Biosynthesis of the fungal glyceraldehyde-3-phosphate dehydrogenase inhibitor heptelidic acid and mechanism of self-resistance

Article abstract of DOI:10.1039/d0sc03805a

Overcoming resistance to bioactive small molecules is a significant challenge for health care and agriculture. As a result, efforts to uncover the mechanisms of resistance are essential to the development of new antibiotics, anticancer drugs and pesticides. To study how nature evolves resistance to highly potent natural products, we examined the biosynthesis and mechanism of self-resistance of the fungal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inhibitor heptelidic acid (HA). HA is a nanomolar inhibitor of GADPH through the covalent modification of the active site cysteine thiol. The biosynthetic pathway of HA was elucidated, which uncovered the enzymatic basis of formation of the epoxide warhead. Structure-activity relationship study using biosynthetic intermediates established the importance of the fused lactone ring system in HA. The molecular basis of HA inhibiting human GAPDH was illustrated through the crystal structure ofHs-GAPDH covalently bound with HA. A GAPDH isozyme HepG encoded in the HA cluster was characterized to be less sensitive to HA, and therefore contribute to self-resistance for the producing host. Comparison of the crystal structures of human GAPDH and HepG showed mutations both within and remote to the active site can contribute to resistance of inactivation, which was confirmed through mutagenesis. Due to the critical role GAPDH plays in aerobic glycolysis and other cellular functions, knowledge of HA mode of action and self-resistance mechanism could accelerate the development of improved inhibitors.

Full text of DOI:10.1039/d0sc03805a

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DOI: 10.1039/D0SC03805A
ARTICLE
Biosynthesis of the fungal glyceraldehyde-3-phosphate
dehydrogenase inhibitor heptelidic acid and mechanism of self-
resistance
Received 00th January 20xx,
Accepted 00th January 20xx
Yan Yan, #ad Xin Zang, Cooper S. Jamieson, Hsiao-Ching Lin, Houk, K. N., Jiahai Zhou,* Yi Tang*
#b
c
ae
c
b
ac
DOI: 10.1039/x0xx00000x
Overcoming resistance to bioactive small molecules is a significant challenge for health care and agriculture. As a result,
efforts to uncover the mechanisms of resistance are essential to the development of new antibiotics, anticancer drugs and
pesticides. To study how nature evolves resistance to highly potent natural products, we examined the biosynthesis and
mechanism of self-resistance of the fungal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inhibitor heptelidic acid
(HA). HA is a nanomolar inhibitor of GADPH through the covalent modification of the active site cysteine thiol. The
biosynthetic pathway of HA was elucidated, which uncovered the enzymatic basis of formation of the epoxide warhead.
Structure-activity relationship study using biosynthetic intermediates established the importance of the fused lactone ring
system in HA. The molecular basis of HA inhibiting human GAPDH was illustrated through the crystal structure of Hs-GAPDH
covalently bound with HA. A GAPDH isozyme HepG encoded in HA cluster was characterized to be less sensitive to HA, and
therefore contribute to self-resistance for the producing host. Comparison of the crystal structures of human GAPDH and
HepG showed mutations both within and remote to the active site can contribute to resistance of inactivation, which were
confirmed through mutagenesis. Due to the critical role GAPDH plays in aerobic glycolysis and other cellular functions,
knowledge of HA mode of action and self-resistance mechanism could accelerate the development of improved inhibitors.
how microbial hosts self-protect against biosynthesized, potent
natural products (NPs). Often referred to as the chemical agent
in microbial warfare, NPs are synthesized by microbes including
Introduction
The rapid emergence of resistance to bioactive small molecules
both bacteria and fungi, to target essential housekeeping
enzymes in antagonistic organisms. To protect against the NPs,
the producing hosts have developed different strategies,
including the ingenious use of a mutated and functional version
of the housekeeping enzyme that is insensitive to the NP, and
1
has become a major challenge for health care and agriculture.
Once effective antibiotics, antifungal, anticancer drugs and
pesticides are gradually becoming obsolete due to the rise of
resistant strains that have acquired mutations in the cellular
targets.2 Therefore, uncovering and overcoming the resistance
mechanisms are of paramount importance for civilization. One
approach that can provide insights into how Nature evolves
resistance in essential housekeeping enzymes is by examining
,3
4
, 5
hence self-resistant.
Gene encoding the self-resistance
enzyme is frequently colocalized with the biosynthetic gene
cluster (BGCs) of a NP, and has been used as a predictive tool
for the bioactivity of the NP synthesized by the BGC.6 Clearly,
Nature has evolved self-resistance enzymes from housekeeping
enzymes to become resistant to potent NPs. Understanding the
molecular differences between sensitive and resistant enzymes
of the same catalytic function can therefore provide immense
information on the origins and mechanisms of resistance.
The central metabolism of microorganisms is a frequent
target of NPs. Notably, both bacteria and fungi have evolved
terpenoids to inhibit the highly conserved glyceraldehyde-3-
, 7
a. Department of Chemical and Biomolecular Engineering, University of California
Los Angeles, CA 90095 (USA)
State Key Laboratory of Bio-organic and Natural Products Chemistry, Center for
b.
Excellence in Molecular Synthesis, Shanghai Institute of Organic Chemistry,
University of Chinese Academy of Sciences, Shanghai 200032 (China)
Department of Chemistry and Biochemistry, University of California Los Angeles,
c.
