Coupling between D-3-phosphoglycerate dehydrogenase and D-2-hydroxyglutarate dehydrogenase drives bacterial L-serine synthesis

Article abstract of DOI:10.1073/pnas.1619034114

L-Serine biosynthesis, a crucial metabolic process in most domains of life, is initiated by D-3-phosphoglycerate (D-3-PG) dehydrogenation, a thermodynamically unfavorable reaction catalyzed by D-3-PG dehydrogenase (SerA). D-2-Hydroxyglutarate (D-2-HG) is traditionally viewed as an abnormal metabolite associated with cancer and neurometabolic disorders. Here, we reveal that bacterial anabolism and catabolism of D-2-HG are involved in L-serine biosynthesis in Pseudomonas stutzeri A1501 and Pseudomonas aeruginosa PAO1. SerA catalyzes the stereospecific reduction of 2-ketoglutarate (2-KG) to D-2-HG, responsible for the major production of D-2-HG in vivo. SerA combines the energetically favorable reaction of D-2-HG production to overcome the thermodynamic barrier of D-3-PG dehydrogenation. We identified a bacterial D-2-HG dehydrogenase (D2HGDH), a flavin adenine dinucleotide (FAD)-dependent enzyme, that converts D-2-HG back to 2-KG. Electron transfer flavoprotein (ETF) and ETF-ubiquinone oxidoreductase (ETFQO) are also essential in D-2-HG metabolism through their capacity to transfer electrons from D2HGDH. Furthermore, while the mutant with D2HGDH deletion displayed decreased growth, the defect was rescued by adding L-serine, suggesting that the D2HGDH is functionally tied to L-serine synthesis. Substantial flux flows through D-2-HG, being produced by SerA and removed by D2HGDH, ETF, and ETFQO, maintaining D-2-HG homeostasis. Overall, our results uncover that D-2-HG–mediated coupling between SerA and D2HGDH drives bacterial L-serine synthesis.

Full text of DOI:10.1073/pnas.1619034114

Coupling between D-3-phosphoglycerate dehydrogenase
and D-2-hydroxyglutarate dehydrogenase drives
bacterial L-serine synthesis
Wen Zhanga,b, Manman Zhanga, Chao Gaoa,1, Yipeng Zhanga, Yongsheng Gea, Shiting Guoa, Xiaoting Guoa,
Zikang Zhouc, Qiuyuan Liua, Yingxin Zhanga, Cuiqing Maa, Fei Taoc, and Ping Xuc,1
aState Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People’s Republic of China; bCenter for Gene and Immunotherapy, The
Second Hospital of Shandong University, Jinan 250033, People’s Republic of China; and cState Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong
University, Shanghai 200240, People’s Republic of China
Edited by Joshua D. Rabinowitz, Princeton University, Princeton, NJ, and accepted by Editorial Board Member Gregory A. Petsko July 26, 2017 (received for
review November 17, 2016)
L-Serine biosynthesis, a crucial metabolic process in most domains
of life, is initiated by D-3-phosphoglycerate (D-3-PG) dehydrogena-
tion, a thermodynamically unfavorable reaction catalyzed by D-3-PG
dehydrogenase (SerA). D-2-Hydroxyglutarate (D-2-HG) is traditionally
viewed as an abnormal metabolite associated with cancer and
neurometabolic disorders. Here, we reveal that bacterial anabo-
lism and catabolism of D-2-HG are involved in L-serine biosynthesis
in Pseudomonas stutzeri A1501 and Pseudomonas aeruginosa PAO1.
SerA catalyzes the stereospecific reduction of 2-ketoglutarate (2-KG) to
D-2-HG, responsible for the major production of D-2-HG in vivo. SerA
combines the energetically favorable reaction of D-2-HG production
to overcome the thermodynamic barrier of D-3-PG dehydrogenation.
We identified a bacterial D-2-HG dehydrogenase (D2HGDH), a flavin
adenine dinucleotide (FAD)-dependent enzyme, that converts D-2-HG
back to 2-KG. Electron transfer flavoprotein (ETF) and ETF-ubiquinone
oxidoreductase (ETFQO) are also essential in D-2-HG metabolism
through their capacity to transfer electrons from D2HGDH. Further-
more, while the mutant with D2HGDH deletion displayed decreased
growth, the defect was rescued by adding L-serine, suggesting that
the D2HGDH is functionally tied to L-serine synthesis. Substantial flux
flows through D-2-HG, being produced by SerA and removed by
D2HGDH, ETF, and ETFQO, maintaining D-2-HG homeostasis. Overall,
our results uncover that D-2-HG–mediated coupling between SerA
and D2HGDH drives bacterial L-serine synthesis.
Certain enzymes exhibit, beyond their classical functions, 2-KG–
reducing activity for D-2-HG production. These enzymes include
hydroxyacid-oxoacid transhydrogenase (HOT) in H. sapiens (20,
21), 4-phospho-D-erythronate dehydrogenase (PdxB) in E. coli (22),
UDP-N-acetyl-2-amino-2-deoxyglucuronate dehydrogenase (WbpB)
in Pseudomonas aeruginosa (23), 2-hydroxyacid dehydrogenase
(McyI) in Microcystis aeruginosa (24), and D-3-phosphoglycerate
dehydrogenase (SerA, also named PHGDH) in H. sapiens (25)
and S. cerevisiae (15). SerA in E. coli is capable of producing 2-HG
from 2-KG; however, the enantiomer of its product (2-HG) needs
to be verified (26, 27).
SerA catalyzes the initial reaction for L-serine biosynthesis, a
critical metabolic pathway that is essential for the survival of
most organisms (26–29). Approximately 15% of the carbon flux gen-
erated from glycolysis of glucose passes through L-serine biosynthesis
in E. coli (27). However, the starting point of L-serine biosynthesis,
D-3-phosphoglycerate (D-3-PG) dehydrogenation catalyzed by SerA,
is quite thermodynamically unfavorable (27, 30). The feasible but
puzzling reaction of D-2-HG production contrasts with the unfa-
vorable but critical physiological reaction of D-3-PG dehydrogena-
tion. This intriguing phenomenon prompts us to study the poorly
Significance
D-3-Phosphoglycerate dehydrogenase (SerA) is a key enzyme in
L-serine biosynthesis. It couples the dehydrogenation of D-3-
phosphoglycerate to 3-phosphohydroxypyruvate and the re-
duction of 2-ketoglutarate to D-2-hydroxyglutarate (D-2-HG). This
provides an example of how nonenergetically favorable and
energetically favorable reactions are linked together to allow
metabolic processes to proceed. D-2-HG is often considered as an
abnormal metabolite produced by several enzymes with “pro-
miscuous” activities. Our findings offer insights into how an
enzymatic reaction that was considered promiscuous or acci-
dental plays a key role in metabolism. We have identified a
bacterial D-2-hydroxyglutarate dehydrogenase (D2HGDH), which
converts D-2-HG produced during L-serine biosynthesis back to
2-ketoglutarate. D-2-HG is a normal metabolite that is simulta-
neously produced and catabolized without accumulation in
bacterial metabolism.
