phosphotransacetylase structural domains and analysisof key compounds involved in activity regulation Valeria Alina Campos-Bermudez, Federico Pablo Bologna, Carlos Santiago Andreo and Ma
Trang 1phosphotransacetylase structural domains and analysis
of key compounds involved in activity regulation
Valeria Alina Campos-Bermudez, Federico Pablo Bologna, Carlos Santiago Andreo and
Marı´a Fabiana Drincovich
Centro de Estudios Fotosinte´ticos y Bioquı´micos (CEFOBI), Universidad Nacional de Rosario, Argentina
Introduction
The successful adaptation of Escherichia coli to
nutri-tional changes depends primarily on metabolic
switches from programs that allow rapid growth on
abundant nutrients to others that permit survival in
their absence One important switch, called ‘the acetate
switch’, involves the transition from the production to the utilization of acetate from the medium [1] During exponential growth on rich medium, E coli cells excrete acetate into the environment as a way, among other reasons, to recycle CoA and regenerate NAD+,
Keywords
acetyl-phosphate; activity regulation;
Escherichia coli; phosphotransacetylase;
protein domain
Correspondence
M F Drincovich, Suipacha 531, 2000
Rosario, Argentina
Fax: +54 341 4370044
Tel: +54 341 4371955
E-mail: drincovich@cefobi-conicet.gov.ar
(Received 15 January 2010, revised
11 February 2010, accepted 12 February
2010)
doi:10.1111/j.1742-4658.2010.07617.x
Escherichia coli phosphotransacetylase (Pta) catalyzes the reversible inter-conversion of acetyl-CoA and acetyl phosphate Both compounds are critical in E coli metabolism, and acetyl phosphate is also involved in the regulation of certain signal transduction pathways Along with acetate kinase, Pta plays an important role in acetate production when E coli grows on rich medium; alternatively, it is involved in acetate utilization
at high acetate concentrations E coli Pta is composed of three different domains, but only the C-terminal one, called PTA_PTB, is specific for all Ptas In the present work, the characterization of E coli Pta and deletions from the N-terminal region were performed E coli Pta acetyl phosphate-forming and acetyl phosphate-consuming reactions display dif-ferent maximum activities, and are difdif-ferentially regulated by pyruvate and phosphoenolpyruvate These compounds activate acetyl phosphate production, but inhibit acetyl-CoA production, thus playing a critical role in defining the rates of the two Pta reactions The characterization
of three truncated Ptas, which all display Pta activity, indicates that the substrate-binding site is located at the C-terminal PTA_PTB domain However, the N-terminal P-loop NTPase domain is involved in expres-sion of the maximal catalytic activity, stabilization of the hexameric native state, and Pta activity regulation by NADH, ATP, phosphoenol-pyruvate, and pyruvate The truncated protein Pta-F3 was able to com-plement the growth on acetate of an E coli mutant defective in acetyl-CoA synthetase and Pta, indicating that, although not regulated by metabolites, the Pta C-terminal domain is active in vivo
Abbreviations
AckA, acetate kinase; Acs, acetyl-CoA synthetase; CDD, Conserved Domain Database; IPTG, isopropyl thio-b- D -galactoside;
PEP, phosphoenolpyruvate; Pta, phosphotransacetylase.
Trang 2producing ATP [1] On the other hand, during the
transition to the stationary growth phase, the
machin-ery responsible for acetate assimilation is activated,
and the cells begin to utilize acetate instead of
excret-ing it
Acetate production and utilization are catalyzed by
different metabolic pathways in E coli Whereas
ace-tate utilization depends primary on acetyl-CoA
synthe-tase (Acs; EC 6.2.1.1), acetate production is catalyzed
by two enzymes: acetate kinase (AckA; EC 2.7.2.1)
and phosphotransacetylase (Pta; EC 2.3.1.8) (Fig 1A)
Acs is the high-affinity system for acetyl-CoA
synthe-sis, and the enzyme catalyzes an irreversible pathway,
owing to intracellular pyrophosphatases that remove
pyrophosphate (Fig 1) [2] However, the Pta–AckA
pathway is reversible, acetyl phosphate being an
inter-mediate of this pathway (Fig 1A) On the other hand,
the reversible Pta–AckA pathway can also assimilate
acetate [3], but only at high concentrations of this
compound
Two classes of Ptas can be found among
micro-organisms: PtaIs, which are nearly 350 amino acids
in length; and PtaIIs, which are twice as long as
PtaIs (nearly 700 amino acids in length) [4,5] These
two types of protein share about 40% identity
Although several crystal structures of PtaIs have
been analyzed [6–8], there is as yet no crystal study
on the larger isoenzymes From sequence alignment
among the different Ptas, it is clear that PtaIs share
homology with the C-terminal domain of PtaIIs
Thus, the active site of PtaIIs is probably located at the C-terminal end of the protein, and the role of the PtaII N-terminal domain has not yet been com-pletely resolved
Recently, PtaII from Salmonella enterica and sev-eral single amino acid variants were characterized [5] With regard to the biochemical characterization
