Elucidation of a Self-Sustaining Cycle in Escherichia coli l -Serine Biosynthesis That Results in the Conservation of the Coenzyme, NAD+

Article abstract of DOI:10.1021/acs.biochem.8b00074

The equilibrium of the reaction catalyzed by d-3-phosphoglycerate dehydrogenase (PGDH), the first enzyme in the l-serine biosynthetic pathway, is far in the direction away from serine synthesis. As such, the enzyme is usually assayed in this direction. To

Full text of DOI:10.1021/acs.biochem.8b00074

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Article
Elucidation of a Self-Sustaining Cycle in E. coli l-Serine Biosynthesis
that Results in the Conservation of the Coenzyme, NAD+.
Gregory A. Grant
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00074 • Publication Date (Web): 01 Mar 2018
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Elucidation of a Self-Sustaining Cycle in E. coli
L
-Serine Biosynthesis that Results in the
Conservation of the Coenzyme, NAD+.
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Gregory A. Grant
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From the Departments of Developmental Biology and Medicine, Washington University School
of Medicine, 660 S. Euclid Avenue, Box 8103, St. Louis, Missouri 63110.
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Running Title: Conservation of NAD+ in E.coli Serine Biosynthesis
To whom correspondence should be addressed: Prof. Gregory A. Grant, Department of Developmental
Biology, Box 8103, Washington University School of Medicine, St. Louis, MO 63110. Phone 314-362-
3367, FAX 314-362-4698, email ggrant@wustl.edu.
Keywords:
ABSTRACT
The equilibrium of the reaction catalyzed by D-3-Phosphoglycerate Dehydrogenase (PGDH), the
first enzyme in the
L-serine biosynthetic pathway, is far in the direction away from serine synthesis. As
such, the enzyme is usually assayed in this direction. In order to easily assay it in the direction of
L-serine
synthesis, it can be coupled to the next enzyme in the pathway, phosphoserine aminotransferase (PSAT),
with the activity monitored by the conversion of NAD+ to NADH by PGDH. However, when PGDH from
several different species were coupled to PSAT, it was found that one of them, ecPGDH, conserves
coenzyme in the production of L
-serine by utilizing an intrinsic cycle of NAD+/NADH interconversion
coupled with the conversion of α-ketoglutarate (αKG) to α-hydroxyglutarate (αHG). Furthermore, the
cycle can be maintained by production of αKG by the second enzyme in the pathway, PSAT, and does
not require any additional enzymes. This is not the case for PGDH from another bacterial source, M.
tuberculosis, and a mammalian source, human liver, where net consumption of NAD+ occurs. Both NAD+
and NADH appear to remain tightly bound to ecPGDH during the cycle, effectively removing a
requirement for the presence of an exogenous coenzyme pool to maintain the pathway and significantly
reducing the energy requirement needed to maintain this major metabolic pathway.
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Introduction
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Nearly all organisms, from bacteria to mammals, contain a pathway for the production of
from the glycolytic intermediate, D-3-phosphoglycerate (PGA, 1-9). D-3-Phosphoglycerate
dehydrogenase (PGDH) initiates the entry of PGA into the pathway (2, 3, 5-9) by converting it to
L-serine
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phosphohydroxypyruvate (PHP) with the concomitant reduction of NAD+ (Scheme 1). PHP is then
converted to phosphoserine (PS) along with the conversion of glutamate (Glu) to α-ketoglutarate (αKG)
by phosphoserine aminotransferase (PSAT). Lastly, phosphoserine is converted to
in an irreversible reaction by phosphoserine phosphatase (PSP).
L-serine and phosphate
Scheme 1. The
phosphohydroxypyruvate; PS, phosphoserine; Ser,
dinucleotide; Glu, -glutamic acid; αKG, alpha-ketoglutarate; PGDH, D-3-phospoglycerate
dehydrogenase; PSAT, phosphoserine aminotransferase; PSP, phosphoserine phosphatase.
L
-Serine Biosynthetic Pathway. PGA, D-3-phosphoglycerate; PHP,
L
-serine; NAD+/NADH, nicotinamde adenine
L
L
-Serine occupies a pivotal position in carbon metabolism due to its role as a precursor for a wide
array essential metabolites (8-9). In addition to incorporation into proteins, -serine is central to the
production of many other metabolites such as glycine, cysteine, tryptophan, phosphatidyl -serine,
L
L
sphingolipids, purines, porphyrins and glyoxalate. As the major precursor to glycine, it also contributes
the one-carbon unit (C1) that is the donor in methylation reactions mediated by derivatives of
tetrahydrofolate and S-adenosyl methionine.