D-2-Hydroxyglutarate 2-ketoglutarate D-3-phosphoglycerate
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L-serine biosynthesis bacterial metabolism
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s a critical intermediate in tricarboxylic acid cycle (TCA
cycle), 2-ketoglutarate (2-KG) can be converted into succinyl-
A
CoA (1), succinate (2), L-glutamate (3), or 2-hydroxyglutarate (2-
HG) (4). 2-HG exists in two stereoisomeric conformations: L-2-
hydroxyglutarate (L-2-HG) (5, 6) and D-2-hydroxyglutarate (D-2-HG)
(7, 8) (SI Appendix, Fig. S1). Mutations in isocitrate dehydrogenase
(IDH), which result in strong 2-KG–reducing activity and accu-
mulation of excess D-2-HG, have been detected in numerous types
of cancer. D-2-HG is a key effector of IDH mutations that promote
oncogenesis (7–10). Another severe disease involving D-2-HG is
inborn D-2-hydroxyglutaric aciduria (D-2-HGA), with increased
D-2-HG levels in body fluids as the biochemical hallmark (4). Thus,
D-2-HG has been viewed as an abnormal metabolite (4, 11, 12).
D-2-HG has been detected in various organisms, including Homo
sapiens (4, 13), Arabidopsis thaliana (14), and Saccharomyces cerevisiae
(15). In animals, plants, and yeasts, D-2-HG can be converted
into 2-KG by D-2-HG dehydrogenase (D2HGDH) (13–15). The
role of D-2-HG catabolism has been extensively studied, particu-
larly in patients with D-2-HGA (4). 2-HG has also been found in
bacteria such as Pseudomonas (16) and Escherichia coli (17).
However, even though D2HGDH activities have been reported in
crude preparations of Pseudomonas putida (18) and Rhodospirillum
rubrum (19), bacterial D2HGDH has never been characterized.
Author contributions: W.Z., C.G., C.M., and P.X. designed research; W.Z., M.Z., C.G.,
Yipeng Zhang, Y.G., S.G., X.G., Z.Z., Q.L., and Yingxin Zhang performed research; W.Z.,
C.G., C.M., F.T., and P.X. analyzed data; and W.Z., C.G., C.M., F.T., and P.X. wrote
the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. J.D.R. is a guest editor invited by the
Editorial Board.
1To whom correspondence may be addressed. Email: jieerbu@sdu.edu.cn or pingxu@sjtu.
edu.cn.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1619034114/-/DCSupplemental.
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understood roles of D-2-HG production by SerA in L-serine
biosynthesis.
properties of the D2HGDH were also investigated in this study
(Table 1 and SI Appendix, Fig. S5 C–F).
D-2-HG was barely detectable in the P. stutzeri A1501 growth
medium during glucose utilization. For A1501-Δd2hgdh, the grad-
ual accumulation of extracellular D-2-HG (SI Appendix, Fig. S3B)
reached an unexpectedly high concentration (0.42 g·L−1, ≈2.8 mM)
from 3.5 g·L−1 of glucose (Fig. 2D), implying continuous and robust
D-2-HG production. By contrast, A1501-Δd2hgdh-d2hgdh+ regained
the ability to catabolize D-2-HG (SI Appendix, Fig. S3C), and
therefore, prevented the accumulation of D-2-HG (Fig. 2D).
Accumulation of intracellular D-2-HG was observed in A1501-
Δd2hgdh (Fig. 2E), further confirming the function of D2HGDH
in D-2-HG catabolism. Although complementation of A1501-Δd2hgdh
with the gene d2hgdh eliminated the D-2-HG accumulation, the
delayed growth kinetics was not reversed (SI Appendix, Fig. S3 A
and C). This may be due to the inhibitory effect of added antibiotic
(gentamicin) and inducer (isopropyl β-D-thiogalactoside, IPTG),
which are required for the expression of d2hgdh.
In this study, we discovered a robust coupling between SerA and
D2HGDH, which constructs the interconversion between D-2-HG
and 2-KG, and drives bacterial L-serine synthesis in Pseudomonas
(Fig. 1). SerA combines the reaction of D-2-HG production from
2-KG to overcome the thermodynamic barrier of D-3-PG de-
hydrogenation. We have identified a bacterial D2HGDH, which
converts D-2-HG to 2-KG, and is functionally tied to L-serine
biosynthesis. The coupling between SerA and D2HGDH also es-
tablishes a linkage among the intermediates of glycolysis (D-3-PG),
TCA cycle (2-KG), and L-serine biosynthesis (Fig. 1).
Results
D2HGDH Is Involved in D-2-HG and Glucose Utilization. All repre-
sentative Pseudomonas species, including Pseudomonas stutzeri
A1501, P. aeruginosa PAO1 and P. putida KT2440, possess ho-
mologous proteins that share more than 30% identity with the
D2HGDH from H. sapiens (SI Appendix, Table S1). This study
focuses on the bacterial metabolism of D-2-HG, using Pseudo-
monas species as the model system. Given their known genomic
information and tractable genetics, P. stutzeri A1501 (31, 32) and
P. aeruginosa PAO1 (33) were selected for further studies.
The gene d2hgdh (Gene ID: 5094894) that encodes the predicted
D2HGDH in P. stutzeri A1501 was deleted and then complemented.
The resulting strains were named as A1501-Δd2hgdh and A1501-
Δd2hgdh-d2hgdh+, respectively. Compared with the wild-type (WT)
strain and A1501-Δd2hgdh-d2hgdh+, A1501-Δd2hgdh is barely able
to use D-2-HG (SI Appendix, Fig. S2 A and B). Disruption of d2hgdh
to impair D-2-HG utilization also caused a relatively slower growth
of P. stutzeri A1501 in the medium containing glucose (SI Appendix,
Fig. S3 A and B). The maximal biomass of A1501-Δd2hgdh was
lower than that of A1501-WT (SI Appendix, Fig. S3 A and B),
implying that D-2-HG metabolism might be involved in glucose
utilization.
Electron Transfer Flavoprotein and Electron Transfer Flavoprotein-
Ubiquinone Oxidoreductase also Participate in D-2-HG Conversion to
2-KG. D2HGDH is not a membrane protein and cannot directly
transfer electrons to the electron transport chain (ETC). Both
cytochrome c and electron transfer flavoprotein (ETF) have
been shown to function as soluble electron carriers between
flavoprotein dehydrogenases and electron transport chains (34).
