Structural and functional evolution of isopropylmalate dehydrogenases in the leucine and glucosinolate pathways of Arabidopsis thaliana

Article abstract of DOI:10.1074/jbc.M111.262519

The methionine chain-elongation pathway is required for aliphatic glucosinolate biosynthesis in plants and evolved from leucine biosynthesis. In Arabidopsis thaliana, three 3-isopropylmalate dehydrogenases (AtIPMDHs) play key roles in methionine chain-elongation for the synthesis of aliphatic glucosinolates (e.g. AtIPMDH1) and leucine (e.g. AtIPMDH2 and AtIPMDH3). Here we elucidate the molecular basis underlying the metabolic specialization of these enzymes. The 2.25 A° resolution crystal structure of AtIPMDH2 was solved to provide the first detailed molecular architecture of a plant IPMDH. Modeling of 3-isopropylmalate binding in the AtIPMDH2 active site and sequence comparisons of prokaryotic and eukaryotic IPMDH suggest that substitution of one active site residue may lead to altered substrate specificity and metabolic function. Sitedirected mutagenesis of Phe-137 to a leucine in AtIPMDH1 (AtIPMDH1-F137L) reduced activity toward 3-(2′-methylthio)-ethylmalate by 200-fold, but enhanced catalytic efficiency with 3-isopropylmalate to levels observed with AtIPMDH2 and AtIPMDH3. Conversely, the AtIPMDH2-L134F and AtIPMDH3-L133F mutants enhanced catalytic efficiency with 3-(2′-methylthio)ethylmalate ～100-fold and reduced activity for 3-isopropylmalate. Furthermore, the altered in vivo glucosinolate profile of an Arabidopsis ipmdh1 T-DNA knock-out mutant could be restored to wild-type levels by constructs expressing AtIPMDH1, AtIPMDH2-L134F, or AtIPMDH3-L133F, but not by AtIPMDH1-F137L. These results indicate that a single amino acid substitution results in functional divergence of IPMDH in planta to affect substrate specificity and contributes to the evolution of specialized glucosinolate biosynthesis from the ancestral leucine pathway.

Full text of DOI:10.1074/jbc.M111.262519

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 33, pp. 28794–28801, August 19, 2011
© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Structural and Functional Evolution of Isopropylmalate
Dehydrogenases in the Leucine and Glucosinolate Pathways
of Arabidopsis thaliana*
Received for publication, May 18, 2011, and in revised form, June 10, 2011 Published, JBC Papers in Press, June 22, 2011, DOI 10.1074/jbc.M111.262519
Yan He‡, Ashley Galant§1, Qiuying Pang‡, Johanna M. Strul‡, Sherifat F. Balogun§2, Joseph M. Jez§, and Sixue Chen‡3
From the ‡Department of Biology, Genetics Institute, Plant Molecular, and Cellular Biology Program, University of Florida,
Gainesville, Florida 32610 and the §Department of Biology, Washington University, St. Louis, Missouri 63130
The methionine chain-elongation pathway is required for ali-
phatic glucosinolate biosynthesis in plants and evolved from
leucinebiosynthesis.InArabidopsisthaliana,three3-isopropyl-
malate dehydrogenases (AtIPMDHs) play key roles in methio-
nine chain-elongation for the synthesis of aliphatic glucosino-
lates (e.g. AtIPMDH1) and leucine (e.g. AtIPMDH2 and
AtIPMDH3). Here we elucidate the molecular basis underlying
To compensate for their sessile nature, plants evolved mech-
anisms to cope with rapid environmental changes and chal-
lenges (1). The production of specialized metabolites is one of
the important mechanisms for the survival and fitness of plants
(2). The molecular diversity of these specialized compounds
arises from differential modification of common backbone
structures, which necessitates the evolution of homologous
enzymes with varied specificities (1). In plants, glucosinolates
constitute a diverse group of sulfur-containing specialized
metabolites (3–4). Biosynthesis of methionine-derived glucosi-
nolates is initiated by the sequential addition of methylene
groups to produce chain-elongated methionine derivatives via
an iterative three-step chain-elongation process that mimics
the chemistry of leucine synthesis (Fig. 1A).