CA 90095 (USA)
d.
Current Address: Key Laboratory of Tropical Marine Bio-resources and Ecology,
Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute
of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road,
Guangzhou 510301, China. Southern Marine Science and Engineering Guangdong
Laboratory (Guangzhou), 1119 Haibin Rd. Nansha District, Guangzhou, 511458,
China
8, 9
phosphate dehydrogenase (GAPDH),
which catalyses the
sixth step of glycolysis to convert D-glyceraldehyde 3-phosphate
10
(
GAP) to D-glycerate 1,3-bisphosphate (1,3-DPG) (Fig. 1A). The
e.
Current Address: Institute of Biological Chemistry, Academia Sinica, Taipei 115,
Taiwan
mechanism of GAPDH catalysis has been intensively studied and
involves a catalytic cysteine in the active site that participates
covalently in the oxidative phosphorylation of GAP with the
#.
These authors contribute equally: Yan Yan and Xin Zang
yitang@ucla.edu, jiahai@mail.sioc.ac.cn
*.
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
+
10-12
concomitant reduction of NAD to NADH.
As an example of
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Figure 1. Biosynthetic gene clusters (BGCs) of fungal and bacterial derived GAPDH inhibitors. A. The reaction mechanism of human GAPDH.
B. BGCs of heptelidic acid from three producing strains (left), and chemical structure of heptelidic acid (1). C. BGC of pentalenolactone (left)
and chemical structure of pentalenolactone (2).
convergent evolution, the fungal-derived heptelidic acid (HA, 1, us to identify key mutations in Hs-GAPDH and HepG that play critical
8
, 13
also known as koningic acid and avocettin) and the bacterial- roles in conferring resistance.
1
4
derived pentalenolactone (2) are both potent GAPDH
inhibitors that use epoxide warheads to covalently target the
1
5, 16
Results
thiolate of the cysteine (Fig. 1B and 1C).
inhibition of human GAPDH (Hs-GAPDH) by HA has been used
The potent
1
7
Characterization of heptelidic acid biosynthetic pathway
to limit glycolytic flux associated with the Warburg physiology
The HA BGC encodes a sesquiterpene synthase (hepA), four
P450s (hepC, hepD, hepE and hepH) and a putative antibiotic
biosynthesis monooxygenase (hepB) (Fig. 1B and Table S1). The
functions of two transcription factors (hepR and hepS) in regulating
HA biosynthesis have been verified. Bioinformatics analysis showed
HA BGC is conserved in other producing strains, including
1
8
19
in human cancer cell lines and in activated immune cells.
2
0-23
While the structures of GADPH from different organisms,
including E. coli, yeast, rat and human, have been solved to
reveal the overall tetrameric fold and the catalytic environment
surrounding the cysteine, there is no structure of GAPDH bound
with a specific covalent inhibitor such as HA to date.
2
9
1
3, 31
Trichoderma virens Gv29-8 and Anthostoma avocetta (Fig. 1B).
The chemical structures of HA and pentalenolactone contain
similar features, such as the exo-epoxide warhead, fused lactone and
α, β-unsaturated carboxylic acid. However, the differences in the ring
systems clearly indicate divergent biosynthetic pathways that start
with the formation of different terpene scaffolds. The biosynthetic
To elucidate the functions of the biosynthetic enzymes, and isolate
potential biosynthetic intermediates, we chose to work with the T.
virens hep BGC. This is due to our ability to observe production of HA
when the strain was grown in potato dextrose broth (PDB) at 28˚C
while shaking at 250 rpm (Fig. 2A). When the culturing condition was
switched to stationary incubation, we observed the appearance of
three new metabolites 3-5 with molecular weights that indicate
these may be biosynthetic intermediates of HA (Fig. 2A). These new
compounds were isolated, and their structure were determined by
NMR (Fig. 2D, Fig. S3 and Table S5). All three compounds are decalin
terpenoids that are less oxidized compared to HA. Based on the
levels of oxidative modifications, we reason the exo-olefin containing
2
4-29
pathway of 2 is well-studied by Cane and co-workers.
During the
course of these work, a GAPDH homolog Gap1, that is 64% identical
to housekeeping enzyme in S. avermitilis, was found in the BGC (Fig.
1
C). This enzyme was shown to be insensitive to 2, and was proposed
2
4
to be the self-resistance enzyme. The BGC of HA was recently
reported from the fungal producer Aspergillus oryzae (Fig. 1B), which
contains a sesquiterpene cyclase and set of cytochrome P450
enzymes. The fungal BGC also contains a GAPDH homolog HepG
that is 72% identical to the housekeeping GAPDH in A. oryzae. The
IC50 of HA towards A. oryzae HepG was ~ 60 times higher compared
to that of the housekeeping GAPDH, implying HepG may also play
a self-resistance role during the biosynthesis of HA. Similarly, two
GAPDH isozymes from Trichoderma koningii, a different producer of
2
9
3
4
is the earliest intermediate of the three, and can be epoxidized to
. Oxidation of the C2 hydroxyl group in 4 gives the ketone 5, which
can be ring-expanded to 1 by Baeyer-Villiger oxidation. These
modifications are proposed to be catalysed by cytochrome P450s
enzymes encoded in the hep BGC, requiring molecular oxygen as the
2
9
3
2, 33
oxidant.