The Km and Vmax values of D2HGDH for cytochrome c were
210.11 18.40 μM and 0.84 0.07 U·mg−1, respectively (Table
1), implying that cytochrome c might not be an efficient electron
acceptor (because of the high Km value). The ETF from P. stutzeri
A1501 was expressed and purified (SI Appendix, Fig. S4B). When
the purified ETF was added, increased activities of D2HGDH
toward dichloroindophenol (DCIP) reduction were detected (SI
Appendix, Fig. S4C). As shown in Fig. 2F, the addition of ETF
strongly favored D-2-HG oxidation. As the ETF concentration
increased, the Vmax values also increased continuously. When ETF
was considered as the substrate for D2HGDH, the Km and Vmax
D2HGDH Contributes to D-2-HG Conversion to 2-KG. The predicted
D2HGDH in P. stutzeri A1501 was expressed and purified as a
His-tagged protein (SI Appendix, Fig. S4A). The purified protein
was identified as a homodimer (SI Appendix, Figs. S4A and S5A)
and bound one flavin adenine dinucleotide (FAD) per subunit
(Fig. 2A and SI Appendix, Fig. S5B). Substrate screening revealed
that only the D-isomers of 2-HG and malate could function as
substrates (Fig. 2B). HPLC analysis showed that D-2-HG was
converted into 2-KG by the enzyme in the presence of artificial
electron acceptors (Fig. 2C), confirming the identification of a
bacterial D2HGDH from P. stutzeri A1501. Other enzymatic
values were measured as 7.71 1.83 μM and 7.50 1.10 U·mg−1
,
respectively (Table 1).
ETF uses ETF-ubiquinone oxidoreductase (ETFQO) as its
essential partner to transfer electrons from flavoprotein dehy-
drogenases to the ubiquinone pool in ETC (34). Coenzyme Q1
(CoQ1) reduction was observed in the presence of D-2-HG,
D2HGDH, ETF, and ETFQO (expressed and purified as shown
in SI Appendix, Fig. S4D) (Fig. 2G). The rate of D-2-HG oxida-
tion increased upon the addition of ETFQO (Fig. 2G), further
demonstrating the role of ETFQO in D-2-HG oxidation and the
accompanied ETF-mediated electron transfer to CoQ1.
An in-frame deletion of etf abolished growth of the mutant in
the media containing D-2-HG, and complementation with the
plasmid containing etf restored growth of the mutant (SI Ap-
pendix, Fig. S2 A and B). The strains were also tested for growth
on the medium containing glucose as the sole carbon source.
Intracellular and extracellular D-2-HG concentrations were also
determined. The results (Fig. 2 D and E and SI Appendix, Fig. S3
D and E) were similar to those obtained with the d2hgdh deletion
mutant and complemented strain (Fig. 2 D and E and SI Ap-
pendix, Fig. S3 B and C). These results indicated that D2HGDH,
ETF, ETFQO, and ETC efficiently maintain D-2-HG homeo-
stasis through the conversion of D-2-HG to 2-KG (Fig. 2H).
Fig. 1. The coupling between SerA and D2HGDH makes the robust inter-
conversion between D-2-HG and 2-KG (green dotted box) to overcome the
thermodynamic barrier of D-3-PG dehydrogenation and facilitate the bio-
synthetic process of L-serine (purple pathway). The coupling establishes a
linkage among the intermediates of glycolysis (D-3-PG), TCA cycle (2-KG), and
L-serine biosynthesis. The black dotted box indicates the already known
straightforward connections between the intermediates of glycolysis (PEP
and pyruvate) and TCA cycle (OAA). OAA, oxaloacetate; PEP, phosphoenol-
pyruvate; 3-PHP, 3-phosphohydroxypyruvate.
SerA Contributes to 2-KG Conversion to D-2-HG. The d2hgdh gene is
adjacent to the D-3-phosphoglycerate dehydrogenase (SerA, also
named PHGDH)-encoding gene serA in all representative spe-
cies of Pseudomonas, including P. stutzeri, P. aeruginosa, P. putida,
P. fluorescens, and so on (Fig. 3A). SerA was disrupted in A1501-
Δd2hgdh to generate the double-mutant A1501-Δd2hgdhΔserA. As
expected, the strain exhibited L-serine auxotrophy (SI Appendix,
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Fig. 2. D2HGDH plus ETF and ETFQO convert D-2-HG to 2-KG. (A) Full-wave scanning of the purified D2HGDH. (B) Substrate screening (D-2-HG in reaction
mixture, 1 mM; other chemicals, 5 mM) for the purified D2HGDH (0.2 μM). D-2-HBA, D-2-hydroxybutyrate; D-MA, D-mandelate; D-PLA, D-phenyllactate; TA,
tartrate. (C) HPLC analysis of the product of D2HGDH-catalyzed D-2-HG oxidation. The reaction mixtures containing D-2-HG (5 mM), 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT, 8 mM), PMS (0.8 mM), and native or heat-inactivated D2HGDH (11.4 μM) in 50 mM Tris·HCl (pH 7.4) were incubated at
37 °C for 40 min. (D) The maximal concentrations of extracellular D-2-HG. (E) The intracellular concentrations of D-2-HG. The strains in D and E were grown in
medium with glucose as the sole carbon source. (F) The effect of ETF (0, 0.5, 1.0, and 3.0 μM) on the kinetics of D2HGDH (0.14 μM) for D-2-HG was studied in the
presence of a series of D-2-HG concentrations (0–1 mM) and DCIP (200 μM). The data were fitted to a hyperbolic curve. (G) The effect of ETFQO on the D2HGDH-
catalyzed CoQ1 reduction. The reaction mixtures containing 50 mM Hepes (K+) (pH 7.4), dodecamaltoside (0.2 mM), D2HGDH (1 μM), D-2-HG (1 mM), ETF (4 μM),
CoQ1 (53.2 μM), and ETFQO (0–12 μM) were monitored at 275 nm at room temperature. (H) Schematic diagram for the process of D-2-HG conversion to 2-KG.
ETFox, oxidized ETF; ETFred, reduced ETF. Values are the average SD (n = 3).
Fig. S2 C and D), and the addition of external L-serine triggered
some growth (SI Appendix, Fig. S3F). A1501-Δd2hgdhΔserA
could grow relatively well (SI Appendix, Fig. S3G) in LB medium,
a rich medium containing the amino acids required for bacterial
growth (including L-serine). Compared with A1501-Δd2hgdh, the
double-mutant A1501-Δd2hgdhΔserA in LB medium showed no
accumulation of extracellular and intracellular D-2-HG (Fig. 3 B
and C), indicating that SerA catalyzes D-2-HG production in vivo.