of PtaII from E coli, an earlier investigation showed activity regulation by nucleotides, NADH, and pyru-vate [9] The study of this enzyme is relevant because, together with AckA, it catalyzes the conver-sion of acetyl-CoA to acetate via acetyl phosphate Acetyl phosphate participates in the regulation of certain two-component signal transduction pathways, and also protects cells against carbon starvation [1,10] Moreover, Pta has been suggested to act as a sensor and⁄ or response regulator for the intracellular acetyl-CoA⁄ CoA concentration ratio [3] Thus, in this work, we focused on the biochemical character-ization of E coli Pta and set out to investigate the function of its N-terminal domain by the construc-tion and analysis of three E coli Ptas with deleconstruc-tions from the N-terminal region The results obtained indicate that, although the substrate-binding site is located in the C-terminal domain, the E coli Pta N-terminal domain is involved in stabilization of the hexameric native structure, in expression of the max-imum catalytic activity, and in allosteric regulation
by NADH, ATP, pyruvate, and phosphoenolpyruvate (PEP)
Acetyl-CoA
Acetate
Pta
Acetyl-AMP Acetyl-P
Acs
Pi
CoA
ADP
ATP
AMP CoA
PPi
ATP
2Pi
PPasa
Glucose
PEP
Pyruvate
Acetyl-CoA
Acetate
CoA
NADH NAD +
CO2
Acetyl-P
Pta
Pi
CoA
Pi
CoA
ADP ATP
Fig 1 (A) Pathways of acetate activation and production in E coli Acs catalyzes an irreversible pathway for high-affinity acetate activation, and AckA and Pta catalyze a reversible pathway involved in acetate production or assimilation at high acetate concentration (B) Regulation of the forward and reverse Pta reactions Pta catalyzes both the synthesis and degradation of acetyl-CoA These two reactions are differentially regulated by pyruvate and PEP, which activate acetyl-CoA degradation and inhibit acetyl-CoA synthesis Acetyl-P, acetyl phosphate.
Trang 3Expression and purification of E coli Pta and
truncated Ptas containing the C-terminal end
By analysis of the protein domain architecture of
E coli Pta, three conserved domains can be detected
[Conserved Domain Database (CDD)] [11]: the P-loop,
containing NTPase at the N-terminal end
(CDD cl09099; Fig 2); a DRTGG domain
(CDD pfam07085; Fig 2); and a domain shared by
the phosphate acetyl⁄ butaryl transferases (PTA_PTB;
CDD cl00390; Fig 2) at the C-terminal end The
members of the P-loop NTPase domain superfamily
(N-terminal domain in E coli Pta; Fig 2) are
charac-terized by a conserved nucleotide phosphate-binding
motif, and are involved in diverse cellular functions
The second domain found in Pta (DRTGG
domain; Fig 2) has been associated with
cystathione-beta-synthase domain pfam00571 and cobyrinic acid
a,c-diamide synthase domain pfam01656 This domain
has been named according to some of the most
conserved residues, but its function is unknown
Finally, the domain at the C-terminal end (PTA_PTB;
Fig 2) is found in phosphate acetyltransferase and
phosphate butaryltransferase Moreover, PtaI-type
Ptas, found in several microorganisms, are composed
only of this PTA_PTB protein domain Thus, this is
the only domain in E coli Pta that can be directly
associated with the catalytic activity of the enzyme In
this way, in order to elucidate the functionality of the
N-terminal end of E coli Pta, three different truncated
Ptas that span the C-terminal domain of this protein
were generated (Pta-F1, Pta-F2, and Pta-F3; Fig 2)
Pta-F1 was designed in order to contain only the
domain found in phosphate acetyltransferase and to
exclude the extra domains with unknown function in
E coli Pta Pta-F2 is 30 amino acids longer than Pta-F1, whereas Pta-F3 was designed to contain the DRTGG domain and the PTA_PTB domains, while excluding the P-loop NTPase domain (Fig 2)
E coli recombinant Pta fused to a His-tag was purified to homogeneity by an affinity approach, using an Ni2+–agarose column The monomer molec-ular mass of the purified protein was 77 kDa, which corresponds to the predicted molecular mass of the protein [12] (Fig 3A) The three truncated Ptas (Pta-F1, Pta-F2, and Pta-F3) were also successfully overexpressed as N-terminal fusion proteins with His-tags The truncated Ptas were purified to homo-geneity, and the molecular mass of each of them, assessed by SDS⁄ PAGE, was in agreement with that predicted from the protein constructs, i.e 36 kDa for Pta-F1, 38 kDa for Pta-F2, and 51 kDa for Pta-F3 (Fig 3A)
CD spectra of the truncated Ptas Besides the good expression levels as soluble proteins
of the truncated Ptas, their folding state was evaluated with CD spectroscopy Despite the absence of an important portion of the protein, all of the truncated Ptas conserved the secondary structure (Fig 4) In this respect, CD spectra for Pta-F1, Pta-F2 and Pta-F3 were comparable, but not identical, to the spectrum of the entire protein (Fig 4) The differences among the spectra may be due to the lack of different regions of the N-terminal end in the truncated Ptas
Pta-F1 Pta-F2 Pta-F3
100 200 300 400 500 600 700
Pta
PTA_PTB DRTGG
P-loop NTPase
Fig 2 Recombinant E coli Pta and truncated Ptas characterized in
the present work The ruler indicates the number of amino acids in
each protein In boxes, the putative conserved domains (CDD
pro-tein classification) in E coli Pta: P-loop NTPase domain; DRTGG
domain; and PTA_PTB domain The truncated Ptas, Pta-F1, Pta-F2,
and Pta-F3 (326, 352 and 470 amino acids, respectively), have the
C-terminal domain alone or the C-terminal domain plus 30 amino
acids of the DRTGG domain or the complete DRTGG domain,
respectively.