˚
the metabolic specialization of these enzymes. The 2.25 A reso-
lution crystal structure of AtIPMDH2 was solved to provide the
first detailed molecular architecture of a plant IPMDH. Model-
ing of 3-isopropylmalate binding in the AtIPMDH2 active site
and sequence comparisons of prokaryotic and eukaryotic
IPMDH suggest that substitution of one active site residue may
lead to altered substrate specificity and metabolic function. Site-
directed mutagenesis of Phe-137 to a leucine in AtIPMDH1
(AtIPMDH1-F137L) reduced activity toward 3-(2؅-methylthio)-
ethylmalate by 200-fold, but enhanced catalytic efficiency
with 3-isopropylmalate to levels observed with AtIPMDH2
and AtIPMDH3. Conversely, the AtIPMDH2-L134F and
AtIPMDH3-L133F mutants enhanced catalytic efficiency with
To date, all the genes involved in the methionine chain-elon-
gation process have been identified and characterized in Ara-
bidopsis thaliana (5–14). The different enzymes of the methi-
onine chain-elongation pathway for glucosinolate synthesis
have evolved from leucine synthesis by gene duplication and
functional specification (14–15). For example, four genes in
3-(2؅-methylthio)ethylmalate ϳ100-fold and reduced activity for Arabidopsis encode isopropylmalate synthases (IPMS)4 with
3-isopropylmalate. Furthermore, the altered in vivo glucosinolate two (IPMS1 and IPMS2) serving in leucine biosynthesis and the
profile of an Arabidopsis ipmdh1 T-DNA knock-out mutant could other two genes encoding methylthioalkylmalate (MAM) syn-
be restored to wild-type levels by constructs expressing thases (MAM1 and MAM3), which catalyze the committed
AtIPMDH1, AtIPMDH2-L134F, or AtIPMDH3-L133F, but not by step in methionine chain-elongation (5–6, 16). A recent study
AtIPMDH1-F137L. These results indicate that a single amino acid showed that loss of a C-terminal regulatory domain and a few
substitution results in functional divergence of IPMDH in planta amino acid exchanges can covert IPMS into MAM (14). Spe-
to affect substrate specificity and contributes to the evolution of cialization of the Arabidopsis isopropylmalate isomerases
(IPMI) for different catalytic properties occurs by changes in
the oligomeric composition of these enzymes. IPMI are het-
erodimeric enzymes consisting of a large subunit encoded by a
single gene and a small subunit encoded by one of three genes
(8–9, 12). Metabolic profiling of the large subunit mutant
revealed accumulation of intermediates in both the leucine
pathway and the methionine chain-elongation pathway, dem-
onstrating the dual function of this subunit in both leucine and
glucosinolate biosynthesis (10). In contrast, the small subunits
are specialized to either leucine biosynthesis or methionine
chain-elongation (2, 10, 12). Furthermore, among the six
branched-chain aminotransferases (BCATs) in Arabidopsis,
BCAT4 in the cytosol is specifically involved in glucosinolate
specialized glucosinolate biosynthesis from the ancestral leucine
pathway.
* This work was supported by the National Science Foundation (Grants MCB-
0845162 (to S. C.) and MCB-0904215, to (J. M. J.)). Portions of this research
were carried out at the Argonne National Laboratory Structural Biology
Center at the Advanced Photon Source, a national user facility operated by
the University of Chicago for the Dept. of Energy Office of Biological and
Environmental Research (DE-AC02-06CH11357).
The atomic coordinates and structure factors (code 3R8W) have been deposited
in the Protein Data Bank, Research Collaboratory for Structural Bioinformat-
ics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
1 Supported by an American Society of Plant Biologists Pioneer Hi-Bred Grad-
uate Research Fellowship.
2 Recipient of an National Institutes of Health-MARC uSTAR Undergraduate
Fellowship.
4 The abbreviations used are: IPMS, isopropylmalate synthase (EC 2.3.3.13);
BCAT, branched-chain aminotransferases; IPMDH, isopropylmalate dehy-
drogenase (EC 1.1.1.85); IPMI, isopropylmalate isomerase (EC 4.2.1.33);
MAM, methylthioalkylmalate synthase (EC 2.3.3.-).
3 To whom correspondence should be addressed: Department of Biology,
University of Florida, Gainesville, FL 32610. Tel.: 352-273-8330; E-mail:
schen@ufl.edu.
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Structure and Function of Arabidopsis Isopropylmalate Dehydrogenases
TGGAACT-3Ј; AtIPMDH3-F, 5Ј-dCCATGGCGGCGTTTT-
TGCAAACTAA-3Ј; and AtIPMDH3-R, 5Ј-dCACGTGTTAA-
ACAGGAACTTTGGAG-3Ј. PCR products were firstly cloned
into pSC-B-amp/kan vector using StrataClone Blunt PCR
Cloning Kit (StrataClone), and then sequenced. Correct frag-
ments were subcloned into the AtIPMDH1pro::GUS vector
(11) to generate constructs for each isoform and/or mutant
under control of the AtIPMDH1 promoter. The resulting con-
structs were introduced into Agrobacterium tumefaciens strain
C58C1 followed by transformation into atipmdh1 plants.
Transgenic plants were selected for hygromycin resistance and
homozygous plants used for subsequent analysis.
Glucosinolate Analysis—Rosette leaves of 4-week-old plants
and mature seeds were used for glucosinolate analysis. Glucosi-
nolates were analyzed using HPLC-mass spectrometry, as pre-
FIGURE1. IPMDHpathways. A, overviewofthemethioninechain-elongation
viously described (11, 13).
pathway of aliphatic glucosinolate biosynthesis in A. thaliana. Note that an
elongated 2-oxo acid can serve as a substrate for MAM1 and MAM3 in subse-
quent rounds through the pathway to yield longer side-chain products.