The genes encoding PGDH, PSAT, and PSP in Proteobacteria are designated serA1, serC, and
serB2, respectively. In Mycobacteria tuberculosis (Mtb) they are designated Rv2996c, Rv0884c, and
Rv3042c, respectively.
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Many bacteria also contain genes designated serA2 and serB1in Proteobacteria and Rv0728c and
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Rv0505c in Mtb. However, only serA1 and serB2 have been shown to be essential (10, 11). SerA2 is a
glycerate dehydrogenase (also called hydroxypyruvate reductase or glyoxylate dehydrogenase) that is
often claimed to be a second, non-phosphorylated pathway for serine biosynthesis that interconverts
D-
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hydroxypyruvate and D
-glycerate with the concomitant conversion of NADH and NAD+. However, as
pointed out by Snell (12) for mammalian systems, this is a misconception. This enzyme is located in the
mitochondria in mammals (13) and in the peroxisome in plants (9) and functions in a catabolic role in a
pathway that converts L-serine to D-2-phosphoglycerate in which hydroxypyruvate and D-glycerate are
intermediates. The serA2 gene in E. coli is also a hydroxypyruvate reductase (Figure S1) and is also not
likely to be involved in de novo serine biosynthesis in E. coli. This is supported by the observation that
when serA1 is knocked out, the bacteria require exogenous serine for growth (14). The "phosphorylated"
pathway, utilizing serA1, is therefore the only anabolic source of
Escherichia coli, for example, approximately 15% of the carbon assimilated when it is grown on glucose
passes through -serine before incorporation into biosynthetic products (15). serB1 has proven difficult to
L-serine in most organisms. In
L
express and isolate and has not been studied further (16, 17), but as mentioned earlier, it is not essential
for growth.
Interestingly, E. coli PGDH (ecPGDH) can utilize αKG as a substrate in the reverse direction (the
direction opposite that toward serine synthesis), converting it to α-hydroxyglutarate (αHG) with the
conversion of NADH to NAD+ (14). In addition to ecPGDH, the ability to use αKG as a substrate has also
been reported for Pseudomonas stutzeri (psPGDH) (19), Pseudomonas aeruginosa (paPGDH) (19), S.
cerevisiae (scPGDH) (20), and human PGDH (hsPGDH) (21). However, this is apparently not the case
for other PGDHs such as that from Mycobacterium tuberculosis (mtPGDH) (22) and Rattus norvegicus
(23).
NADH binds very tightly to ecPGDH and psPGDH. For ecPGDH, the Kd of binding has been
estimated to be less than 10 nM and NADH binding induces a conformational change that essentially
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locks the coenzyme in the protein (18). The Kd of binding for psPGDH is reported to be ~ 0.1 ꢀM by
isothermal titration calorimetry (19). The binding of substrate and coenzyme is ordered, but the order
differs among species. The order of binding for ecPGDH (34, 35) and psPGDH (19) is coenzyme before
substrate, while that for mtPGDH is substrate before coenzyme (36).
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Consistent with this, ecPGDH (34, 37) and psPGDH (19) contain at least 2 mols of very tightly
bound NADH per mol of enzyme tetramer, as measured by the fluorescence of NADH, when they are
isolated. The NADH bound to isolated ecPGDH cannot be removed by dialysis (34). Furthermore, the
bound NADH cannot be displaced by the addition of NAD+. Since NADH is produced in situ with each
turnover of the enzyme, this would seem to present a problem for the cell since NAD+ must bind to the
enzyme prior to substrate binding for the production of
efficiently produces -serine in vivo, some compensating mechanism must be operational. In a recent
review (38), Gusyatiner and Ziyatdinov hypothesized that αHG production from αKG by ecPGDH is
L-serine to proceed. Therefore, since E. coli
L
necessary to convert the bound NADH to NAD+ in order for the forward reaction to proceed toward
serine biosynthesis. However, this review presented the hypothesis only and no experimental results
L-
confirming it were included. In addition, a recent report by Zhang et. al. suggested that coupling between
psPGDH and a -2-hydroxyglutarate dehydrogenase (D2HGDH) drives -serine production (19).