The emergence of 3-5 under stationary culture may
3
0
HA, were cloned and shown to have different sensitivity to HA.
hence be a result of lower aeration of the stationary culture
compared to under shaking conditions.
We then characterized the biosynthetic enzymes in the BGC,
starting with the terpene cyclase HepA. The FLAG-tagged HepA was
obtained upon heterologous expression in Saccharomyces cerevisiae
RC01. In the presence of 1 mM farnesyl pyrophosphate (FPP) and 5
To understand how HA inhibits Hs-GAPDH, and identify
mutations that may increase resistance to HA, we obtained the
crystal structures of Hs-GAPDH covalently modified with HA, as well
as that of HepG. We also delineated the biosynthetic pathway of HA
and used the biosynthetic intermediates to probe the structural-
activity relationship in Hs-GAPDH inhibition. These studies enabled
mM MgCl
2
, HepA was able to convert FPP to 6 as the only product,
2
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With 3 as the substrate, only the yeast strain expressing HepE
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DOI: 10.1039/D0SC03805A
was able to transform the compound into the epoxide 4, and further
oxidize into the ketone 5 (Fig. 2B). Directly supplying 4 led to
transformation by HepE into 5, thereby establishing HepE is a
bifunctional P450 in oxidizing the upper periphery of the terpene
(Fig. 2D). When 5 was supplied to the yeast strains expressing one of
the other P450s, however, the proposed final oxidation of 5 to HA
was not detected. We suspected that formation of HA under in vivo
conditions would inhibit yeast GADPH and arrest metabolism. In
addition, any HA produced would be covalently bound to GADPH and
1
5
preclude detection from the organic extract. Therefore, we
performed the assays using the corresponding yeast microsomal
fractions containing the individual P450s and CPR. We observed
HepD-containing microsomes were able to convert 5 into HA (Fig.
2
C). This result is therefore consistent with previous isotope labelling
experiment, which suggested a Baeyer-Villiger oxidation reaction is
3
5
required to generated the lactone ring of HA. Although P450-
mediated Baeyer-Villiger oxidation has precedence in catalysing
castasterone to brassinolide in biosynthesis of the plant NP
brassinosteroid, HepD represents the first microbial P450 discovered
3
7
to catalyze this reaction. In comparison, formation of the lactone in
is catalysed by a FAD-dependent monooxygenase PntE that is more
2
2
8
commonly associated with Baeyer-Villiger reactions. It’s worth
noting that as in PntE, HepD catalyses migration of the less
substituted methylene from the Criegee intermediate associated
with Baeyer-Villiger oxidation, indicating enzymatic control of
Figure 2. Characterization of heptelidic acid biosynthesis. A.
Fermentation of T. virens (top) in shaking and stationary (bottom)
culturing conditions. B. In vivo bioconversion of 3 and 4 using S.
cerevisiae expressing HepE and cytochrome P450 reductase. The
control strain is S. cerevisiae only expressing cytochrome P450
reductase. C. In vitro biochemical assay of 5 using microsomal
fractions of S. cerevisiae expressing HepD and cytochrome P450
reductase. The control assay is performed using microsome of S.
cerevisiae only expressing cytochrome P450 reductase.
3
8
regioselectivity.
Inhibition kinetics of HA and biosynthetic intermediates
In the first step of GAPDH catalysis, the deprotonated thiolate
of C152 attacks the carbonyl of GAP to form a hemithioacetal
intermediate, which is then oxidized to the thioester via hydride
+
transfer to the cofactor NAD . Following cofactor exchange in which
+
NADH is replaced with a new molecule of NAD , the high energy
which has the identical GC-MS spectrum to germacrene D-4-ol (Fig.
3
4
thioester is then attacked by inorganic phosphate to release 1,3-DPG
S2). The result is consistent with previous isotopically labelled
acetate incorporation experiments, which indicated HA is derived
(Fig. 1A). Upon binding of HA, a proposed attack of the thiolate on
3
5
the exo-epoxide leads to covalent and irreversible inactivation of
from a germacrene-type sesquiterpene precursor. The same
compound 6 was observed in the culturing media of Saccharomyces
cerevisiae expressing HepA (Fig. S2B).
1
5, 16
GADPH.
The structures of both HA and 2 contain a lactone ring
formed by the Baeyer-Villiger reaction, hinting that the ester oxygen
atom plays an important role in binding to GADPH. Furthermore, it is
intriguing that the overall size of HA, with its 6,7-bicyclic ring system
To assign catalytic functions to the P450 enzymes in the hep
BGC, cDNAs of hepC, hepD, hepE, hepH, as well as the T. virens
electron transfer partner cytochrome P450 reductase (CPR) were
obtained. The cytochrome P450s were individually expressed in S.
cerevisiae RC01 together with the CPR, all under the adh2
(
15 carbons), is considerably bulkier than the linear substrate GAP (3
carbons).
To assess the structural-activity-relationship of HA binding to
3
6
GAPDH, we assayed the inhibition parameters of HA, 4 and 5 on Hs-
promoter. Compounds 3-6 were then supplied to the yeast strains
as substrates for biotransformation. After 12 h, the cultures were
extracted with ethyl acetate and subjected to HPLC-MS analysis.