SerA from P. stutzeri A1501 was expressed and purified as a His-
tagged protein (SI Appendix, Fig. S4E). The purified SerA was
found to reduce 2-KG to 2-HG in the presence of NADH (Fig. 3 D
and E), and the results from enzymatic chirality assays clearly
showed that the reaction product is D-2-HG solely (Fig. 3F and SI
Appendix, Fig. S4 F and G); this indicates that SerA can reduce
2-KG with good stereoselectivity to produce D-2-HG (Fig. 3D).
ability to reduce 2-KG to D-2-HG (Fig. 4A). The rate of SerA-
catalyzed 2-KG reduction was considerably higher than that of
SerA-catalyzed D-3-PG dehydrogenation, which was below the
detection limit (Fig. 4B). SerA-catalyzed D-3-PG dehydrogenation
has a high ΔG°′obs value (+33.0 kJ·mol−1) (30). According to the
conventional view, D-3-PG dehydrogenation is driven through
coupling with the reactions catalyzed by L-phosphoserine amino-
transferase (SerC) (ΔG°′obs = −11.5 kJ·mol−1) (30) and L-phosphoserine
phosphatase (SerB) (ΔG°′obs = −10.2 kJ·mol−1) (35) in L-serine
synthesis. The ΔG°′obs of the coupled reactions from D-3-PG to
L-phosphoserine and from D-3-PG to L-serine are +21.5 kJ·mol−1
and +11.2 kJ·mol−1, respectively, indicating that these reactions
are not thermodynamically favorable. However, D-3-PG was ef-
ficiently transformed by SerA and SerC (expressed and purified
as shown in SI Appendix, Fig. S4H) in the presence of D-3-PG,
NAD+, and L-glutamate (Fig. 4C), which are the substrates for
SerA and SerC (Fig. 4A). Importantly, D-2-HG was also detected
in the reaction mixture (Fig. 4C). This result was unexpected but
is theoretically possible: The reaction catalyzed by SerC would
2-KG Is Converted to D-2-HG During the Coupled Reaction of SerA and
SerC. SerA catalyzes the dehydrogenation of D-3-PG to 3-phos-
phohydroxypyruvate (3-PHP), but it also possesses the puzzling
Table 1. Kinetic parameters of D2HGDH for D-2-HG, ETF and cytochrome c
Substrate
Km, mM
Vmax, U·mg−1
Kcat, min−1
Kcat/Km, min−1·mM−1
D-2-HG
ETF
Cytochrome c
0.17 0.02
4.56 0.60
7.50 1.10
0.84 0.07
473.83 62.87
779.72 114.85
87.38 6.98
2,723.55 91.15
(1.03 0.11) × 105
416.08 5.76
(7.71 1.83) × 10−3
(210.11 18.40) × 10−3
Zhang et al.
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D-3-PG dehydrogenation. When 2-KG was added to the solution
containing as-isolated SerA, the absorbance at 325 nm decreased
dramatically, indicating oxidation of the tightly bound NADH to
NAD+ by 2-KG (Fig. 5A).
Therefore, we hypothesized that 2-KG conversion to D-2-HG
is not a meaningless side reaction, but a reaction that couples
and drives D-3-PG dehydrogenation through oxidizing NADH that
binds to SerA and reducing the total ΔG°′obs of the SerA-
catalyzed reaction. The sum of ΔG°′obs values of these two reac-
tions is +4.5 kJ·mol−1, which is a value considerably lower than
that of a single D-3-PG dehydrogenation reaction (Fig. 4A).
Then, we analyzed SerA-catalyzed reactions containing D-3-PG,
2-KG, and NAD+. D-3-PG was very poorly oxidized in the absence
of 2-KG, but when 2-KG was added, the dehydrogenation of D-3-PG
was enhanced and the residual D-3-PG in the reaction mixture was
diminished (SI Appendix, Fig. S6A), confirming that the coupling
with 2-KG reduction is needed for the SerA-catalyzed D-3-PG
dehydrogenation.
D-3-PG and 2-KG were also incubated with as-isolated SerA
but without external NAD+ or NADH. As shown in Fig. 5B, the
amount of the oxidized D-3-PG (estimated from the peak height)
far exceeds that of the NADH contained in the enzyme (≈17 μM),
suggesting that as-isolated SerA can oxidize D-3-PG with multi-
ple turnovers by recycling of its bound coenzyme through re-
ducing 2-KG into D-2-HG. The coupled reactions can also be
simplified into a single reaction (Fig. 5C), which employs D-3-PG
and 2-KG as the substrates and 3-PHP and D-2-HG as the products.
The stoichiometry analysis of the SerA-catalyzed coupled reaction
showed the equimolar conversion of D-3-PG and 2-KG to 3-PHP
and D-2-HG (SI Appendix, Fig. S7). Due to the SerA-catalyzed
D-3-PG dehydrogenation and the coupled D-2-HG production,
efficient CoQ1 reduction was observed in the presence of D-3-PG,
2-KG, as-isolated SerA, D2HGDH, ETF, and ETFQO (Fig. 5D).
Fig. 3. SerA contributes to 2-KG conversion to D-2-HG. (A) SerA-encoding gene
serA is adjacent to the predicated D2HGDH encoding gene d2hgdh. (B) The
concentrations of extracellular D-2-HG during growth. (C) The intracellular
concentrations of D-2-HG (sampled at 12 h). The strains in B and C were grown
in LB medium. Values are the average SD (n = 3). (D) The possible configu-
rations of 2-HG produced by SerA-catalyzed 2-KG reduction. (E) HPLC analysis
of the product of SerA-catalyzed 2-KG conversion in presence of NADH. The
mixtures containing 2-KG (5 mM), NADH (5 mM), native SerA, or heat-
inactivated SerA (5.6 μM) in 50 mM Tris (pH 7.4), were incubated at 30 °C for
3 h. (F) The configuration of the SerA-produced 2-HG was determined using an
enzymatic method. The reactions using authentic D-2-HG (squares), L-2-HG
(circles), or SerA-produced 2-HG (triangles) as substrates were catalyzed by the
D2HGDH purified in this study (red lines) or L-2-hydroxyglutarate oxidoreduc-
tase (LGRO, blue lines), respectively. The purification and characterization of
LGRO are presented in SI Appendix, Fig. S4 F and G.
produce 2-KG as the end product, which could then be reduced
by SerA. The ΔG°′obs value of the 2-KG reduction to D-2-HG is
approximately −28.5 kJ·mol−1 (36). The total ΔG°′obs of SerA-
catalyzed D-3-PG dehydrogenation, SerC-catalyzed reaction,
and SerA-catalyzed 2-KG reduction would be −7.0 kJ·mol−1 (Fig.
4A), suggesting that D-3-PG dehydrogenation catalyzed by SerC
and SerA is feasible.
