36NISR Research GRANT
2010 Research Grant

Development of transporter-control technology
for fermentation
Keietsu ABE
Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University

Research aims
In some strains of the lactic acid bacterium Tetragenococcus halophilus, a proton-motive force (PMF) is generated by the combined action of an intracellular L-aspartate
decarboxylation reaction, catalyzed by an L-aspartate-4-decarboxylase (AspD, EC 4.1.1.12), and an electrogenic aspartate1 : alanine0 exchange reaction, catalyzed by an aspartate : alanine antiporter (AspT, TC# 2.A.81.1.1). The
PMF generated is sufficiently high to drive ATP synthesis
via the bacterial F0F1-ATPase. This combination of PMF
and ATP synthesis has been proposed as a proton-motive
metabolic cycle (Fig. 1). Such decarboxylation reactions
are thought to be advantageous for cells because they generate metabolic energy and regulate intracellular pH1,2). In
previous work with proteoliposomes, we found that the aspartate:alanine exchange catalyzed by AspT is electrogenic1–3). AspT is classified as a conventional secondarytransport protein and belongs to the newly classified aspartate:alanine exchanger (AAEx) family (TC# 2.A.81) oftransporters (http://www.tcdb.org/index.php). AspT is amembrane protein containing 543 amino acids (57.2 kDa).
AspT has a unique topology (Fig. 2)3) and the transmem-brane domain (TM) 3 participates in the formation of a hydrophilic cleft in the membrane, implicating the TM3 in theligand-induced conformational changes4). Previously, wedeveloped a solubilization and purification scheme of AspT
by using n-dodecyl-b -D-maltoside (DDM)4). Here, we characterized, in detail, the kinetic properties of AspT by analyzing the transport kinetics of reconstituted AspT undervarious conditions. In addition, to reveal the substrate
recognition mechanism of AspT, we performed competition
analysis with a series of amino acids and the aspartate
analogs. The competition analysis suggested that the putative L-aspratate and L-alanine binding sites are independently located in the substrate translocation pathway ofAspT.
Methods
Reconstitution and Transport of Purified AspT-His6
Purified AspT-His6 was reconstituted in a final volume of1 ml with 800 m l of detergent DDM extracts (10 to 20 m g ofpurified AspT protein) (or control lipid extract), 130 m l ofbath-sonicated liposomes (5.9 mg of E. coli phospholipid),and 18 m l of 15% 1-O-n-Octyl-b -D-glucopyranoside, withFig. 1. (A) Energy generation coupled with L-aspartate decarboxylation, and (B) the structure of the asp operon encoding L-aspartate-4-decarboxylase andL-asparate : L-alanine antiporter.

37Fig. 2. Membrane topology of AspT and 3D-model of the large cytoplasmic loop region of AspT. AspT possesses 10 transmembrane helices, a large cytoplasmic loop between TM5 and TM6, and N- and C-termini facing periplasm.

the balance made up by 50 mM potassium phosphate (pH7). After incubation for 20 min on ice, proteoliposomes (orcontrol liposomes) were formed at room temperature (RT)by rapid injection of the mixtures into 20 ml of loading
buffer containing buffer solution and suitable counter substrate such as 100 mM L-aspartate or L-alanine as the potassium salt for L-aspartate or L-alanine exchange reaction.
Buffer components were changed in each experiment. The
substrate-loaded proteoliposomes (or liposomes) were kept
at RT for 20 min. To assess L-aspartate transport by L-[3H]labeled aspartate or alanine-loaded particles, proteoliposomes were diluted 20-fold from the concentrated stock
into an appropriate volume of assay buffer (50 mM potassium phosphate [pH 7], 100 mM K2SO4). After 1 to 3 minof pre-incubation at 25°C, L-[3H]aspartate or L-[3H]alanine
was added to a final concentration of 100 m M; at various
times, 50- to 100-m l aliquots were removed for membrane
filtration with 0.22-m m-pore-size GSTF Millipore filter(Millipore Co., Billerica, MA). The membrane filters werewashed twice with 5 ml of assay buffer5).
Filtration Assay of Substrate Transport
Initial rates of L-[3H]aspartate or L-[3H]alanine entry
were measured in duplicate at 25°C by means of a filtration
assay5). Proteoliposomes were applied directly to the centerof a 0.22-m m pore-size GSTF Millipore filter and washedtwice with 5 ml of chilled assay buffer (50 mM potassium
phosphate [pH 7], 100 mM K2SO4). Upon release of thevacuum, the proteoliposomes were covered with assay
buffer that was pre-incubated at 25°C with 0.1 mM L[3H]aspartate or L-[3H]alanine, and the reaction was terminated after 1 min by use of filtration and washing.
Results
Kinetic Analysis of AspT Transport
A kinetic study was undertaken, relying on samples fil-Table 1.