Unexpectedly, none of the strains were able to modify 6 into a more
oxidized substrate, such as 3. Repeated attempts with coexpression
of multiple P450s, addition of other hep genes, and the use of P450-
containing microsomal fractions were also unsuccessful, thereby
precluding the determination of the initial oxidative steps. We
proposed that unidentified or unclustered enzymes may be involved
in modification of 6, or 6 may be an off-pathway shunt product
formed under in vitro and yeast assay conditions.
GAPDH (accession number: NP_001276674.1) using the Kitz-Wilson
3
9
method. Purified Hs-GAPDH was first incubated with the
compound individually at various concentrations (Fig. S4A). This was
followed by a 20-fold dilution of the GAPDH-inhibitor mixture and
initiating the GAPDH reaction via addition of GAP. Reaction progress
was measured by the appearance of NADH with UV at λ = 340 nm. All
three epoxide-bearing compounds showed time-dependent,
irreversible inhibition of Hs-GAPDH. Semilog plots of the fraction of
residual GAPDH activity against the concentration of the inhibitors
showed pseudo-first-order inactivation (Fig. S5). The binding
constant K
I
, the maximum potential rate of covalent bond formation
that indicates
kinact, and the overall inactivation rate constant kinact/K
I
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Table 1. Catalytic and inhibition kinetics and of various GAPDHs towards heptelidic acid and the biosynthetic intermediates. DVaietwa Aarrteiclme Oenalinne
DOI: 10.1039/D0SC03805A
±
standard deviation (SD) from three biologically independent experiments.
+
the rate to generate covalent GAPDH-inhibitor complex, of each NAD -binding domain (residues 1–150 and 314–335) and a C-
+
inhibitor were determined and are shown in Table 1.
terminal catalytic domain (residues 149–313). The N-terminal NAD -
of 40 μM binding domain has an α/β dinucleotide-binding fold, while the C-
of inhibition terminal catalytic domain consists of eight-stranded mixed parallel β-
Hs-GAPDH is rapidly inhibited by HA with a K
I
compared to the K
M
of 240 μM towards GAP. The kinact/K
I
of Hs-GAPDH by 5 is 16-fold lower than HA, with nearly four-fold sheets connected by either short α-helices or turns. A molecule of HA
increase in K
I
and four-fold decrease in kinact. Therefore, the inserted with occupancy of 0.8 and 0.7 in chain O and chain Q, respectively, is
+
lactone oxygen atom in HA has a significant effect on inhibitor observed together with a NAD cofactor in the active sites (Fig. 3A).
binding and subsequent inactivation. A more significant, 1750-fold The exo-epoxide ring of HA is opened by the C152 thiolate to form a
decrease in kinact/K
I
of 4 compared to HA, which corresponds to over thioether linkage, which verifies the mode of action of the inhibitor
+
1
00-fold decrease compare to that of 5, was observed. This indicates (Fig. 3B and C). The conformation of NAD cofactor in Hs-GAPDH
the importance of the C2 ketone group in binding to GAPDH, in bound with HA is identical to the reported holo human liver GAPDH,
addition to serving as a prerequisite functional group for the Baeyer- suggesting that inhibitor binding does not alter the conformation of
+
Villiger oxidation.
We next assayed the properties of the proposed self-resistance
NAD (Fig. S13A).
In addition to the covalent thioether adduct, the oxidized
enzyme HepG 29 in the hep BGC. We cloned and expressed both HepG portion of HA interacts extensively with the two anion recognitions
and the putative housekeeping copy of GAPDH in T. virens (Tv- sites in Hs-GAPDH that binds to inorganic phosphate and the
1
1
i
GAPDH, accession number: XP_013958680.1), which share 73% phosphate group of GAP (Fig.3C and D). The P site is formed by the
sequence identity (Fig. S12). HA showed time-dependent and 209-215 loop (Fig. 3B and D), and in previous GAPDH structures,
irreversible inhibition towards both T. virens enzymes (Fig. S6 and binds inorganic phosphate when adopting conformation A, while
S7), indicating HA can bind and form covalent adduct with the switching to conformation B that can bind the C3 phosphate group
+
21, 41
cysteine residues. However, the overall inhibition rate constants of the thioester after hydride transfer to NAD .
of HepG and Tv-GAPDH are 233 and 100-fold lower than that of Hs-GAPDH-HA complex, the 209-215 loop has 0.175 Å RMSD for 7
of Hs-GAPDH, respectively (Table 1). In the absence of HA, both Cα atoms to conformation A (PDB: 1NQA) and 1.189 Å RMSD for 7 Cα
enzymes exhibited similar kcat/K (with respect to GAP) to that of Hs- atoms to conformation B (PDB: 1DC4), which indicates this loop
In the structure
kinact/K
I
M
GAPDH (Table 1). This indicates the amino acid residue(s) adopts conformation A in binding to HA (Fig. S13B and C). The
responsible for decreased sensitivity of HA in T. virens enzymes may carboxylate of HA forms an anion-dipole interaction with hydroxyl of
be orthogonal to those responsible for the catalytic efficiency of T211 and backbone amide of G212 (Fig. 3B and C), both of which are
1
1
GAPDH (Table 1).