SerA Couples D-3-PG Dehydrogenation with Conversion of 2-KG to
D-2-HG. Purified SerA (as-isolated SerA) exhibited a prominent
spectral signal at 325 nm (Fig. 5A), suggesting the presence of
tightly bound NADH. The ratio of NADH to subunit of as-isolated
SerA is ≈0.4–0.5 (SI Appendix, Fig. S4I). This was further sup-
ported by the determined high affinity of the apoprotein (SI Ap-
pendix, Fig. S4I) of SerA for NADH (equilibrium dissociation
constant: KD ≈ 0.1 μM) using isothermal titration calorimetry (SI
Appendix, Table S2). Additional NAD+ binding to SerA in this
state (as-isolated or preincubated with NADH) was not preferred
(SI Appendix, Table S2). Thus, reoxidation of the bound NADH
to regenerate NAD+ timely is a prerequisite for NAD+-dependent
Fig. 4. 2-KG conversion to D-2-HG couples and drives D-3-PG dehydroge-
nation. (A) Thermodynamic analysis of the reactions in the L-serine bio-
synthesis pathway indicates that D-2-HG production serves SerA to facilitate
D-3-PG dehydrogenation and L-serine biosynthesis. The ΔG°′obs values were
calculated using the following formula: ΔG°′obs = −RTlnKobs. The Kobs value
is the observed equilibrium constant of the reaction performed at 38 °C,
pH 7.0 in previous studies. 3-PS, 3-phosphoserine. (B) The activities of 2-KG
reduction reaction (2-KG, 5 mM; NADH, 0.2 mM) and D-3-PG dehydrogenation
reaction (D-3-PG, 10 mM; NAD+, 1 mM) catalyzed by SerA (0.28 μM). (C) HPLC
analysis of the products of the reaction coupled by SerA and SerC (heat-inactivated
enzymes as a control). The mixtures containing NAD+ (4 mM), L-glutamate
(10 mM), D-3-PG (20 mM), SerC (1.2 mg·mL−1), and SerA (1.03 mg·mL−1) in
50 mM Tris (pH 7.4) were reacted at 30 °C for 3 h.
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Fig. 5. The reactions of D-2-HG production and D-3-PG dehydrogenation are coupled by redox transitions of NAD+ and NADH. (A) Full-wave scanning of SerA.
Black line, as-isolated SerA; red line, as-isolated SerA treated with 5 mM D-3-PG; blue line, as-isolated SerA treated with 1.25 mM 2-KG. (B) HPLC analysis of the
coupled reaction of D-3-PG dehydrogenation and 2-KG reduction catalyzed by SerA. The reaction mixtures containing as-isolated SerA (8.3 μM), D-3-PG
(5 mM), and 2-KG (10 mM) as the substrates were reacted at 30 °C for 3 h. (C) Schematic diagram for the redox state transitions of NADH/NAD+ in the coupling
and the stoichiometry of SerA-catalyzed coupled reaction. According to SI Appendix, Fig. S7, D-3-PG and 2-KG decrease 1.06 0.04 mM and 1.04 0.04 mM,
respectively, while D-2-HG increases 1.01 0.03 mM. Since the D-3-PG can only be converted into 3-PHP under the reaction conditions, the result can dem-
onstrate the equimolar conversion of D-3-PG and 2-KG to 3-PHP and D-2-HG. (D) Enzymatic assays of the coupled reaction using an enzyme system including
the essential elements for the HG-KG interconversion. The reaction mixtures containing 50 mM Hepes (K+) (pH 7.4), dodecamaltoside (0.2 mM), D2HGDH
(1 μM), ETF (4 μM), CoQ1 (53.2 μM), 2-KG (5 mM), D-3-PG (5 mM), ETFQO (1.32 μM), and as-isolated SerA (1.66 μM) were monitored by CoQ1 reduction at
275 nm. (E) Effects of coenzyme on the activities of the coupled reaction. Apoenzyme of SerA, 0.19 μM; NAD+, 12.5 μM; NADH, 12.5 μM. (F) Schematic di-
agram for the multiple enzyme system assembled by SerA, D2HGDH, ETF, and ETFQO.
When as-isolated SerA was replaced with apoenzyme, CoQ1
reduction disappeared. The CoQ1 reduction could be restored
by adding NAD+ or NADH (Fig. 5E), suggesting the indispensable
role of the coenzyme in the coupled reaction. Thus, SerA, D2HGDH,
ETF, and ETFQO can be assembled into a multiple enzyme system
that converts D-3-PG to 3-PHP for L-serine production and trans-
fers the electrons produced during D-3-PG dehydrogenation to
CoQ in the ETC (Fig. 5F).
(PA0317; Gene ID: 878323) in P. aeruginosa PAO1 (ΔPA0317) also
yielded a slower growth (Fig. 6A) and accumulation of extracellular
and intracellular D-2-HG (Fig. 6 B and C). Interestingly, external
addition of L-serine (2 mM) was able to rescue the growth defect
(Fig. 6A), suggesting that the knockout of D2HGDH decreases the
flux for L-serine synthesis and that D2HGDH is functionally tied to
L-serine synthesis.
D-2-HG inhibited the SerA-catalyzed reduction of 2-KG and the
inhibitory effect increased with D-2-HG concentrations (SI Appendix,
Fig. S6 A and B). As 2-KG reduction is a prerequisite for D-3-PG
dehydrogenation, D-2-HG would also inhibit D-3-PG dehydrogena-
tion in the direction of L-serine synthesis. Since L-2-HG did not
inhibit the activity of SerA-catalyzed 2-KG reduction (SI Ap-
pendix, Fig. S6C), the inhibitory effect is truly enantiomer-specific
for D-2-HG. The real intracellular concentrations of D-2-HG (Figs.
L-Serine Rescues the Growth Defect of D2HGDH Knockout. While the
knockout of D2HGDH slowed the growth of P. stutzeri A1501,
L-serine exhibited a strong inhibitory effect on the growth of
P. stutzeri A1501 (SI Appendix, Fig. S3 H and I). Therefore,
P. stutzeri A1501 is not suitable to study the capacity of L-serine to
rescue the effects of D2HGDH knockout. P. aeruginosa PAO1 grows
normally in the presence of a relatively high concentration of L-serine
(2 mM) (Fig. 6A). The deletion of the D2HGDH-encoding gene 2E and 6C) were far below extracellular ones (Figs. 2D and 6B).
Fig. 6. L-Serine rescues the growth defect of D2HGDH knockout in P. aeruginosa PAO1. (A) The growth curves of the P. aeruginosa strains in the medium
containing glucose (G) or glucose plus 2 mM L-serine (G+S). (B) The concentrations of extracellular D-2-HG during growth. (C) The concentrations of intra-
cellular D-2-HG at 12 h. Values are the average SD (n = 3).
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This means that most of the produced D-2-HG accumulated in the
intermediates involved in glycolysis, TCA cycle, and amino acid
extracellular medium of A1501-Δd2hgdh (Figs. 2D and 6B), indi- metabolism (SI Appendix, Fig. S8 A and C) were detected, which
cating the existence of an unknown efflux system for D-2-HG.
indicates that the dysfunction of the HG-KG interconversion in-
fluences these core metabolisms.