B, IPMDH catalyze the conversion of 3-isopropylmalate to 4-methyl-2-ox-
ovalerate in leucine synthesis and the conversion of 3-(2Ј-methylthio)ethyl-
malate to 5-methylthio-2-oxopentoate in glucosinolate synthesis.
Protein Expression, Purification, Assays, Crystallization, and
Structure Determination—Expression and purification of wild-
type and mutant AtIPMDHs as histidine-tagged proteins for
functional analysis was performed using nickel-affinity chro-
matography, as previously described (11). IPMDH assay condi-
tions using either 3-isopropylmalate or 3-(2Ј-methylthio)ethyl-
malate as a substrate and the analysis of steady-state kinetic
parameters were as previously described (11). All kinetic
parameters were determined by directing fitting data to the
Michaelis-Menten equation in SigmaPlot.
biosynthesis, whereas BCAT3 in the plastids functions in both
amino acid and glucosinolate biosynthesis (7, 9). The molecular
changes that tailor BCAT activity are unclear.
Previously, we showed that A. thaliana isopropylmalate
dehydrogenase 1 (AtIPMDH1) catalyzes the oxidative decar-
boxylation step in the methionine chain-elongation of glucosi-
nolate biosynthesis and that AtIPMDH2 and AtIPMDH3 are
primarily involved in leucine biosynthesis (Fig. 1B) (11, 13).
These studies highlight the functional specialization of these
isoforms, but do not reveal how these activities evolved.
Here we examine the molecular basis for the functional evo-
lution of the IPMDH family in Arabidopsis. The crystal struc-
ture of AtIPMDH2, the first determined for a plant IPMDH,
reveals an active site structure similar to that of the bacterial
enzymes and provides a template for modeling substrate bind-
ing in the active site. Analysis of the AtIPMDH2 structure,
sequence comparisons, and site-directed mutagenesis demon-
strates that a single residue difference in the active site drasti-
cally alters substrate specificity of the AtIPMDH isoforms both
in vitro and in vivo. This work demonstrates the basis for func-
tional divergence of an AtIPMDH isoform for glucosinolate
biosynthesis from those of leucine biosynthesis.
For crystallization of AtIPMDH2, the histidine tag was
removed by thrombin digestion and the protein further puri-
fied using size-exclusion chromatography (17). Crystals of
AtIPMDH2 were obtained in 5 ␮l hanging drops of a 1:1 mix-
ture of protein and crystallization buffer (0.16 M ammonium
sulfate, 0.08 M sodium acetate trihydrate, 20% PEG 4000, 20%
glycerol) at 4 °C over a 0.7 ml reservoir. Data collection (100 K)
was performed at beamline 19-ID at the Advanced Photon
Source Argonne National Laboratory. Diffraction data were
integrated and reduced using HKL3000 (18). The structure of
AtIPMDH2 was solved by molecular replacement performed
with PHASER (19) using the structure of IPMDH from Salmo-
nella typhimurium (20) as a search model. Model building was
performed in COOT (21) and all refinements were performed
with PHENIX (22). Data collection and refinement statistics are
reported in Table 1. The atomic coordinates and structure fac-
tors for AtIPMDH2 have been deposited in the Protein Data
Bank (PDB ID code 3R8W).
EXPERIMENTAL PROCEDURES
Site-directed Mutagenesis and Mutant Protein Analysis—
Plants and Growth—Seeds of A. thaliana ecotype Columbia Site-directed mutagenesis was performed using the
(Col-0) and SALK mutant atipmdh1 (Salk_063423C) were QuikChange PCR method (Stratagene). Bacterial expression
obtained from the Arabidopsis Biological Resource Center vectors for each AtIPMDH (11, 13) were used as templates
(ABRC). Seed germination and plant growth conditions were as with specific oligonucleotide pairs, as follows: AtIPMDH1-
previously described (11, 13).