Although they demonstrated that psPGDH was capable of converting αKG to αHG, they concluded that
robust coupling between psPGDH and D2HGDH drives -serine production in Pseudomonas.
Furthermore, since they were unable to find a D2HGDH homolog in E. coli, they concluded that other
unknown enzymes were involved in -2-HG catabolism in E. coli.
The investigation reported here presents experimental in vitro evidence that demonstrates that
αKG is not required to initiate -serine biosynthesis in E. coli and that a D2HGDH counterpart is also not
required for efficient recycling of αKG or the synthesis of -serine. On the contrary, ecPGDH utilizes an
D
L
L
D
L
L
internal cycle that preserves NAD+ by constantly interconverting the NADH produced back to NAD+
through a reaction with αKG that is produced by the next coupled enzyme in the pathway, PSAT. This
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investigation also demonstrates that in addition to tightly bound NADH, ecPGDH also contains tightly
bound NAD+ so that exogenous NAD+ is not required for the cycle to be functional in vitro. This work
also demonstrates that this is not the case for tbPGDH or hsPGDH, and that hsPGDH does not efficiently
utilize αKG as a substrate to produce αHG.
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EXPERIMENTAL PROCEDURES
PGDH, PSAT, and PSP from E. coli and M. tuberculosis are produced with hexa-histidine tags,
expressed in Escherichia coli BL21 DE3 cells, and purified as previously described (39). D-glycerate
dehydrogenase from E. coli (serA2) was cloned from E.coli DNA by PCR amplification as previously
described for tbPGDH (36). All reagents were purchased from Sigma Aldrich, Inc. The assays presented
in Table 1 were run in duplicate in 100 mM KPO4 buffer, pH 7.0, initiated with the addition of PGA, and
monitored at 340 nm for the production of NADH. Assays contained, 2.5 mM PGA, 1.25 mM NAD+, 200
mM Glu, and 6.8 mM αKG when present. Enzyme concentrations were 3.4 ꢀM ecPGDH, 0.4 ꢀM
tbPGDH, 1.8 ꢀM hsPGDH, and 0.5 ꢀM PSAT. The colorimetric phosphate assays were performed by a
modification of the assay of Chen et.al. (40) as reported on-line by Dunham
(Dunham.gs.washington.edu/MDphosphateassay.htm). NADH bound to ecPGDH was converted to
NAD+ by reaction with αKG followed by washing with 10 volumes of 50 mM MOPS buffer, pH 7.0
using an Amicon Ultra centrifugal filter with 10 kDa molecular weight cutoff. NAD+ bound to ecPGDH
was converted to NADH by reaction with PGA after washing with 10 volumes of 50 mM MOPS buffer,
pH 7.0 using an Amicon Ultra centrifugal filter with 10 kDa molecular weight cutoff. Fluorescence due to
NADH was measured by scanning from ~ 370-490 nm with excitation at 340 nm. The assays presented in
Table 2 were run in duplicate in 50 mM MOPS buffer pH 7.0, 5 mM MgCl2. When present, PSP was 1
ꢀM and all other reagent and enzyme concentrations were as listed for Table 1. Reactions in Table 2 were
initiated with the addition of PGA and allowed to run for 10 min. Then PSP was added and allowed to
incubate for 1 min before addition of phosphate assay reagent which stopped the reaction. The data in
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Figure 2 was produced in the same manner except the reaction was allowed to proceed for various times
prior to the addition of PSP. The data in Figure 5 was produced with PGDH, PSAT, and PSP present at
the beginning and allowed to run for 10 minutes before adding assay reagent to stop the reaction. Color
development proceeded for 1.5 h at 37ºC and the absorbance at 820 nm was recorded. Phosphate
concentration was determined by comparison to a standard curve. Absorbance due to NADH was
monitored at 340 nm in separate experiments.
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Kinetic parameters for PGDH in the forward reaction direction were determined by coupling
PGDH and PSAT and monitoring the production of NADH at 340 nm. The coupled reaction was
optimized so that the initial enzyme, PGDH, was rate limiting and the rate was determined with the linear
portion of the curve where the intermediate, PHP, reaches a steady state following an initial lag phase.
Plots of activity versus substrate concentration were fit to the Michaelis-Menten equation for a
cooperative process,
n
v = Vm[S]n / Km +[S]n
(Eq. 1)
where v is the velocity at substrate concentration S, Vm is the maximum velocity, Km is the substrate
concentration at half-maximal velocity, and n is the Hill coefficient.