L-Asp
L-Ala
D-Asp
D-Ala

Kinetic parameters of AspT

Km
[mM]

Vmax
[m mol/min/mg protein]

Vmax /Km

0.350.03
262
0.0980.000
3.30.2

175211559
3.80.3
3.30.4

500
6.039
1.0

tered after a 10 s reaction, to estimate the initial velocitiesof the L-aspartate and L-alanine self-exchange reactions. A
Hanes-Woolf plot was used to determine the Michaelis constant (Km) and the maximum reaction (Vmax) (Table 1). We
found that the L-aspartate self-exchange reaction had a
Michaelis constant (Km) of 0.350.03 mM and a maximum
velocity of 17521 m mol min1 mg1 of protein and that
the L-alanine self-exchange reaction had a Km of 262 mM
and a maximum velocity of 1559 m mol min1 mg1 ofprotein (meanS.E.). L-Aspartate had a higher affinity thanL-alanine for AspT, but the L-aspartate and L-alanine selfexchange reactions had almost the same maximal velocities(Vmax). Thus, the L-alanine self-exchange reaction showedalmost the same transport rate as that of the L-aspartate
self-exchange reaction in the presence of sufficiently highconcentrations of L-alanine. A kinetic study of D-aspartateand D-alanine self-exchange reactions was also undertaken;
the results are summarized in Table 1. D-Asparate and Dalanine showed a higher affinity than L-aspartate and L-alanine, respectively, for AspT. On the other hand, the L-isomers had higher Vmax values than those of the D-isomers.
These results suggest that the D-isomers form a tight andstable complex with AspT, with slow dissociation rates andlow Vmax values. Thus, AspT recognizes not only L-aspartate and L-alanine, but also their D-isomers, as substrates.