51-
-66 -45 -35 kDa
-25 -18
-660 -440 -232
-140
-66
kDa
Fig 3 Purified recombinant E coli Pta and truncated Ptas (A) Coomassie Blue-stained SDS ⁄ PAGE (5 lg of each protein) of recombinant purified Pta (lane 1), Pta-F3 (lane 2), Pta-F2 (lane 3), and Pta-F1 (lane 4) The calculated molecular masses of the purified proteins are indicated on the left Molecular mass markers (MM) were loaded on the right (B) Coomassie Blue-stained native gel (5 lg of each protein) of purified recombinant Pta (lane 1), Pta-F1 (lane 2), Pta-F2 (lane 3), and Pta-F3 (lane 4) Native molecular mass markers (MM) were loaded on the right.
Trang 4Kinetic characterization of E coli Pta and
truncated Ptas
The three truncated Ptas displayed Pta catalytic
activ-ity Thus, the kinetic parameters of the entire Pta and
Pta-F1, Pta-F2 and Pta-F3 were determined using the
conditions in which the in vitro Pta activity was
opti-mal, and compared for both the forward (acetyl-CoA
synthesis) and reverse (acetyl phosphate synthesis)
directions of the Pta reaction (Fig 1B)
Kinetic parameters for the Pta forward reaction
(acetyl-CoA-forming)
Different kinetic responses of E coli Pta were observed
for acetyl phosphate and CoA Whereas the kinetic
response in the case of acetyl phosphate was hyperbolic,
sigmoidal kinetics were observed with respect to CoA, with a Hill coefficient of 1.7 (Table 1) The enzyme displayed measurably higher affinity for CoA than for acetyl phosphate, with a relatively high kcat value (227.6 s)1; Table 1)
On the other hand, despite the absence of the N-ter-minal end in the three truncated Ptas, the affinity for the two substrates, CoA and acetyl phosphate, was almost the same when Pta was compared with the three truncated Ptas (Table 1) This result indicates that the binding site for the substrates has not been significantly modified by the deletions Moreover, in the case of Pta-F3, the sigmoidal response when the CoA concentration was varied was maintained, with a Hill coefficient of 1.6 (Table 1) However, in the case
of Pta-F2 and Pta-F1, the sigmoidal response was lost (Table 1) On the other hand, the kcat values for Pta-F1, Pta-F2 and Pta-F3 were significantly reduced with respect to the complete Pta, displaying values lower than 1% of the kcat estimated for the complete Pta (Table 1)
Kinetic parameters for the E coli Pta reverse reaction (acetyl phosphate-forming)
With regard to the reverse reaction catalyzed by E coli Pta, a nearly eight-fold lower kcatvalue than for ace-tyl-CoA synthesis was observed (Table 1) Sigmoidal kinetics with respect to acetyl-CoA were obtained, with
a Hill coefficient of 1.3 (Table 1)
On the other hand, when the truncated Ptas were analyzed, very low kcat values were measured, from 1.5% to 0.1% of the estimated kcat for the complete Pta (Table 1) However, as the case of the acetyl-CoA synthesis reaction, the affinity for the substrate was
Fig 4 Comparative CD spectra of E coli Pta and truncated Ptas.