F137L-F, 5Ј-dCTGAGACCTGAGATGGCTCTGCTTTACC-
Plasmid Construction and Plant Transformation—The full- TTAGAAGAGATCTC-3Ј; AtIPMDH1-F137L-R, 5Ј-dGAGA-
length coding sequences of AtIPMDH1, AtIPMDH2, and TCTCTTCTAAGGTAAAGCAGAGCCATCTCAGGTCT-
AtIPMDH3 were amplified using the Platinum Pfx DNA CAG-3Ј; AtIPMDH2-L133F-F, 5Ј-dCTGAGGCCTGAGAAG-
Polymerase (Invitrogen) with appropriate primer pairs, as GGGTTATTTCAGATTCGTGCAGCTCTC-3Ј; AtIPMDH2-
follows: AtIPMDH1-F, 5Ј-dCCATGGCGGCGTTTTTGCAA- L133F-R, 5Ј-dGAGAGCTGCACGAATCTGAAATAACCCC-
3Ј; AtIMPDH1-R, 5Ј-dCACGTGTTAAACAGTAGCTGG- TTCTCAGGCCTCAG-3Ј; AtIPMDH3-L134F-F, 5Ј-dCTG-
AAC-3Ј; AtIMPDH2-F; 5Ј-dCCATGGCGGCGGCTCTGCA- AGACCTGAGATGGGTCTGTTTAACATTCGAAGAGA-
GACG-3Ј; AtIMPDH2-R, 5Ј-dCACGTGTTAAACAGAAGC- 3Ј; AtIPMDH3-L134F-R, 5Јd-TCTCTTCGAATGTTAAACA-
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Structure and Function of Arabidopsis Isopropylmalate Dehydrogenases
FIGURE 2. Glucosinolate profile analysis. Glucosinolates in seeds (A) and leaves (B) from wild-type, atipmdh1 mutant, and transgenic plants harboring each
AtIPMDH driven by the AtIPMDH1 promoter were analyzed. Levels of aliphatic glucosinolates with varied methylene chain length (C3-C8) are shown. All indole
glucosinolates are combined into a single group. Data are mean Ϯ S.D. (n ϭ 3).
TABLE 1
GACCCATCTCAGGTCTCAG-3Ј (mutated codon in under-
Data collection and refinement statistics
lined and mutation shown in bold). Mutant protein expression,
purification, and assays were performed as described above for
wild-type enzyme.
Crystal
Space group
P21
Cell dimensions
a ϭ 76.81 Å, b ϭ 211.0 Å,
c ϭ 76.90 Å; ␤ ϭ 90.15°
Data Collection
RESULTS
Wavelength
0.979 Å
Resolution range (highest shell)
Reflections (total/unique)
Completeness (highest shell)
ϽI/␴Ͼ (highest shell)
37.0–2.25 Å (2.29–2.25 Å)
433,041/114,991
99.7% (100%)
31.2 (1.7)
Differential Expression and AtIPMDH Metabolic Spe-
cialization—The three IPMDH genes in Arabidopsis have
overlapping, yet distinct expression patterns. AtIPMDH1
(At5g14200) is highly expressed in leaves and roots; AtIPMDH2
(At1g80560) is weakly expressed throughout the plant; and
AtIPMDH3 (At1g31180) is constitutively expressed at high lev-
els in all tissues (11, 13, 23). To test the possible contribution of
differential expression to the specialization of AtIPMDHs, each
gene was placed under control of the native AtIPMDH1 pro-
moter and then transformed into an atipmdh1 mutant line (11).
As shown in Fig. 2, the altered glucosinolate profile of the
atipmdh1 mutant could only be rescued by expression of
AtIPMDH1. In addition, the atipmdh1 glucosinolate pheno-
type could not be rescued if expression was driven using either
AtIPMDH2 or AtIPMDH3 promoter (data not shown). These
results indicate that AtIPMDH1 sequence and differential
expression are important in AtIPMDH specialization.
Rsyma (highest shell)
5.5% (58.4%)
Refinement
c
Rcrystb/Rfree
17.5%/20.7%
10,885
No. of protein atoms
No. of water molecules
459
No. of ligand atoms
64
R.m.s. deviation, bond lengths
R.m.s. deviation, bond angles
Avg. B-factor - protein, water, ligand
Stereochemistry: most favored, allowed,
generously allowed, prohibited
0.007 Å
0.98°
57.0, 60.3, 80.1 Å2
97.1, 2.9, 0, 0%
a Rsym ϭ ⌺͉Ih Ϫ ϽIhϾ͉/⌺Ih, where ϽIhϾ is the average intensity over symmetry.
b Rcryst ϭ ⌺͉Fo Ϫ ϽFcϾ͉/⌺Fo, where summation is over the data used for
refinement.
c Rfree is defined the same as Rcryst, but was calculated using 5% of data excluded
from refinement.
1.3–1.7 Å2 over ϳ350 residues. Because the plant and bacterial
Structure of AtIPMDH2—To determine the molecular archi- IPMDHs share ϳ50% sequence identity, conservation of key
tecture of a plant IPMDH, the 2.25 Å resolution x-ray crystal residues defines the active site region situated in a cleft between
structure of AtIPMDH2 was solved by molecular replacement the two domains of each monomer (Fig. 3A).