RESULTS
E. coli PGDH does not show net production of NADH in the forward direction when coupled
with PSAT. The equilibrium of the reaction of PGDH interconverting PGA/NAD+ and PHP/NADH lies
far in the direction of PGA/NAD+ production (19, 41). It is very difficult to assay the enzyme in the
forward direction, and historically, fluorescence monitoring was necessary for sensitivity and hydrazine
was added, presumably to trap the PHP produced (41). At 25º C, the ratio of the rates of the reverse to the
forward reaction was reported to be 70 for ecPGDH (41). Because of this, catalytic activity assays are
most often conducted in the reverse reaction direction by measuring the disappearance of NADH.
However, theoretically, if the PGDH forward reaction is coupled to the second enzyme in the pathway,
PSAT (Scheme 1), a facile forward reaction for PGDH should be able to be observed by monitoring the
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absorbance of NADH produced. Coupled reactions between PGDH and PSAT have been utilized for
hsPGDH (42, 43) in screening chemical libraries for inhibitors and for psPGDH (19). However, neither
study reported the kinetic parameters. In order to better quantify the parameters of the forward coupled
reaction, this study investigated the forward kinetic parameters of PGDH from E. coli, M. tuberculosis,
and human liver. The first efficiently utilizes αKG as a substrate, the second does not, and the third has
been reported to use it (21), but only inefficiently. The results are shown in Table 1.
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Table 1
Kinetic Parameters of PGDH Forward Reaction Coupled with PSATa
Enzyme
Variable Substrate
Km
(mM)
kcat
kcat / Km
nH
(s-1)
(M-1s-1)
tbPGDH
PGA
NAD
0.54 ± 0.08
0.06 ± 0.01
1.3 ± 0.1
1.5 ± 0.1
2.4 x 103
2.5 x 104
0.8 ± 0.1
1.2 ± 0.1
hsPGDH
PGA
NAD
0.48 ± 0.10
0.49 ± 0.14
0.38 ± 0.02
0.42 ± 0.03
0.8 x 103
0.9 x 103
0.63 ± 0.06
0.67 ± 0.10
ecPGDH
PGA
NAD
NATBDb
NATBDb
a 50 mM MOPS pH 7.0.
b NATBD, not able to be determined by monitoring A340.
The forward reaction of mtPGDH and hsPGDH could be easily monitored by following the
production of NADH using its absorbance at 340 nm (Figure 1, Table 1, and Table 2, reaction 1, 2 and 3).
The kinetic parameters for the two enzymes are similar, however, hsPGDH displays a higher requirement
for NAD+ and marked negative cooperativity. On the other hand, the forward reaction assay for ecPGDH
showed no change in absorbance at 340 nm (Figure 1 and Table 2, reactions 4 and 5) even though the
reverse reaction using either αKG or PHP showed robust conversion of NADH to NAD+ (not shown),
indicating a very active enzyme preparation. The same thing is observed using ecPGDH that has been
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depleted of NADH (Table 2, reaction 6). Interestingly, when tbPGDH is added to the reaction mixture
containing ecPGDH, a robust increase in absorbance at 340 nm is observed (Figure 1, and Table 2,
reaction 7). Furthermore, the coupled reactions are not specific for the origin of the PSAT as ecPSAT and
tbPSAT yield similar results (not shown). It is particularly interesting to note that when ecPGDH is added
to the reaction mixture with tbPGDH (reaction 7), the rate of NADH production appears to be reduced
from that with tbPGDH alone (reactions 1 and 2), and it is completely eliminated with the addition of
αKG (reaction 8). This is likely due to the ability of ecPGDH to use αKG to convert NADH back to
NAD+.
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Figure 1. Left: Generation of NADH monitored by absorbance at 340 nm. Blue, reaction contains PGA,
NAD+, and either ecPGDH or ecPGDHapo; Red, reaction contains PGA, NAD+, ecPGDH, PSAT, and Glu;
Green, reaction contains PGA, NAD+, ecPGDH, PSAT, Glu, and tbPGDH. Abbreviations are the same as
used in the text. Right: Fluorescence emission spectra of ecPGDH excited at 340 nm with bound NADH
(blue) and after NADH has been oxidized by the addition of αKG (red).
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Table 2
Catalytic Activity of PGDH Reaction Mixtures
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Rx#
Reagentsa
Enzymesa
ꢀM/minb
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1.
2.
3.
4.