HG-KG Interconversion Works Robustly in Metabolism. Coupling
between SerA and D2HGDH generates an HG-KG intercon-
version (the interconversion between D-2-HG and 2-KG) system
(SI Appendix, Fig. S8A). D2HGDH activities were detectable in
cells grown in these distinct types of media and at any growth
period (SI Appendix, Fig. S8B). D-2-HG was found to accumulate
Phylogenetic Analysis of the Coupling Between SerA and D2HGDH.
The distribution of D2HGDH and SerA in 4,929 completely
sequenced bacterial genomes was studied (Fig. 7A and Datasets
S2 and S3) and is illustrated in SI Appendix, Table S4. The co-
existence of homologs of D2HGDH and SerA is found in
to high concentrations during the culture of A1501-Δd2hgdh 660 bacterial genomes (Fig. 7A and Dataset S4), many of which
with each carbon source investigated here (glucose, glycerol,
pyruvate, succinate, 2-KG, and L-glutamate) or LB medium (SI
Appendix, Table S3). These results indicated that substantial flux
flows through the ongoing HG-KG interconversion during the
metabolism under various conditions. Next, the effects of impaired
also have adjacent genomic localization of D2HGDH and SerA
homologs (marked in Dataset S4). Thus, the coupling between
SerA and D2HGDH is likely to be widespread in bacteria.
Phylogenetic analysis revealed that D2HGDHs from eukary-
otic microorganisms, animals, and plants (SI Appendix, Table S1)
D2HGDH function on the profiles of intracellular metabolites form a cluster (Fig. 7B). It is very clear that D2HGDHs found in
were investigated by GC-MS–based metabolomic analysis with
glucose as the sole carbon source (SI Appendix, Fig. S8C and
Dataset S1). 2-HG exhibits the most striking fold change (SI
H. sapiens (13), A. thaliana (14), and S. cerevisiae (15) catalyze
the conversion of D-2-HG to 2-KG. The SerA homologs from
eukaryotic microorganisms (SI Appendix, Table S1) constitute a
Appendix, Fig. S8C). Glycine is the metabolite that is directly re- phylogenetic group with SerA proteins from Pseudomonas (Fig.
lated to L-serine through the action of L-serine hydroxymethyl- 7C), revealing the close relationship among them. SerA proteins
transferase. There was no decrease in serine; however, the level of from animals and plants (SI Appendix, Table S1) group into one
glycine decreased by fourfold (SI Appendix, Fig. S8D), indicating
that the downstream pathway of L-serine biosynthesis was af-
fected. Additionally, statistically significant changes in levels of
separate cluster (Fig. 7C and SI Appendix, Table S1). Interestingly,
SerA proteins from H. sapiens (6, 25) and S. cerevisiae (15) have
been found to have the ability to produce D-2-HG from 2-KG.
Fig. 7. The type of HG-KG interconversion may exist universally across from bacteria to eukaryotes. The distribution of the homologs of D2HGDH and SerA in
bacteria were analyzed (A). Phylogenetic bootstrap consensus trees of D2HGDH (B) and SerA (C) in various organisms were constructed. According to the previous
reports, the organisms in which SerA can reduce 2-KG to 2-HG were marked with red color. The sequence details were shown in SI Appendix, Table S1.
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Combining the results obtained from these analyses, the coupling substantial flux flowing through D-2-HG in physiological me-
between D2HGDH and SerA may also take place in eukaryotic
microorganisms, animals, and plants; however, a further detailed
verification is still needed.
tabolism (SI Appendix, Table S3). In addition, there might be an
efflux system for D-2-HG in Pseudomonas. If this efflux does not
exist, the intracellular D-2-HG would accumulate, inhibit SerA,
and cause serine auxotrophy in the absence of D2HGDH. We
speculate that animal cells also have such efflux system since
high levels of D-2-HG can be detected in the plasma and urine of
patients with D-2-HG–related cancer and aciduria (4, 38). De-
spite the existence of the efflux system, the disruption of D2HGDH
in Pseudomonas also led to some growth defects. Serine levels
remained unchanged, but the level of glycine, the downstream
metabolite of L-serine, decreased significantly (SI Appendix, Fig.
S8 C and D), indicating that the process of L-serine synthesis was
affected. Exogenous L-serine may replenish the cellular L-serine
pool, inhibit the 2-KG reduction reaction catalyzed by SerA (SI
Appendix, Fig. S6D), reduce D-2-HG production and accompanied
loss of 2-KG, and as a result, rescue the growth defect of the
D2HGDH mutant. Exogenous 2-KG could only replenish the 2-KG
pool and, therefore, only partly improve the growth of the D2HGDH
mutant (SI Appendix, Fig. S10). L-Serine biosynthesis relies on
2-KG (a substrate for SerA) and L-glutamate (a substrate for SerC
that is converted to 2-KG) (Fig. 4A). Although glutamate and
2-KG were not detected directly, it is worth noting that the levels
of glutamate-related amino acids (ornithine, proline, putrescine,
and pyroglutamate) were dramatically reduced in A1501-Δd2hgdh
(SI Appendix, Fig. S8 A and C). The impaired back conversion of
D-2-HG into 2-KG and then L-glutamate may result in a short supply
of these substrates for L-serine biosynthesis in the D2HGDH mu-
tant. Thus, D2HGDH is functionally tied to L-serine biosynthesis
possibly through preventing the inhibitory effect of D-2-HG on
SerA and maintaining sufficient pools of substrates (2-KG and
L-glutamate) for the L-serine biosynthesis.
There is no physical association between SerA and D2HGDH
(SI Appendix, Fig. S11), implying that 2-KG produced from D-2-
HG during L-serine biosynthesis will get mixed with the cellular
2-KG pool. P. stutzeri A1501 could use D-2-HG as the sole car-
bon source for growth, further supporting that 2-KG produced
by D2HGDH should enter major metabolic pathways to support
D-2-HG–dependent growth and other 2-KG–related metabolic
processes. Notably, SerA from E. coli (also some other strains
marked in light red in Fig. 7C) shows a close relationship with
SerA from P. stutzeri A1501 (Fig. 7C) and was reported to have
the ability to produce 2-HG. However, there was no D2HGDH
homolog in E. coli, suggesting the existence of other unknown
enzymes involved in the D-2-HG catabolism.
A metabolic network is a complete set of metabolites, en-
zymes, reactions, and pathways cross-linked with each other.
Glycolysis and TCA cycle are connected by pyruvate or phos-
phoenolpyruvate through pyruvate carboxylase or phosphoenol-
pyruvate carboxykinase, respectively (the black dotted box in Fig.
1) (1, 39–41). TCA cycle and amino acid synthesis are connected
by 2-KG through glutamate dehydrogenase or transaminases
(1, 3). In this study, the HG-KG interconversion integrates D-3-
PG (generated by glycolysis), L-serine, 2-KG (a key intermediate
of the TCA cycle), and L-glutamate (SI Appendix, Fig. S8A). The
HG-KG interconversion works as a bridge that closely connects
glycolysis, TCA cycle, and amino acid metabolism to achieve a
more sophisticated metabolic network.