(Table 1). There were four molecules in the asymmetric unit
The active site (Fig. 3B) is roughly delineated by ␣8 at the
representing two AtIPMDH2 dimers. The monomers of each bottom and with ␣4 of one monomer and ␣7 of the adjacent
dimer are related by non-crystallographic symmetry. Each monomer forming opposite sides of the site. Within the active
AtIPMDH2 monomer consists of two domains (Fig. 3A). site, all of the residues previously identified in structures of
Domain 1 contains seven ␣-helices (␣1–4 and ␣9–11) and five bacterial IPMDHs in complex with isopropylmalate and Mg2ϩ
␤-strands (␤1–3 and ␤11–12), along with the N and C termini. are also conserved in AtIPMDH2 (24, 25). Because efforts to
Four ␣-helices (␣5–8) and seven ␤-strands (␤4–10) comprise obtain a structure of AtIPMDH2 in complex with ligands did
domain 2. Between the two domains, ␤4 and ␤5 form the inter- not yield crystals, 3-isopropylmalate and Mg2ϩ were manually
domain region. The second domain also serves as the dimeriza- modeled into the plant enzyme based on the positions of these
tion interface with ␤6 and ␤7 of each monomer as part of an ligands observed in the bacterial structures (Fig. 3, B and C) (24,
inter-subunit ␤-sheet and ␣7 and ␣8 of each monomer forming 25). This comparison shows that Asp264* (asterisk denotes a
a four-helix bundle at the dimer interface. The overall structure residue from the adjacent non-crystallographic symmetry
of AtIPMDH2 is similar to those of the IPMDHs from various related monomer), Asp-288, and Asp-292 are positioned to
bacteria, including Salmonella typhimurium and Thermus interact with a catalytically essential divalent metal (i.e. Mg2ϩ
thermophilus (20, 24, 25), with a root mean square deviation of or Mn2ϩ) and that a trio of arginines (Arg-136, Arg-146, and
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Structure and Function of Arabidopsis Isopropylmalate Dehydrogenases
FIGURE 3. Structure of AtIPMDH2. A, ribbon diagrams of the AtIPMDH2 dimer. Monomer A is shown with gold ␣-helices and blue ␤-strands and monomer B
is drawn with rose ␣-helices and green ␤-strands. Secondary structure features are labeled on the A monomer. The right view is rotated 90° to show the two
domains of each monomer. The position of the active site cleft is indicated. B, active site view and model of 3-isopropylmalate (IPM) and divalent metal (M2ϩ).
Side chains of active site residues are shown with those from the adjacent subunit (gray) indicated by an asterisk. The positions of the substrate and metal are
modeled based on the bacterial structures (20, 24, 25). The active site difference among the AtIPMDH isoforms is highlighted in gold. C, schematic of the active
site model. D, sequence comparison of the region including residue 133 (AtIPMDH2 numbering).
Arg-174) is poised to form charge-charge interactions with the
carboxylate groups of the substrate. Residues corresponding to
Leu-132, Leu-133, Tyr-181, Lys-232*, Asn-234*, and Val-235*
form a largely hydrophobic region around the isopropyl group
of the substrate.
FIGURE 4. Proposed mechanism for AtIPMDH. The conversion of 3-isopro-
Although all of these amino acids are invariant in the bacte-
pylmalate to 4-methyl-2-oxovalerate in leucine synthesis and the conversion
of 3-(2Ј-methylthio)ethylmalate to 5-methylthio-2-oxopentoate in glucosi-
nolate synthesis utilize the same chemistry but with different R-groups.
rial and plant IPMDHs involved in leucine biosynthesis, the
side-chain corresponding to Leu-133 is replaced with a pheny-
lalanine in AtIPMDH1 (Fig. 3D), which is the isoform previ-
ously shown to be primarily involved in glucosinolate synthesis single amino acid exchange from the leucine found in
in Arabidopsis (11). Mechanistically, the conversion of 3-isopro- AtIPMDH2 and AtIPMDH3 to the phenylalanine in the active
pylmalate to 4-methyl-2-oxovalerate in leucine synthesis and the site of AtIPMDH1 may contribute to the functional divergence
conversion of 3-malate derivatives (e.g. 3-(2Ј-methylthio)- of this isoform for glucosinolate biosynthesis.
ethylmalate) to 2-oxo acids (e.g. 5-methylthio-2-oxopentoate)
Biochemical Analysis of Wild-type and Mutant AtIPMDHs—
in glucosinolate synthesis likely use a common metal-depen- Previous studies on the AtIPMDHs demonstrate that each iso-
dent reaction (Fig. 4); however, different substrate side chains form accepts 3-isopropylmalate as a substrate (11, 13), but a
of 3-malate derivatives (Fig. 1B) must fit in the plant IPMDH kinetic comparison with a glucosinolate pathway substrate has
active site for production of aliphatic glucosinolates with six not been reported. Using both 3-isopropylmalate and 3-(2Ј-
different chain lengths (C3-C8). Thus, we hypothesize that this methylthio)ethylmalate, the steady-state kinetic parameters for
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Structure and Function of Arabidopsis Isopropylmalate Dehydrogenases
TABLE 2
Kinetic parameters of wild-type and mutant AtIPMDHs
All reactions were performed as described under “Experimental Procedures.” All kcat and Km values are expressed as a mean Ϯ S.E. for an n ϭ 3.