5.
6.
7.
8.
PGA NAD+ Glu
PGA NAD+ Glu
PGA NAD+ Glu
PGA NAD+ Glu
PGA NAD+ Glu
PGA NAD+ Glu
PGA NAD+ Glu
PGA NAD+ Glu
PSAT
PSAT
PSAT
PSAT
PSAT
PSAT
PSAT
PSAT
tbPGDH
tbPGDH
hsPGDH
38±1
34±1
45±1
<0.1
<0.1
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αKG
αKG
αKG
ecPGDH
ecPGDH
ecPGDHapo <0.1
tbPGDH
tbPGDH
ecPGDH
ecPGDH
22±1
<0.1
a PGA, D-3-phosphoglycerate; NAD+, oxidized nicotinamide adenine dinucleotide; Glu, glutamic acid;
αKG, α-ketoglutarate; PSAT, phosphoserine aminotransferase; tbPGDH, D-3-phosphoglycerate
dehydrogenase from M. tuberculosis; ecPGDH, D-3-phosphoglycerate dehydrogenase from E. coli;
ecPGDHapo, D-3-phosphoglycerate dehydrogenase from E. coli that has been enzymatically depleted of
NADH. hsPGDH, D-3-phosphoglycerate dehydrogenase from human liver.
c Generation of NADH measured by an increase in absorbance at 340 nm.
Production of phosphoserine in the forward reaction direction. Based on the above
observations, one can hypothesize that when ecPGDH is present, net production of NADH is not
observed because it is efficiently converted back to NAD+ in the presence of αKG. The reduced
production of NADH in reaction 7 is likely due to ecPGDH using the relatively low levels of αKG
produced by the PSAT reaction to convert some, but not all, of the NADH produced back to NAD+. The
net production of NADH is eliminated if excess αKG is supplied (reaction 8). If this conclusion is correct,
even if no net production of NADH is observed spectrophotometrically, phosphoserine should be
produced along with αKG in the PSAT reaction (Scheme 1). To test this, phosphoserine phosphatase
(PSP) is added to various reaction mixtures after an initial period of incubation and the amount of
phosphate produced as a consequence of phosphoserine conversion to serine (Scheme 1) is measured
colorimetrically. The net production of NADH is monitored by change in absorbance at 340 nm (Table 3).
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Table 3
Phosphoserine and Net NADH Production by Reaction Mixtures
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Substratesa
Enzymesa
[PO4] ꢀMb
ꢀM/minc
8
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1. PGA NAD+ Glu
PSAT
PSAT
PSAT
PSAT
PSAT
ecPGDH PSP
ecPGDH PSP
ecPGDH PSP
tbPGDH PSP
tbPGDH PSP 111.0 ± 2.1
ecPGDH
PSP
PSP
ecPGDH
ecPGDH PSP
30.2 ± 2.4
62.8 ± 1.6
23.3 ± 0.3
90.6 ± 4.2
<0.1
<0.1
<0.1
65.6 ± 1.8
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2. PGA NAD+ Glu αKG
3. PGA
Glu
4. PGA NAD+ Glu
5. PGA NAD+ Glu αKG
<0.1
6. PGA
0.25 ± 0.06
1.0 ± 0.1
0.91 ± 0.02
1.9 ± 0.3
7. PGA NAD+ Glu
8. PGA NAD+ Glu
9. PGA NAD+ Glu
αKG
αKG
αKG
PSAT
PSAT
<0.1
<0.1
<0.1
a PGA, D-3-phosphoglycerate; NAD+, oxidized nicotinamide adenine dinucleotide; Glu, glutamic acid;
αKG, α-ketoglutarate; PSAT, phosphoserine aminotransferase; tbPGDH, D-3-phosphoglycerate
dehydrogenase from M. tuberculosis; ecPGDH, D-3-phosphoglycerate dehydrogenase from E. coli; PSP,
phosphoserine Phosphatase.
b Phosphate determined colorimetrically.
c Generation of NADH measured by an increase in absorbance at 340 nm.
In Table 3, the principal reactions are in bold print with the other reactions serving as controls. Reaction 1
shows that even without added αKG, the reaction with ecPGDH produces significant levels of
phosphoserine but net production of NADH is not observed spectrophotometrically. Reaction 2 shows
similar results when αKG is added and shows that the level of phosphoserine produced increases.