involved in binding to inorganic phosphate. The α,β-unsaturated
double bond conjugated with the carboxylate further lowers the pK
of HA and facilitates the formation of carboxylate anion. The P site
a
Crystal structure of Hs-GAPDH and heptelidic acid complex
s
binds the phosphate group in GAP prior to oxidative thioester
formation, and also after cofactor exchange, to allow inorganic
To understand the mechanism of action of HA, we solved the
crystal structure of HA–bound Hs-GAPDH complex at 1.82 Å
resolution through molecular replacement method (Fig. 3A and
Table S6). Similar to the previously reported human liver GAPDH
crystal structure (PDB: 1ZNQ, RMSD = 0.304 Å for 332 Cα atoms in
subunit O), the complex contains four identical subunits (chain O, P,
Q and R) arranged as homotetramer in an asymmetric unit, in which
phosphate binding in the P
a key interaction in the P
i
site (Fig. 3D). In the HA-bound structure,
site is the hydrogen bond between the
s
ester oxygen atom in the lactone ring in HA and 2’-hydroxyl group of
+
ribose adjacent to the nicotinamide in the NAD cofactor (Fig. 3B and
1
1
C). In addition, the backbone amide of C152 also participates in
hydrogen-bonding to the lactone C2 carbonyl of HA (Fig. 3B and C).
These observed interactions are consistent with the results of
inhibition assays performed with 4 and 5: the seven-membered
+
chain O and chain Q are in holo form and each has a NAD cofactor
4
0
in the active site (Fig. 3A). Each subunit consists of an N-terminal
4
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koningii.30 T177 and the general base H179, which is proposed to
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DOI: 10.1 402 39/D0SC03805A
deprotonate C152 to generate the reactive thiolate, are located on
the same side of the β-sheet although no directly hydrogen bonding
between the two side chains is observed. (Fig. 4C). In HepG, the
T177A substitution results in slight change in orientation of the
imidazole side chain of H179, which could affect the kcat and kinact of
the enzyme. The Hs-GAPDH-T177A mutant was constructed, and the
inhibitory effect of HA against the mutant was measured (Fig. S4D
and S8). As predicted, kinact was decreased by ~ 4 fold, while the kcat
is decreased by 1.7 fold (Table 1). While the K
for the mutant, the K of the mutant towards GAP unexpectedly
decreased by 6.2-fold, resulting in an overall 4-fold increase in kcat/K
Table 1). Hence, the T177A mutation in Hs-GAPDH increases
I
remained unchanged
M
M
(
resistance to HA through enhanced catalytic efficiency towards GAP.
The imidazole side chain of the H179 is also proposed to interact with
the phosphate group in GAP, which may be enhanced by the nearby
2
1
T177A mutation. However, compare to HepG, the Hs-GAPDH-
T177A mutant remains ~ 64-fold more sensitive to HA, which
indicates the change from Thr to Ala is not the primary determinant
of resistance in HepG.
We also identified A232 that is conserved in GAPDH active site
is substituted with serine in HepG. A232 interacts with the
hydrocarbon portion of HA, and replacement with serine could lower
the binding affinity of HA. The next residue F233 of which the side
chain points away from the active site is substituted by valine in
HepG, which may also affect the active site geometry and interaction
with HA (Fig. 4D). Both residues were mutated to give a double
Figure 3. Crystal structure of Hs-GAPDH with HA bound in the
active sites. A. Overview of the Hs-GAPDH homotetramer bound
with HA (PDB: 6M61, 1.82 Å). Chain O is in green, chain P is in cyan,
chain Q is in salmon, chain R is in grey, and HA is shown in yellow.
B. View of the active site in Hs-GAPDH-HA complex. 2Fo-Fc (light
gray mesh, contoured at 1.0 σ) electron density map of HA and
C152 are shown in grey mesh. The distances (Å) between the atoms
involved in HA binding is labelled in orange (hydrogen bond) and
purple (ion-dipole interaction). C. Detailed interactions between
the active site of Hs-GAPDH and inhibitor HA. Hydrogen bond and
charge-dipole interactions are shown with orange and purple
dashed lines, respectively. D. The proposed model of Hs-GAPDH
bound with native substrate GAP.
mutant Hs-GAPDH-A232S-F233V (Fig. S4E and S9). Although the K
towards HA increased by 1.5-fold after mutation, the K towards
GAP is also increased by 2-fold, and the overall inactivation rate
constant kinact/K remained similar. Therefore these two residues are
I
M
I
lactone ring and the C2 carbonyl both provide binding energy to the
HA-GAPDH interactions. Collectively, the carboxylate group mimics
not determinants of resistance in HepG (Table 1).
We next examined if resistance to HA may be increased from
subtle changes in the active site conformation caused by more
remote mutations. The Hs-GAPDH-HA complex structure was
superimposed to HepG and distance between HA and key residues in
the inorganic phosphate in the P
phosphate group in GAP in interaction with the P
i
site and the lactone replaces the
site, and
s
synergistically position the inhibitor in the proper orientation in the
active site for the thiolate attack on the epoxide (Fig. 3C and D).