3-Isopropylmalate
3-(2؅-Methylthio)ethylmalate
kcat
min
Km
␮M
k
M
cat/Km
kcat
min
Km
␮M
k
cat/Km
Ϫ1
Ϫ1
Ϫ1
Ϫ1
Ϫ1 s
M
Ϫ1 s
AtIPMDH1
37 Ϯ 4
25.2 Ϯ 2.3
11.4 Ϯ 1.7
10.9 Ϯ 1.3
30.3 Ϯ 2.5
9.2 Ϯ 1.4
24,471
336,257
570,336
20,352
983,696
25,731
51 Ϯ 5
45.3 Ϯ 3.6
323 Ϯ 21
435 Ϯ 32
77.0 Ϯ 8.5
502 Ϯ 35
103 Ϯ 10
18,763
AtIPMDH1-F137L
AtIPMDH2
230 Ϯ 14
373 Ϯ 33
37 Ϯ 5
2.0 Ϯ 0.2
1.0 Ϯ 0.2
22 Ϯ 1
103
38.3
AtIPMDH2-L133F
AtIPMDH3
4,761
33.2
543 Ϯ 36
44 Ϯ 5
1.0 Ϯ 0.1
16 Ϯ 1
AtIPMDH3-L134F
28.5 Ϯ 1.5
2,589
each AtIPMDH were determined (Table 2). Comparison of the (Fig. 6), indicating that the active site substitution impaired
catalytic efficiencies shows that AtIPMDH2 and AtIPMDH3 AtIPMDH1 function for glucosinolate synthesis in vivo. In con-
favor 3-isopropylmalate over 3-(2Ј-methylthio)ethylmalate by trast, the glucosinolate phenotype could be restored to the wild-
14,900- and 29,600-fold, respectively. Moreover, these isoforms type profile by expression of either AtIPMDH2-L133F or
were ϳ20-fold more active with the leucine biosynthesis sub- AtIPMDH3-L134F under the control of AtIPMDH1 native pro-
strate than AtIPMDH1. In comparison, AtIPMDH1 accepts moter (Fig. 6). The in planta findings corroborate the conclu-
both substrates with comparable kcat/Km values, but was ϳ500- sion drawn from the biochemical analysis of recombinant pro-
fold more efficient with the glucosinolate substrate than the teins and provide evidence for the evolution of AtIPMDH1 by
other two isoforms. These catalytic efficiencies agree with the gene duplication and a single critical amino acid substitution.
observed in vivo roles of the AtIPMDH isoforms in glucosino-
DISCUSSION
late and leucine synthesis pathways (11–13).
To investigate the significance of the active site difference in
The evolution of specialized metabolism from primary
the AtIPMDH, a series of point mutants (AtIPMDH1-F137L, metabolism is a common theme across biochemical pathways
AtIPMDH2-L133F, and AtIPMDH3-L134F) were generated. in plants (and microbes). Here we explored the molecular basis
Kinetic analysis of these mutants demonstrates the critical role underlying the divergence of biological function in the IPM-
of this active site change in determining substrate specificity DHs in the leucine and glucosinolate biosynthesis pathways of
(Table 2). In AtIPMDH1, substitution of Phe-137 with a leucine Arabidopsis. Although all three AtIPMDHs accept 3-isopropy-
reduced the kcat/Km of the mutant for 3-(2Ј-methylthio)ethyl- lmalate, AtIPMDH1 is less efficient than the other isoforms (11,
malate to values comparable to those observed for AtIPMDH2 13). Previous work also showed that knock-out mutants of
and AtIPMDH3. This was also accompanied by improved cat- AtIPMDH1 result in reduced levels of leucine and the C4 to C8
alytic efficiency with 3-isopropylmalate, as the AtIPMDH1- aliphatic glucosinolates (11). In contrast, knock-out mutations
F137L mutant was only 2- to 3-fold less efficient with this sub- of the other isoforms did not alter glucosinolate levels but
strate than AtIPMDH2 and AtIPMDH3. The complementary reduced leucine content (11, 13). Interestingly, a double muta-
mutation in either AtIPMDH2 (L133F) or AtIPMDH3 (L134F) tion of AtIPMDH2 and AtIPMDH3 in Arabidopsis plants led to
yields mutant enzymes that were ϳ30-fold less active with defects in pollen and embryo sac development, suggesting that
3-isopropylmalate than the corresponding wild-type proteins, leucine synthesis is essential for gametophyte formation. Using
but still comparable to wild-type AtIPMDH1. Moreover, a combination of structural and functional analysis, this work
AtIPMDH2-L133F and AtIPMDH3-L134F displayed nearly a demonstrates that a single amino acid change in the AtIPMDH
100-fold improvement in activity with 3-(2Ј-methylthio)ethyl- active site leads to functional divergence of these enzymes in
malate as a substrate to kcat/Km values that were 4- and 7-fold leucine synthesis (primary metabolism) and aliphatic glucosi-
less than those observed with AtIPMDH1. In addition, based on nolate synthesis (specialized metabolism).