Remarkably, reaction 3 shows that significant phosphoserine is produced even in the absence of added
NAD+ and αKG. Furthermore, the level of phosphoserine produced is far more than the level of
intrinsically bound NADH on the enzyme. Reaction 4 shows that when tbPGDH is used instead of
ecPGDH, both phosphoserine and net NADH production is observed. However, reaction 5 shows that
when ecPGDH is included along with tbPGDH in the presence of added αKG, phosphoserine production
is essentially the same but no net production of NADH is seen as was the case with reaction 8 in Table 1.
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Reaction 6 demonstrates that PGA is not a substrate for PSP. Reactions 7-9 show that ecPGDH, PSAT,
and PSP are required for phosphate production.
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ecPGDH also contains intrinsically bound NAD+. The unexpected observation that
phosphoserine is produced even in the absence of added NAD+ and αKG was explored further by
monitoring the reaction with respect to time. Figure 2 shows that phosphoserine accumulates as a function
of time and eventually reaches a plateau. Therefore, the color produced cannot be attributed to the
presence of phosphate in the initial reaction mixture. Since NAD+ is required in the net conversion of
PGA to PSP, the only logical explanation for this observation is that ecPGDH contains intrinsically bound
NAD+.
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Figure 2. Time course of phosphate generation by ecPGDH in the absence of added NAD+ and αKG.
Reaction mixtures contained PGA, glutamate, ecPGDH, PSAT, with (green circles) or without (blue
squares) PSP. The reaction is started by the addition of PGA and allowed to proceed. At the designated
times, aliquots are removed and added to the phosphate colorimetric reagent to stop the reaction and
develop color. Data is the average of duplicate determinations and error bars represent ± SEM. If error
bars are not evident, they are within the area of the symbol.
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To test this, the intrinsically bound NADH in a fresh isolate of ecPGDH was converted to NAD+ by the
addition of αKG. When conversion was complete as monitored by fluorescence analysis, the enzyme was
extensively washed by repeated cycles of buffer exchange using an Amicon Ultracentrifugal filter with 10
kDa molecular weight cutoff. After washing, the fluorescence of the resulting protein showed that it did
not contain any appreciable amounts of NADH (Figure 3). However, upon addition of PGA to the sample,
a very strong fluorescence signal was generated indicating the production of NADH. Therefore, since
NAD+ was not added to the reaction but is required for the generation of NADH coupled to PGA
conversion to PHP, it must have been bound to the enzyme initially.
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Figure 3. Fluorescence spectra of extensively washed ecPGDH and PGA. Black, PGA alone; Blue,
ecPGDH alone; Green, ecPGDH with added PGA. Excitation wavelength is 340 nm.
To further test this observation, concentration dependent analyses were performed by varying
either PGA or NAD+. Figure 4 shows that there was substantial production of phosphoserine (as measured
by phosphate production after addition of PSP) with no added NAD+, as previously observed, and that the
phosphoserine production increased only by approximately 20% with the addition of 1250 ꢀM NAD+ and
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then remained level after that. Varying the concentration of PGA in the presence and absence of NAD+
also showed only approximately a 20% difference in maximum level of phosphoserine produced. These
observations indicate that the occupancy of the intrinsically bound NAD+ was approximately 80% in the
isolated and washed ecPGDH used in this example.
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Figure 4. Concentration dependence of the reaction. Left: NAD+ is varied from 0 to 1250 ꢀM in the
presence of 5 mM PGA and 200 mM Glu. Right: PGA is varied from 0 to 5 mM in the presence (3) and
absence (5) of 1250 ꢀM NAD+ with 200 mM Glu in both. The reaction contained ecPGDH and PSAT.
PSP was added after 10 min. Data is the average of duplicate determinations and error bars represent ±
SEM. If error bars are not evident, they are within the area of the symbol.
In the experiments presented so far, PSP was added after the reactions were allowed to progress
for 10 minutes. The experiments were designed in this way to prevent feedback inhibition of the ecPGDH
by the serine produced. Figure 5 shows that if PSP is added at the start of the reaction, substantially less
serine is produced (as measured by phosphate production from conversion of phosphoserine to serine plus
phosphate). This is consistent with the in situ inhibition of the reaction by serine and verifies that the
entire serine biosynthetic pathway is functional in these reactions.