Considering that D-2-HG has been viewed as an abnormal
metabolite, D-2-HG conversion to 2-KG seems to be a process of
metabolite repair. Here, we report that D-2-HG production is not
a meaningless side reaction but serves SerA to facilitate D-3-PG
dehydrogenation. For the key but toxic metabolite, both its pro-
duction and elimination are essential. This is somewhat different
from the reported metabolite repair mechanism, in which repair
enzymes catabolize the toxic products generated from side reac-
tions (11, 12). The findings also offer insights into the mechanism
Discussion
According to the second law of thermodynamics, enzymatic
conversions occur only in the direction with negative Gibbs free
energy. There are several reported strategies to support an en-
zymatic conversion toward the thermodynamically unfavorable
direction: the integration of thermodynamically favorable reac-
tions catalyzed by other enzymes, the coupling with hydrolyzing
reactions of high-energy phosphate bond, and redox coupling
(for example, in complex II, from succinate directly to ubiqui-
none in ETC), and so on. The mechanism for bypassing the
thermodynamic obstacle in this study may be different from the
known ones. Both 2-KG reduction and D-3-PG dehydrogenation
are catalyzed by SerA, and SerA combines them to make the
reaction operated in the desired direction. The redox transition of
NADH to NAD+ is coupled to an interesting metabolic transition
of 2-KG to D-2-HG. In addition, the produced D-2-HG could be
efficiently removed by D2HGDH with the assistance of ETF,
ETFQO, and ETC. This study provides an example of how non-
energetically favorable and energetically favorable reactions are
coupled to allow metabolic processes to proceed forward.
L-Serine is a source of one-carbon units (37) and serves as a
precursor for the biosynthesis of glycine, cysteine, tryptophan,
and phospholipids (27–29). SerA-involved L-serine biosynthesis
pathway is the major anabolic source for L-serine (27), which
could be demonstrated by the occurrence of L-serine auxotroph
due to SerA knockout in this study (SI Appendix, Fig. S2C). The
Gibbs free energy of the SerA-catalyzed D-3-PG dehydrogena-
tion reaction yielding 3-PHP in real cellular environments (ΔG)
has also been assessed (SI Appendix, Table S5) according to the
values of ΔG°′obs (Fig. 4A) and the intracellular concentrations
of related products and reactants. SerA-catalyzed D-3-PG de-
hydrogenation is thermodynamically unfavorable to proceed due
to the high positive free energy in E. coli, MDA-MB-468, and
BT-20 cells (SI Appendix, Table S5). Here, the calculated ΔG
values of the reaction of D-3-PG dehydrogenation coupled with
2-KG reduction (SI Appendix, Table S5) suggest that the coupled
reaction occurs easily in vivo and might be physiologically signifi-
cant for L-serine biosynthesis.
The SerA-catalyzed D-3-PG dehydrogenation and 2-KG re-
duction follow the ordered Bi Bi mechanism (SI Appendix, Table
S2). NAD+ or NADH binds first, followed by the other substrate.
Following the reactions catalyzed by SerA, the products (3-PHP
and D-2-HG) will fall off the enzyme before NAD+ or NADH. The
presence of tightly bound NADH to as-isolated SerA indicates that
NADH is not released after D-3-PG dehydrogenation. 2-KG ad-
dition would oxidize the NADH tightly bound to SerA to NAD+,
and most of the NAD+ generated from NADH still bound to the
enzyme (SI Appendix, Fig. S9A). However, the intracellular con-
centration of NAD+ in P. stutzeri A1501 was more than 100 μM,
which was much higher than the KD (13 μM; SI Appendix, Table
S2) and apparent Km of the apoenzyme for NAD+ (1.31 μM; SI
Appendix, Fig. S9B). Therefore, the enzymatic steps for the SerA-
catalyzed 2-KG reduction and D-3-PG oxidation might not include
the release of NAD+ or NADH, and instead, the coenzymes could
remain bound during the coupled reaction. D-3-PG and 2-KG
(generated from glycolysis and TCA cycle, respectively), would be
incorporated into the coupled reaction of SerA. Then, the products
of SerA, D-2-HG and 3-PHP, would be continuously removed or
used by cells (SI Appendix, Fig. S9C).
D2HGDH has been studied in animals, plants, and eukaryotic
microorganisms (13–15). Here, we have identified a bacterial
D2HGDH from Pseudomonas. Although there is a rather low
level of D-2-HG under normal conditions, our results suggest
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catalyzed D-3-PG oxidation activity was measured by monitoring NADH
formation (340 nm) at 37 °C.
by which an enzymatic reaction previously considered promiscuous
or accidental actually plays a key role in metabolism.
In eukaryotic cells, SerA-catalyzed D-2-HG production from
2-KG originates in the cytosol while D2HGDH-catalyzed D-2-HG
conversion to 2-KG occurs in the mitochondrial matrix. There are
active carriers that facilitate the transport of various metabolic
intermediates across the mitochondrial membrane. The cytoplas-
mic and mitochondrial pools of different metabolites have tight
connections (42, 43). A recent report indicated that deficiency in
SLC25A1, which encodes the mitochondrial citrate carrier, causes
D-2-HG accumulation and D-2-HGA (44), implying the presence
of D-2-HG transport across mitochondrial membrane. Thus, the re-
actions catalyzed by cytoplasmic SerA and mitochondrial D2HGDH
may be coupled through the shuttle of 2-KG and D-2-HG between
the cytosol and mitochondrial matrix.
Major structures of the HG-KG interconversion modules in
Pseudomonas and humans are extremely similar. D2HGDH,
ETF, or ETFQO mutations can block D-2-HG elimination and
lead to increased levels of D-2-HG in patients with D-2-HGA and
glutaric aciduria type II (4, 45). Elevated expression of SerA
results in the increased metabolic flux originating from glucose
into L-serine synthesis pathway, which is essential for the growth
of certain types of cancer, such as breast cancer and melanoma
(28, 29). We speculate that D-2-HG may also be overproduced,
accumulating in these cells, and thereby promoting cancer.
Together with SerA, other enzymes such as PdxB, HOT, and
WbpB also contribute to the production of D-2-HG. These en-
zymes seem to contain tightly bound NADH and may also catalyze
thermodynamically unfavorable reactions (20–23, 46). For exam-
ple, HOT is the key enzyme in the catabolism of 4-hydroxybutyrate
(GHB). Knockdown of HOT reduces the accumulation of D-2-HG
in certain D-2-HG–related cancer cells (47), indicating that HOT
might also participate in D-2-HG production in vivo. Thus, there
may be similar HG-KG interconversion modules involving HOT
and other D-2-HG–producing enzymes (SI Appendix, Fig. S12).
The reaction through which 2-KG is reduced to D-2-HG is coupled
to facilitate these enzymes-catalyzed unfavorable but necessary
metabolic conversions (metabolite A to B). This type of HG-KG
interconversion (SI Appendix, Fig. S12) represents a unique bio-
chemical mechanism enabling reactions in thermodynamically
unfavorable directions.