the currently available plant sequences in GenBankTM
,
Functional specification of AtIPMDHs in leucine and glu-
AtIPMDH1 is the only IPMDH with the unique phenylalanine cosinolate biosynthesis has been observed (11, 13). To evaluate
instead of leucine, which is present in all other species (Fig. 5). if altered expression of AtIPMDH isoforms underlies functional
These results demonstrate the critical role of the residue at specialization, each isoform gene was expressed under control
position 133 (AtIPMDH2 numbering) in the evolution of of the AtIPMDH1 promoter in an atimpdh1 mutant back-
AtIPMDH1 for the methionine chain-elongation reactions of ground. Because the glucosinolate profile in the mutant was
glucosinolate biosynthesis.
rescued only by expression of AtIPMDH1 (Fig. 2), it appears
In Vivo Analysis of AtIPMDH Mutant Function—To test that gene duplication and subsequent mutation to a new func-
whether the amino acid substitution that occurred in tion is the underlying evolutionary mechanism.
AtIPMDH1 contributes to its specific function in vivo,
The three-dimensional structure of AtIPMDH2 (Fig. 3) and
atipmdh1 mutant plants were transformed with each of the functional analysis (Table 2) of the AtIPMDHs provides insight
mutant AtIPMDH genes driven by the AtIPMDH1 promoter. on the specific changes required to alter the metabolic roles of
After isolation of homozygous lines, the glucosinolate profile in these enzymes. A common chemical transformation is required
each mutant was examined. In comparison to the results shown to convert 3-isopropylmalate to 4-methyl-2-oxovalerate in leu-
in Fig. 2, the pronounced glucosinolate phenotype in the cine synthesis and 3-malate derivatives to 2-oxo acids in glu-
atipmdh1 mutant could not be rescued by AtIPMDH1-F133L cosinolate synthesis (Fig. 1). The AtIPMDH active site includes
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Structure and Function of Arabidopsis Isopropylmalate Dehydrogenases
FIGURE 5. Alignment of IPMDH sequences from different plant species. Amino acid residues with high consensus value (90%) are colored in red. The
residues interacting with the malate backbone of the substrates are shaded in yellow and residues interacting with the ␥ moiety of the substrates are
highlighted in light blue. The LF residues in AtIPMDH1 and conserved LL residues in other IPMDH homologs were marked with asterisks. Species abbreviation:
Bn, Brassica napus (rape); Ca, Capsicum annuum (pepper); Gm, Glycine max (soybean); Os, Oryza sativa (rice); Pp, Physcomitrella patens (moss); Pt, Populus
trichocarpa (poplar); Rc, Ricinus communis (castor); Sb, Sorghum bicolor (sorghum); Vv, Vitis vinifera (grape); and Zm; Zea mays (corn).
invariant residues for binding of either Mg2ϩ or Mn2ϩ (Asp- by spontaneous decarboxylation, stabilization of the resulting
288, Asp-292, Asp-264*) and for charge-charge interactions enolate by the metal ion, and protonation to yield the final
with the substrate carboxylate groups (Arg-136, Arg-146, and product.
Arg-174). Likewise, Tyr181 and Lys232*, which are proposed to
Leucine and glucosinolate synthesis requires the same chem-
perform general acid-base chemistry in the reaction mecha- istry, but the AtIPMDH active site must accommodate reac-
nism (26), are conserved. For both 3-isopropylmalate (leucine tants with different side-chains (i.e. isopropyl versus elongated
synthesis) and 3-malate derivatives (glucosinolate synthesis), methionine side-chain groups). The AtIPMDH2 structure and
the overall reaction (Fig. 4) involves oxidation of the alcohol by sequence analysis reveals a single amino acid difference of a
ϩ
deprotonation and hydride transfer to NAD . This is followed leucine (AtIPMDH2 and AtIPMDH3) versus a phenylalanine
AUGUST 19, 2011•VOLUME 286•NUMBER 33
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Structure and Function of Arabidopsis Isopropylmalate Dehydrogenases
FIGURE6. Glucosinolateprofilesofpointmutants. Glucosinolatesinseeds(A)andleaves(B)ofwild-type, atipmdh1mutant, andtransgenicplantsexpressing
AtIPMDH1-F137L, AtIPMDH2-L133F and AtIPMDH3-L134F driven by AtIPMDH1 native promoter were analyzed. Levels of aliphatic glucosinolates with varied
methylene chain length (C3-C8) are shown. All indole glucosinolates are combined into a single group. Data are mean Ϯ S.D. (n ϭ 3).