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Figure 5. Concentration dependence of the reaction in the presence and absence of phosphoserine
phosphatase at the beginning of the reaction. The reactions contained 1250 ꢀM NAD+ and 200 mM Glu in
both as well as ecPGDH and PSAT. PGA is varied in the presence (5) and absence (3) of PSP at the
start of the reaction. Data is the average of duplicate determinations and error bars represent ± SEM. If
error bars are not evident, they are within the area of the symbol.
DISCUSSION
A simple attempt to monitor the activity of PGDH in the forward reaction direction by coupling
its activity to PSAT resulted in several unexpected observations that further emphasize the differences in
serine metabolism among species. Two major conclusions are evident. First, ecPGDH conserves
coenzyme in the production of L
-serine by utilizing an intrinsic cycle of NAD+/NADH interconversion
that can be maintained by production of αKG by the second enzyme in the pathway, PSAT (Scheme 2).
This is not the case for PGDH from another bacterial source, M. tuberculosis, and a mammalian source,
human liver, where net consumption of NAD+ occurs. Second, both NAD+ and NADH remain bound to
ecPGDH during the cycle, effectively removing a requirement for the presence of an exogenous
coenzyme pool to maintain the pathway. That NADH remained tightly bound to ecPGDH was already
known, but this is the first demonstration that the same is true for NAD+.
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PSAT
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Glu
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PS
NADH
αKG
αHG
PHP
PGA
PSP
PGDH
PO4
NAD+
Ser
Scheme 2. The proposed NAD+/NADH cycle in the production of
phosphoglycerate; PHP, phosphohydroxypyruvate; PS, phosphoserine; Ser,
nicotinamde adenine dinucleotide; Glu,
L
-serine by ecPGDH. PGA, D-3-
-serine; NAD+/NADH,
-glutamic acid; αKG, alpha-ketoglutarate; αHG, alpha-hydroxy
L
L
glutarate; PGDH, D-3-phospoglycerate dehydrogenase; PSAT, phosphoserine aminotransferase; PSP,
phosphoserine phosphatase. The box represents E. coli PGDH with bound NADH and NAD+ as isolated.
Thus, the ability of ecPGDH to utilize αKG as a substrate provides for a mechanism where
biosynthesis of L
-serine proceeds efficiently without a net consumption of NAD+. In addition, for every
molecule of PGA consumed a molecule of αKG is formed by PSAT, the second enzyme in the pathway.
Under the conditions of the reaction used in this investigation, the level of αKG produced by PSAT is
sufficient to maintain the cycle. As a consequence of this, although αKG is not strictly required to initiate
serine synthesis as suggested by Gusyatiner and Ziyatdinov, its conversion to αHG is an indispensable
reaction in maintaining the production of -serine in E. coli. The intracellular concentration of αKG in E.
L
coli has been measured at 440 ꢀM (45) and the Km for αKG in phosphate buffer is 88 ꢀM (18). So,
although the data suggest that an αKG pool is not required to initiate the cycle, there is ample αKG in
vivo to drive the cycle. This cycle does not occur with tbPGDH and presumably other enzymes that
cannot utilize αKG as a substrate.
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A question that is relevant to this observation is whether or not the enzymes in the pathway form
a complex that allows the efficient transfer of αKG from one to another without being released into
solution. The fact that PSAT from E. coli and M. tuberculosis can be used interchangeably in vitro,
without any appreciable difference in the results, would seem to suggest not. However, one cannot
dismiss the possibility that there has been an evolutionary conservation of the structural elements that
function in the formation of a putative enzyme complex between PGDH and PSAT. Studies are ongoing
to address this.
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ecPGDH is isolated with essentially irreversibly bound, but sub-stoichiometric amounts of
NADH, and the remaining subunits that do not contain NADH, contain NAD+ and catalyze the
conversion of PGA to PHP. In this respect, the tightly bound coenzyme in ecPGDH acts somewhat like a
prosthetic group in that it is essentially irreversibly bound to the enzyme as opposed to being transiently
associated with the enzyme as is usually the case for NADH with dehydrogenases. However, since the
reverse reaction utilizing either αKG or PHP in the presence of NADH is robust and continuously turns
over, resulting in net consumption of NADH, it would appear that NADH is capable of displacing bound
NAD+ while it has been shown that the opposite is not the case (18).
ecPGDH can be viewed as a dimer of dimers, where residues from each subunit of the dimer
contribute to the catalytic site. Previous studies (44) used engineered asymmetric hybrid tetramers to
demonstrate that the catalytic dimers can act independently of each other. Therefore, the mechanism is
likely one where two active sites can catalyze the conversion of PGA and NAD+ to PHP and NADH,
while the other two can catalyze the conversion of αKG and NADH to αHG and NAD+ and vice-versa as
the reactions proceed. This proposal is illustrated schematically in Figure 6.
