ser139	Ser141	Ser192
Bacteria	Tyr	Lys	X†	Ser	Ser	Ser
Sulfolobales	Tyr	Lys	Asn	Ser	Ser	Ser
Eukarya	Tyr	Lys	Ile/Asn	Ser	Ser	Ser
MPD-like active site residues
R.castenholzii	Tyr74	Leu77	Arg83	Ala201	Ser203	Thr255
Haloarchaea/Thermoplasmatales	Tyr /Phe	Met/Tyr	Arg	Ser	Ser	Polar‡
Chloroflexi	Tyr	Leu	Arg/Thr	Ala	Ser	Thr