38

Substrate Screening of AspT
To examine the substrate specificity of AspT, substratecompetition experiments were performed by measuring theuptake of L-[3H]aspartate and L-[3H]alanine in self-exchange reactions in the presence of 15 mM of various unlabeled amino acids. L-Asparagine and L-cysteine strongly inhibited L-[3H]aspartate uptake (relative accumulation of L[3H]aspartate: 10% to 30%) whereas L-glutamate and Ltryptophan moderately inhibited L-[3H]aspartate uptake(relative accumulation of L-[3H]aspartate: 70% to 80%).
None of the amino acids completely inhibited L-[3H]alanine
uptake. L-Asparagine and L-serine considerably inhibited L[3H]alanine uptake (relative accumulation of L-[3H]alanine:40% to 60%). L-Phenylalanine, L-arginine, L-leucine, Lthreonine, D-serine, L-methionine, b -alanine, and D-cycloserine moderately inhibited L-[3H]alanine uptake (relative accumulation of L-[3H]alanine: 60% to 80%). These results suggest that AspT recognizes as substrates not only Laspartate, L-alanine, D-aspartate, and D-alanine, but also
many other D- and L-amino acids, such as L-serine and Lcysteine. L-cysteine and L-serine had unique inhibitory effects. L-[3H]aspartate uptake was strongly inhibited by Lcysteine; however, L-[3H]alanine uptake was not inhibited.
On the other hand, L-serine apparently inhibited L-[3H]alanine uptake and barely inhibited L-[3H]aspartate uptake.
Inhibitory effect of L-cysteine, L-serine, and aspartate
analogs
To evaluate the inhibitory effects of L-cysteine and L-serine, we performed a Dixon–Webb plot analysis of data fromsamples that were filtered after a 10-s reaction to estimate
initial velocities. External substrate concentrations were adjusted to 0.07 mM, 0.35 mM (= Km value), and 0.7 mM for
L-aspartate self-exchange, and 5.8 mM, 26 mM (=Kmvalue), and 58 mM for L-alanine self-exchange. The threelines in the Dixon plot crossed at one point, demonstratingcompetitive inhibition of L-aspartate and L-alanine by Lcysteine and L-serine, respectively. The calculated inhibition constant values (Ki) were 2.00.2 mM (L-aspartate exchange inhibited by cysteine), 202.7 mM (L-alanine exchange inhibited by cysteine), and 5.31.7 mM (L-alanineexchange inhibited by serine) (Table 2). The Ki of L-serine
for L-aspartate self-exchange was too large to measure, suggesting that serine is a specific inhibitor of alanine self-exchange but not of aspartate self-exchange. These analyses
reveal that the mechanism of inhibition of L-aspartate selfexchange and L-alanine self-exchange by L-cysteine and Lserine is competitive inhibition. Because the Km of L-serine
to AspT was larger than the Km of L-aspartate to AspT but
smaller than the Km of L-alanine to AspT, the apparent selective inhibitory effect of L-serine on L-alanine self-exchange can be explained by the difference in the Km valuesof L-serine and L-alanine. However, the Km of L-cysteine
was larger than that of L-aspartate; this cannot explain theapparent selective inhibition of L-aspartate self-exchangewith L-cysteine.
To further examine the selective inhibition of L-aspartate
self-exchange with L-cysteine, we used substrate analogs
for both cysteine and aspartate, namely L-CSA, L-CA, andTable 2.

Ki values for compounds competitive for L-Asp or L-Ala uptake

L-cysteine
L-serine
L-CSA
L-CA
D-CA

L-Asp self-exchange

L-Ala self-exchange

Ki [mM]

IC50 [mM]

Ki [mM]

IC50 [mM]

2.00.2
N.D.*
0.590.08
0.290.05
0.140.02

8.9
N.D.*
0.83
0.47
0.19

202.7
5.31.7280140
5.00.2

652828169.0* N.D., not determined. The Ki value of L-Ser for L-Asp self-exchange
was too large.
D-CA.

We measured L-aspartate and L-alanine self-exchange in their presence. L-CSA, D-CA, and L-CA inhibited
both the L-aspartate and the L-alanine self-exchange reaction via AspT (Table 2). Dixon plot analyses revealed thecompetitive inhibition of L-aspartate and L-alanine by theanalogs. D-CA was the strongest inhibitor of both the L-aspartate and the L-alanine self-exchange reactions. All threeanalogs markedly inhibited L-aspartate self-exchange but
only weakly inhibited L-alanine self-exchange.
Conclusion
T. halophilus AspT catalyzes the electrogenic exchangeof L-asparate1 with L-alanine0. Here we show that thebinding sites of L-aspartate and L-alanine are independently
present in AspT by means of the kinetic analyses. We purified AspT-His6 and characterized its kinetic properties
when reconstituted in liposomes (Km0.350.03 mM forL-aspartate, Km0.0980 mM for D-aspartate, Km262mM for L-alanine, Km3.30.2 mM for D-alanine). Competitive inhibition by various amino acids of L-aspartate orL-alanine in self-exchange reactions revealed that L-cysteine
selectively inhibited L-aspartate self-exchange but only
weakly inhibited L-alanine self-exchange. Additionally, Lserine selectively inhibited L-alanine self-exchange butbarely inhibited L-aspartate self-exchange. The aspartate
analogs L-CSA, L-CA, and D-CA competitively andstrongly inhibited L-aspartate self-exchange compared with
L-alanine self-exchange. Taken together, these kinetic datasuggest that the putative binding sites of L-aspartate and Lalanine are independently located in the substrate translocation pathway of AspT.
References
1)

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