Analytical Methods. The reaction mixtures mentioned above were analyzed by
HPLC (Agilent 1100 series; Hewlett-Packard) using a refractive index detector, as
described (48). Generally, a Bio-Rad Aminex HPX-87H column was used and the
mobile phase (10 mM H2SO4) was pumped at 0.4 mL·min−1 (55 °C). For quan-
tification of D-3-PG, 2-KG, and D-2-HG, 1 mL·L−1 of formic acid (≥88%; HPLC
grade; Kermel) solution was used as the mobile phase to obtain good peak
shape for HPLC or liquid chromatography-tandem mass spectrometry (LC-MS;
negative electrospray ionization mode). Glucose and L-glutamate concentra-
tions were determined with a bio-analyzer (SBA-40D; Shandong Academy of
Sciences).
Identification of the Conformation of the SerA-Produced 2-HG. An enzymatic
method was used to identify the configuration of the SerA-produced 2-HG.
L-2-hydroxyglutarate oxidase (LGO) from E. coli showed strong stereospecificity
toward L-2-HG (49). We identified the homologous L-2-hydroxyglutarate oxi-
doreductase (LGOR) in P. stutzeri strain SDM-LAC (50) through a BLASTX search
of the whole genome (AGSX01000142.1) with the known LGO sequence
(YP_490875.1). Subsequently, the putative LGOR (31% identity with LGO) was
expressed and purified (SI Appendix, Fig. S4F). To examine the substrate speci-
ficity, the reaction mixtures containing 50 mM Tris, 200 μM DCIP, 200 μM PMS,
5 mM substrate, and 0.045 mg·mL−1 LGOR were assayed at 37 °C. The obtained
enzyme exclusively catalyzes L-2-HG using PMS and DCIP as electron acceptors
(SI Appendix, Fig. S4G). Thus, the purified D2HGDH and LGOR were used to
define the configuration of the SerA-produced 2-HG. The reaction mixtures
(0.8 mL) contained 200 μM PMS, 75 μM DCIP, and 16 μg of D2HGDH or 1.6 μg of
LGOR in 0.1 M phosphate buffer at 37 °C. To initiate the reactions, 40 μL of
1 mM authentic D-2-HG, 1 mM authentic L-2-HG, or SerA-produced 2-HG was
added, and the reactions were monitored at 600 nm using a UV/visible spec-
trophotometer (Ultrospec 2100 Pro; Amersham Biosciences).
Quantification of D-2-HG. To quantify intracellular D-2-HG, three separate cell
cultures were sampled (15 mL) and then rapidly transferred to prechilled
centrifuge tubes. The supernatant was removed after centrifugation at
3,000 × g and −9 °C for 9 min, before washing the pellet with prechilled PBS
(5 mL). Intracellular D-2-HG was extracted using the hot ethanol method (17).
Ethanol (1 mL) was added to resuspend the precipitate, and the suspension
was boiled for 15 min in a closed tube. After the cell debris was removed by
centrifugation at 12,000 × g and 4 °C for 5 min, the supernatant was dried in
a vacuum centrifuge (60 °C). The dried residue was resuspended in distilled
water, and the supernatant obtained after centrifugation, as described
above, was stored at −80 °C until analysis. To measure extracellular D-2-HG,
aliquots of the cultures (1 mL) were sampled, and the supernatants after
centrifugation were saved for further analysis.
Quantitative analysis of D-2-HG was performed as described (51). The
NAD+-dependent D-2-hydroxyglutarate dehydrogenase gene from Acid-
aminococcus fermentans (52) was synthesized and expressed, and then the
produced protein was purified. Diaphorase from Clostridium kluyveri was
purchased from Sigma–Aldrich. We assumed that the cell volume of a 1-mL
Pseudomonas sample at an optical density (600 nm) of 1 was 3.5 μL (53).
Then, the intracellular D-2-HG concentration was estimated by dividing the
amount of D-2-HG in the sample by the total cell volume.
In summary, we have uncovered that the coupling between
SerA and D2HGDH sits in the hub of L-serine biosynthesis and
establishes a linkage among intermediates in glycolysis, TCA
cycle, and amino acid biosynthesis. This study also illustrates that
core metabolic processes might work together in a more sophisti-
cated manner than we thought, raising the significance of digging
the ancient and classic metabolic network.
Materials and Methods
Bioinformatics. The phylogenetic trees of SerA and D2HGDH were constructed
from the alignment of multiple proteins, followed by neighbor-joining
analysis using the MEGA software program (version 6.06) with bootstrap
analysis for 1,000 replications (cutoff value: 50%). The resulting trees were
processed by iTOL (54). The distributions of SerA and D2HGDH in bacteria
were vigorously checked by searching the sequenced bacterial genomes from
GenBank (updated until April 6, 2016) with the SerA and D2HGDH in P. stutzeri
A1501 as the queries. Individual genomes showing query coverages more than
90%, E values lower than e−30, and maximum identity levels higher than 30%
with the query protein, were selected and further evaluated.
Primers, plasmids, and strains used in this study are listed in SI Appendix,
Table S6. The full details for bacteria culture, enzyme preparation, cofactor
or coenzyme analysis, gene manipulation, metabolome analysis, isothermal
titration calorimetry, and the related analytical methods are described in SI
Appendix, SI Materials and Methods.
Enzyme Assays. The enzyme assay used to assess D2HGDH activity was
performed by recording the reduction of DCIP spectrophotometrically at
600 nm in a reaction mixture containing 50 mM Tris·HCl buffer (pH 7.4),
phenazine methosulfate (PMS; 200 μM), DCIP (generally 100 μM; for the
study of ETF effect on D2HGDH kinetics, 200 μM of DCIP was used), and
variable concentrations of substrate incubated at 37 °C. e value of DCIP is
22 cm−1·mM−1. The double-reciprocal plot method was used to estimate
the kinetic constants of D2HGDH. One unit (U) of enzyme activity was
defined as the amount that reduces 1 μmol of DCIP per min. The reduction
of 2-KG by SerA was assayed by measuring the absorbance of NADH at
340 nm in 50 mM Tris·HCl (pH 7.4) at 37 °C. One U of enzyme activity was
defined as the amount that oxidizes 1 μmol of NADH per min (26). SerA-
ACKNOWLEDGMENTS. We thank Dr. Haijun Liu at Washington University in
St. Louis, Dr. Wei Xiong at National Renewable Energy Laboratory, and
Dr. Luying Xun at Washington State University for their helpful discussion
and assistance in preparing the manuscript and the editor and anonymous
reviewers for their critical comments that resulted in a significantly improved
manuscript. The work was supported by National Natural Science Foundation
of China Grants 31470199, 31470164, 31270090 and Young Scholars Pro-
gram of Shandong University Grant 2015WLJH25.
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