i s s
P and P sites of HepG were measured (Fig. 4B). In the P site in HepG,
the distance between lactone oxygen of HA and 2’-OH of ribose
moiety of the NAD cofactor is slightly increased from 3.4 Å in Hs-
GAPDH to 3.6 Å (Fig. 3B and 4B). In the P site, the distance between
i
Structure of HepG implies the resistance mechanism at molecular
level
+
While catalytically comparable to Hs-GAPDH, the more resistant carboxylate of HA and side chain hydroxyl of T209 is essentially
HepG has 31-fold increase in K , and 7-fold decrease in kinact towards unchanged (2.7 Å in Hs-GAPDH vs. 2.8 Å in HepG), and the ion-dipole
I
HA. The crystal structure of HepG was determined at 2.06 Å interaction is maintained. The most significant difference is the
resolution (Fig. 4A and Table S6). Each subunit of the tetrameric distance between carboxylate of HA and backbone amide of G210,
+
HepG contains a NAD cofactor, and the overall structure of HepG is which is increased from 3.2 Å in Hs-GAPDH to 3.7 Å in HepG. This
highly similar to Hs-GAPDH, with identity of 66.4% and RMSD of corresponds to a significant disruption to this critical ion-dipole
0
.449 Å for 305 core Cα atoms of chain O. Compared to Hs-GAPDH, interaction between HA and P site (Fig. 3B and 4B). Therefore, we
i
+
the conformation of NAD cofactor, catalytic residues (C150 and hypothesized that weakening the interaction with HA in the P site
i
H177) and anion recognition site residues (T209 and G210) in the may account for increased resistance towards HA.
active site of HepG are nearly all the same (Fig. 4B).
Next, we sought to identify unique amino acid residues in HepG,
The mutation L203A in Hs-GAPDH confers resistance against HA
when introduced into the Hs-GAPDH, could lead to increased
resistance towards inactivation by HA. Guided by sequence
i
Given T209 and G210 in P site are conserved among all GAPDH
alignment and structural comparisons, potential residues in Hs- sequences, we examined if changes in surrounding secondary
GAPDH were mutated. The first candidate residue is T177 in Hs- structures near the 209-215 loop may lead to the observed
GAPDH, which is conserved in nearly all GAPDH sequences except the differences between Hs-GAPDH and HepG. The N-terminus of this
substitution of alanine in HepG and the more resistant copy from T. loop is directly connected to a region defined by residues 180-208,
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Journal Name
View Article Online
DOI: 10.1039/D0SC03805A
Figure 4. Comparing the crystal structure of Hs-GAPDH and HepG. A. Overview of the HepG homotetramer (PDB: 6M5X, 2.10 Å). Chain O
is in purple, chain P is in light green, chain Q is in cyan, and chain R is in pink. B. View of the superimposed active site of Hs-GAPDH (green)
and HepG (purple) in chain O. The carbon backbone of HA is shown in yellow. The distances (Å) between HA and HepG are shown in grey
dashed lines. The residue numbers of HepG are labelled in purple. C. Superimposed HA binding site of Hs-GAPDH and HepG with T177 of
Hs-GAPDH and A175 of HepG shown. D. Superimposed HA binding site of Hs-GAPDH and HepG with S232-F233 in Hs-GAPDH and A230-
V231 in HepG shown. E. Sequence alignment and secondary structures near loop 209-215 (α-helix is labelled with helix, β-sheet is labelled
with arrow, and β-turn is labelled with “T”). The RMSD of corresponding motifs in the black dashed boxes are shown in the table below. F.
Superimposed structures of homotetrameric Hs-GAPDH (green) and HepG (purple). Zoomed in view near loop 209-215 is shown on the
right. The main chain of residues 203-212 in Hs-GAPDH and 201-210 in HepG are shown as ribbons. Residues A201 in HepG and L203 in Hs-
GAPDH from chain R are labelled as A201’ and L203’ respectively.
while the C-terminus is connected to an α-helix (α6)-loop motif mutant is drastically enhanced, as seen in the ~ 25-fold decrease in
(residues 216-228) (Fig. 4E). Superimposing crystal structures of both
K
I
which approaches that of the Hs-GAPDH. Overall, interchanging
Hs-GAPDH-HA complex and HepG shows the 209-215 loop of the P
i
the identity of this residue led to a complete switch of relative
site has RMSD of 0.257 Å for 7 Cα atoms, the C-terminal α-helix-loop sensitivity/resistance between the two enzymes, with the Hs-
motif exhibits RMSD of 0.217 Å for 13 Cα atoms, and the N-terminal GAPDH-L203A mutant now more resistant towards HA than the
region 180-208 shows RMSD of 0.381 Å for 27 Cα atoms (Fig. 4E). This HepG-A201L mutant.
suggests the subtle structural differences of P
i
sites between Hs-
At the secondary and tertiary structure levels, residue L203 in
GAPDH and HepG may result from the altered conformation of the Hs-GAPDH is part of a β-turn region (residues 202-204), which is
N-terminal region. In particular, the Cα atoms of two residues, L203 located on the peripheral of the globular Hs-GAPDH monomer (Fig.
and I181 in Hs-GAPDH showed RMSD deviation of 1.0 Å each to the 4F). At the quaternary structure level, motif 180-208 is located in the
corresponding residues A201 and Y179 in HepG. These distances are centre of the homotetramer (Fig. 4F) and is in close proximity to the
significantly greater than the RMSD of the entire region 180-208 same region from other monomers. The L203 residues from Hs-
(0.381 Å). Interestingly, L203 is well-conserved among primate GAPDH monomers O and R are located close to each other with a Cα
GAPDH, while in other eukaryotic and microbial GAPDHs, the atoms distance of 6.9 Å, and can have steric clashes from certain
position is occupied primarily by alanine (Fig. S12). Therefore, we rotamers of the leucine side chains. On the other hand, residues
propose these two residues, in particular L203, may contribute to the A201 from HepG monomers O and R are separated by Cα distance of
high sensitivity of Hs-GAPDH to HA.