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NAD+
NADH
NAD+
NADH
NAD+
NAD+
NADH
PGA
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=
NADH
αKG
αHG
PHP
NAD+
αHG
NADH
PHP
NADH
αKG
NAD+
PGA
NADH
PHP
NAD+
αHG
NAD+
PGA
NADH
αKG
Figure 6. The ecPGDH tetramer is illustrated as a ribbon diagram (PDB: 1YBA) on the left with the
arrows indicating the active site clefts. The subunits are colored alternately yellow and blue. On the right
is a diagram depicting how the subunits may function in a flip-flop manner to simultaneously catalyze the
conversion of the substrate pairs, NADH/αKG and NAD+/PGA. Note that since the tetramer is
symmetrical, the top two representations are equivalent and can be superimposed with a 180º rotation.
Zhang et. al. recently made similar observations for PGDH from Pseudomonas stutzeri (19). That
investigation showed that psPGDH was capable of using αKG as a substrate, as had been previously
reported for ecPGDH, and suggested that a similar cycle may be operative. However, they concluded that
PGA was poorly oxidized in the absence of added αKG, and that a 2-hydroxyglutarate dehydrogenase
(D2HGDH) was required in Pseudomonas species for efficient PGA oxidation. On the contrary, our data
clearly show that the αKG generated by PSAT, the next enzyme in the pathway, was sufficient to produce
phosphoserine at levels comparable to that produced by tbPGDH, an enzyme that does not utilize αKG,
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under similar conditions when NAD+ is supplied to the latter enzyme. Thus, there is no indication that a
D2HGDH enzyme is required in E. coli. This is also consistent with the observation by Zhang et. al. that
E. coli does not appear to contain a gene that codes for a similar D2HGDH.
9
The reactions catalyzed by PGDH and PSAT in vivo would eventually reach an equilibrium state,
but since the reaction catalyzed by PSP is irreversible, this enzyme acts as a sink to draw the entire
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pathway in the direction of
biosynthetic pathway are present at the outset, a natural attenuation of the production of
accomplished by the feedback mechanism wherein -serine inhibits the activity of PGDH. Under the
conditions of the assay, a steady state level of -serine is produced at the lowest concentration of PGA
used. This is consistent with an apparent Km of 0.15 ± 0.04 mM determined from the data in Figure 4. The
concentration of -serine produced, approximately 10 ꢀM, is equal to the IC50 of -serine for ecPGDH in
non-phosphate buffers. Of course, -serine will continuously be converted to other metabolites in vitro,
L
-serine production. As expected, when all three enzymes of the
L-serine
L-serine is
L
L
L
L
L
so the situation will be much more dynamic.
The relatively large amount of assimilated carbon, estimated at 15% (15), that passes through L-
serine when E. coli is grown on glucose would impose a large demand on the organism for NAD+. This
investigation demonstrates that E. coli has evolved to relieve this metabolic burden by a mechanism
where it efficiently recycles NAD+/NADH during the normal course of
L-serine production by utilizing a
metabolite that is naturally produced by the biosynthetic pathway itself, αKG. Furthermore, the cycle is
essentially self-sustaining and is not dependent on any other enzymatic activity.
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ACKNOWLEDGMENT
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The author thanks Xiao Lan Xu for excellent technical assistance.
CONFLICT OF INTEREST
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The author declares that he has no conflicts of interest with the contents of this article.
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AUTHOR CONTRIBUTIONS
GAG conceived and directed the study and wrote the paper
Funding
Funding was provided by the Department of Developmental Biology, Washington University School of
Medicine.
Supporting Information. Supporting Information is available free of charge on the ACS Publications
website.
A Figure showing the velocity versus substrate concentration plot for SerA2, a D-glycerate
dehydrogenase (also called hydroxypyruvate reductase or glyoxylate dehydrogenase) is provided.
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44. Grant, G. A., Xu, X. L., and Hu, Z. (2004) Quantitative relationships of site to site interaction in
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J. Bio. Chem. 279, 13452-13460.
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Absolute mewtabolic concentrations and implied enzyme active site occupancy in Escherichia
coli. Nature Chem. Biol. 5, 593-599.
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Biochemistry
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