(AtIMPDH1) in the active site. This difference occurs in the set pinnings for this evolution are only beginning to be understood.
of residues proposed to form the substrate interaction surface For example, the substrate specialization of the heterodimeric
in the bacterial and plant IPMDH (20, 24, 25). Both in vitro and IPMI is determined by which small subunit associates with the
in vivo functional analysis of AtIPMDH1-F137L, AtIPMDH2- large subunit (2, 8, 10, 12). Recently, the changes needed to
L133F, and AtIPMDH3-L134F demonstrates that switching convert IPMS from leucine synthesis into a MAM was demon-
this amino acid in each isoform is sufficient to interconvert strated to involve the loss of a C-terminal regulatory domain
catalytic efficiency (Table 2) and to change the aliphatic glu- responsible for feedback inhibition by leucine and a series of
cosinolate profiles in transgenic plants (Fig. 6). These results amino acid mutations (14). In contrast to large remodeling of
suggest that gene duplication of AtIPMDH followed by muta- protein structure in IPMS and MAM, the substrate specificity
tion of one active site residue in AtIPMDH1 leads to its special- of IPMDH requires one amino acid difference.
ized role for glucosinolate synthesis in Arabidopsis.
Interactions between Arabidopsis and its environment may
Sequence alignment of AtIPMDH homologs in other plant have driven the co-evolution of the pathways needed to synthe-
species revealed that the phenylalanine is not present in other size the core glucosinolate structure and the elongation of the
homologs (Fig. 5), suggesting they may be primarily involved in methionine side-chain. The biosynthesis of the glucosinolates
leucine biosynthesis. As more genomic sequences become has been suggested to have evolved from the prevalent system
available, a broad implication of this amino acid substitution of cyanogenic glucoside biosynthesis (28–30). Evidence for this
can be appreciated. It should be noted that the three includes the wide distribution of cyanogenic glucosides in
AtIPMDHs have overlapping substrate specificity, but with dis- plants and arthropods, and the conservation of cytochrome
tinct preferences. AtIPMDH1 is primarily involved in methio- P450s in the biosynthesis of glucosinolates and cyanogenic glu-
nine chain-elongation of aliphatic glucosinolate biosynthesis. cosides. In addition, metabolic engineering using cytochromes
When AtIPMDH1 is mutated, the functions of AtIPMDH2 and P450 involved in cyanogenic glycoside biosynthesis allows for
AtIPMDH3 in glucosinolate biosynthesis become evident (13). the generation of acyanogenic plants that also display altered
On the other hand, all three AtIPMDHs are involved in leucine glucosinolate profiles (28–31). It is evident that when environ-
biosynthesis, with AtIPMDH2 and AtIPMDH3 exhibiting mental challenges such as insect herbivores present themselves,
dominant roles (11, 13). This study has uncovered the molecu- specialization of enzymes from different pathways contributes
lar basis, i.e. a single amino acid substitution underlying sub- to the evolution of methionine-derived glucosinolates for plant
strate preference and functional divergence of the AtIPMDHs. survival (32–34).
The structure-function analysis of the AtIPMDH provides
In summary, we have determined a key molecular change
insight on the molecular basis for altered function, but it is responsible for altering substrate specificity of the IPMDHs in
unclear how the leucine to phenylalanine mutation allows Arabidopsis and the recruitment of an IPMDH from leucine
AtIPMDH1 to accommodate the growing methionine chain in biosynthesis for the specialized synthesis of glucosinolates.
subsequent iterations of the glucosinolate synthesis reactions Future studies need to explore protein level changes in other
(Fig. 1A). Multiple structures of IPMDH from bacteria indicate glucosinolate enzymes to understand how the entire glucosino-
that the structural features around the active site are flexible late pathway evolved.
and that active site dynamics likely plays a potential role in
Acknowledgment—We thank Bing Chen for technical support.
substrate recognition and catalysis (27). Moreover, the effect of
the longer side-chain on the kinetics of the various glucosino-
late biosynthesis pathway enzymes (i.e. BCAT, MAM, IPMI,
and IPMDH) has not been explored. In Arabidopsis, multiple
lines of evidence strongly support the evolution of methionine
chain-elongation process of glucosinolate biosynthesis from
leucine biosynthesis (5–8, 11); however, the molecular under-
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AUGUST 19, 2011•VOLUME 286•NUMBER 33
JOURNAL OF BIOLOGICAL CHEMISTRY 28801

Metabolism:
Structural and Functional Evolution of
Isopropylmalate Dehydrogenases in the
Leucine and Glucosinolate Pathways of
Arabidopsis thaliana
Yan He, Ashley Galant, Qiuying Pang,
Johanna M. Strul, Sherifat F. Balogun, Joseph
M. Jez and Sixue Chen
J. Biol. Chem. 2011, 286:28794-28801.
doi: 10.1074/jbc.M111.262519 originally published online June 22, 2011
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