6.5 Å (Fig. 4F), and the smaller side chains of alanine do not result in
The single-point mutants I181Y and L203A of Hs-GAPDH were steric clashes. In Hs-GAPDH, mutation of L203 to alanine relieves the
constructed and purified (Fig. S4F). Whereas the I181Y mutation potential steric interactions, and results in conformation change of
made the enzyme completely inactive, the L203A mutant has the the motif 180-208. As T209 and G210 are directly connected to this
same kcat/K
mutant showed a 16-fold decrease of the overall inhibition rate be relayed to the P
constant kinact/K , contributed from 4-fold increase in K and ~ 4-fold binding interactions with HA as proposed in HepG. As a result, L203A
decrease in kinact (Table 1). To test if the opposite mutation, A201L, in mutation in Hs-GAPDH caused significant increases in K of HA and
M
as the wild type (Fig. S4G and S10). However, the L203A N-terminal region, conformation change to the 180-208 region can
i
site, and lead to disruption of the anion-dipole
I
I
I
HepG can make the enzyme more sensitive to inhibition by HA, the the opposite is observed in the HepG A201L mutant. The
mutant was constructed and assayed (Fig. S4H and S11). While the interchanging mutations in both enzymes do not affect the catalytic
catalytic efficiency of HepG A201L mutant is comparable to the wild properties, which indicate the binding affinities of inorganic
i
type HepG, the mutant is significantly more sensitive towards HA phosphate in both P sites are not compromised.
inhibition with an overall 48-fold increase in inhibition rate constant
I .
kinact/K (Table 1) In particular, the binding affinity of HA towards the
6
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carboxylate intermediate 3, each subsequent biosynthetic
transformation increases the inhibition potency. Modification of 3 to
Discussion
View Article Online
DOI: 10.1039/D0SC03805A
In this study, we investigated biosynthesis of the fungal GAPDH
inhibitor HA and uncovered the self-resistance mechanism in its
fungal producer at molecular level. Given the central role of GAPDH
4
enables covalent modification by the thiolate, while modification
of 4 to 5, then to 1, allows binding of HA to the P site. One may
s
therefore consider these enzymatic transformations to reflect the
evolutionary steps that Nature took to arrive at the end-product HA.
Because we are not able to reconstitute the initial oxidation of 6,
alternative oxidative sequence may possibly be employed in the
biosynthesis of 2, given some fungal P450s have substrate
promiscuity. Although the oxidation steps in biosynthesis of HA differ
from those of pentalenolactone, in which the epoxide formation is
the last step, the closely mirrored biosynthetic pathways between
the two molecules is a stunning example of convergent evolution.
4
3
in metabolism, both bacterial and fungi have evolved terpene NPs
as inhibitors. The potential use of GAPDH inhibitor in cancer and
inflammatory disease treatment has received attention in recent
1
9, 44
years.
Locasale and coworkers demonstrated that targeting the
Warburg Effect by inhibition of GAPDH using HA was effective in
suppressing the growth of highly glycolytic cancer cell lines in vitro
and in orthotopic tumor models.18 By targeting GAPDH in activated
immune cells, Snyder et al. recently showed HA can suppress
glycolysis and immune responses. Besides its role in glycolysis,
GAPDH has been shown to be involved in other cellular processes
such as regulation of apoptosis, transcription activation and vesicle
1
9
While the kinetic parameters (kcat, K
M
) of HepG and Hs-GAPDH
are comparable, the K of HepG towards HA is ~ 30-fold higher and
I
the kinact is ~ 8 fold slower. Comparison of crystal structure of HepG
and Hs-GAPDH revealed both active site and remote residues
contribute to the difference in sensitivity towards HA between Hs-
GAPDH and HepG. The T177A mutation in the active site was shown
4
5-47
transportation.
For example, Ryu and coworkers showed that
GAPDH can bind Rheb and inhibits mTORC1 signaling under low
glucose concentrations, thereby correlating glycolytic flux and
mTORC1 control of cell growth. The dimer and tetramer interfaces
of GAPDH have also been shown to bind the adenine-uridine rich
elements involved in posttranscriptional regulation of mRNA with
submicromolar affinity. Therefore, understanding the mode of
inhibition of GAPDH by HA and molecular basis of resistance can be
useful for improving inhibitor design.
4
8
to selectively decrease the Hs-GAPDH K
M
towards GAP. Both kcat and
kinact are decreased for the mutant, indicating the threonine residue
which is close to the general base H179, influences catalytic turnover.
Endo et al previously isolated two GAPDH variants from T. koningii
and noted the more resistant copy has an alanine at this position as
in HepG, while the more sensitive copy has a threonine as in Hs-
4
9
The multistep mechanism of GAPDH involves the binding of one
5
0
GAPDH. However, two factors point to this mutation not being the
primary determinant of sensitivity displayed by Hs-GAPDH: 1) the K
+
cofactor (NAD ) and two substrates (GAP and P
i
). The order and the
I
locations of substrate binding in the active site have been postulated
of the Hs-GAPDH-T177A mutant towards HA remained unchanged;
and 2) The Tv-GAPDH housekeeping enzyme which is 100-fold more
resistant than Hs-GAPDH, also contains a threonine at the
corresponding position.
1
1, 21, 40, 41
based on numerous structures and biochemical analysis.
+
NAD is bound first, followed by entry of GAP, formation of the
hemithioacetal and oxidation to the thioester. In these steps, the
phosphate of GAP binds to the P
s
site and interact with the 2’-OH of
This led us to examine differences in the P
between the enzymes to identify the large difference in K
of G210 in the P pocket of HepG disrupts the critical anion-dipole
i
and P
s
binding sites
+
NAD . After thioester is formed, the GAP phosphate flips into the P
i
I
. The shift
+
site, which allows the NADH to be exchanged with NAD . The GAP
i
phosphate then flips back to the P
phosphate to bind to the P site and attack the thioester to form 1,3-
DPG. The P site can adopt two conformations to bind either the
s
site to enable inorganic
interaction with the carboxylate of HA, and weakens the overall
binding of HA in the active site by “loosening” one end of the
interactions. We were able to trace the cause of this structural
difference to a flexible loop at the N-terminal of the P loop, and
i
pinpoint L203 as a key determinant of resistance. The importance of
i
i
phosphate from GAP or inorganic phosphate. The overall active site
is therefore spacious and dynamic during catalysis. From the Hs-
GAPDH structure complexed with HA, it is evident that HA binds to
this residue was validated by mutagenesis in both Hs-GAPDH and
HepG, which led to a dramatic reversal in the K values toward HA.
I
both P
interactions of the carboxylate with the P
the P site anchor the inhibitor in this large active site and properly
i
and P
s
site through the furnished functional groups. The
i
site and the lactone with
We speculate that such subtle differences in active sites may be
exploited in the design of selective inhibitors of microbial GAPDH
over Hs-GAPDH in the search for new antibacterial or antifungal
candidates.
Lastly, our data show the housekeeping GAPDH, Tv-GAPDH, is
also significantly less sensitive to HA compared to Hs-GAPDH, and the
s
position the epoxide within attack range from the thiolate. This
rationalizes the relatively larger size of HA compared to GAP, as well
the common structural features shared between HA and
pentalenolactone.
The observed binding mode of HA showcases the ingenuity of
Nature in evolving potent natural product inhibitors for GAPDH. The
biosynthesis of HA and pentalenolactone both start with the
synthesis of multicyclic hydrocarbon scaffolds derived from isoprene
building blocks. A collection of oxidative enzymes then precisely
modify the hydrocarbon at selective positions to install polar
I
kinact/K is only 2.3-fold higher than that of HepG. Because HA is a
covalent and irreversible inhibitor, the host may had evolutionary
pressure to evolve its housekeeping copy to also be more resistant,
while still requiring the resistant copy in HepG. The increased
resistance displayed by HepG provides further protection to the host,
before HA is transported to the outside of the cell, possibly by the
MFS efflux pump HepF. The less sensitivity measured for the
housekeeping GAPDH here is in contrast to previous reports in which
housekeeping GAPDH from A. oryzae and T. koningii are much more
sensitive towards HA. For example, Endo and coworkers showed the
(lactone), charged (carboxylate) and reactive (epoxide)
functionalities to target the various sites in the active site of GAPDH.
Our biotransformation and biochemical assays suggest a possible
oxidative sequence as shown in Figure 2. Starting with the
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Journal Name
k
inact/K
I
of housekeeping GAPDH in T. koningii was ~ 160-fold greater 11.
S. Moniot, S. Bruno, C. Vonrhein, C. DidierjeaVnie,wSA.rBticolescOhnili-ne
Muller, M. Vas, G. Bricogne, G. BraDnOlaIn: 1t0, .A10. 3M9/oDz0zaSCre0l3li8a0n5dA
C. Corbier, J Biol Chem, 2008, 283, 21693-21702.
3
0
than the proposed resistant isozymes. While Koyama and
coworkers showed the IC50 of HA towards the housekeeping GAPDH
is 60-fold lower than the HepG homolog in A. oryzae. These
differences in the level of sensitivity displayed by the housekeeping
GAPDH may therefore reflect the different extent of evolution in
conferring self-resistances towards HA. While Tv-GAPDH may indeed
have evolved to be more resistant to HA, the different methodology
used for the assays in the prior reports can also contribute to the
1
1
2.
3.
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Gao, R. Baldi, M. Mehrmohamadi, M. O. Johnson, N. S.
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Conflicts of interest
There are no conflicts to declare.
19.
M. D. Kornberg, P. Bhargava, P. M. Kim, V. Putluri, A. M.
Snowman, N. Putluri, P. A. Calabresi and S. H. Snyder,
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Acknowledgements
We thank Dr. Yang Hai and Dr. Mengbin Chen for helpful
discussions. This work was supported by NIH (1R35GM118056)
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Journal Name
The structural basis and self-resistance mechanism of fungal
glyceraldehyde-3-phosphate dehydrogenase inhibitor heptelidic
acid are uncovered.
View Article Online
DOI: 10.1039/D0SC03805A
1
0 | J. Name., 2012, 00, 1-3
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