CD spectra of Pta, Pta-F1 and Pta-F2 were recorded in the far-UV
range (190–260 nm) Five repetitive scans were obtained using
10 l M each enzyme The Pta-F3 CD spectrum (not shown) was
practically the same as those obtained for the other truncated Ptas.
Table 1 Kinetic parameters for the forward reaction (acetyl-CoA-forming) and reverse reaction (acetyl phosphate-forming) of E coli Pta and truncated Ptas Kinetic values are given as average ± standard deviation Each value is averaged over at least two different enzyme prepara-tions Ac-P, acetyl phosphate; Ac-CoA, acetyl-CoA; NA, not applicable.
Acetyl-CoA-forming reaction
K m, Ac-P (m M ) K m, CoA (l M ) Hill constant for CoA V max (UI ⁄ mg) k cat (s)1)
Acetyl phosphate-forming reaction
S 0.5, Ac-CoA (l M ) Hill constant K m , phosphate (m M ) V max (UI ⁄ mg) K cat (s)1)
a Kinetics for these reactions are sigmoidal, and the reported values are S0.5values, not true Kmvalues.
Trang 5not significantly modified in the truncated versions in
relation to the complete Pta (Table 1) Moreover, in
some cases (such as Pta-F1), even higher affinity for
acetyl-CoA was observed, with an increase in the Hill
coefficient value (Table 1)
Regulation of E coli Pta and truncated Pta
activity by metabolic effectors
The effects of several metabolites that acted as
meta-bolic effectors of different Ptas were analyzed for the
recombinant E coli Pta and the three truncated Ptas
in both the forward and reverse reactions (Fig 5A)
NADH and ATP substantially inhibited the
activ-ity of E coli Pta in both directions (Fig 5A) On the
other hand, pyruvate and PEP displayed differential
behavior, depending on the direction of the Pta
reac-tion analyzed (Fig 5A) In this way, these
com-pounds acted as activators of the acetyl
phosphate-forming reaction while inhibiting the formation of
acetyl-CoA (Fig 5A) The activation of the E coli
Pta acetyl phosphate-forming reaction was analyzed
at different pyruvate and PEP concentrations (Fig 5B) The results obtained indicate that the maxi-mum percentage of activation is reached at concen-trations higher than 0.5 mm PEP or 10 mm pyruvate (Fig 5B)
On the other hand, E coli Pta acetyl phosphate-forming activity was measured in the presence of acti-vators (pyruvate or PEP) and inhibitors (NADH or ATP) (Fig 5A) The results indicate that PEP is able
to reverse, in part, the inhibitory effects of both NADH and ATP (Fig 5A) In the case of pyruvate, although partial reversal of NADH inhibition was observed, total reversal of ATP inhibition was found (Fig 5A)
The regulatory properties of the truncated Ptas were also studied (Fig 5A) For the three polypeptides, any
of the compounds analyzed (NADH, pyruvate, ATP, and PEP) was able to modify the enzyme activity, at different concentrations, in both the forward and reverse reactions
Pta-F3
Ac-CoA synthesis activity (%) 0
20 40 60 80 100 120
0 20 40 60 80 100 120
No addition
100
105
110
115
120
0 0.5 1.0 1.5 2.0
0 5 10 15 20 25 30
100 110 120 130 140
Ac-CoA synthesis activity (%) 0
20
40
60
80
100
120
140
0 20 40 60 80 100 120 140
No addition
+ NADH
PEP + NADH
PEP + ATP
Pyr + ATP
Pta
A
B
Fig 5 Regulatory properties of the recombinant E coli Pta and Pta-F3 in the acetyl-CoA (Ac-CoA)-forming or acetyl phosphate (Ac-P)-forming directions (A) The activities of Pta and Pta-F3 in the forward and reverse reactions were monitored in the absence or presence of 0.8 m M NADH, ATP, and ⁄ or PEP, and ⁄ or 15 m M pyruvate (Pyr), as indicated on the axes Substrate concentrations were maintained at the K m for each enzyme (Table 1) Results are presented as percentage activity in the presence of the effectors relative to the activity measured in the absence
of the metabolites Assays were performed at least in triplicate, and error bars indicate standard deviations Similar results to that obtained for Pta-F3 were obtained for Pta-F2 and Pta-F1 (B) Activation of the acetyl phosphate synthesis activity of E coli Pta by different concentrations of pyruvate and PEP Results are presented as percentage of activity in the presence of PEP or pyruvate relative to the activity measured in the absence of the metabolites Assays were performed at least in triplicate, and error bars indicate standard deviations.
Trang 6Oligomeric state of Pta and the truncated Ptas
The native oligomeric state of recombinant E coli Pta
was analyzed by size exclusion chromatography With
this technique, a native molecular mass of
484 ± 5 kDa was obtained, indicating that E coli Pta
assembles as a hexamer (77 kDa per subunit; Fig 3A)
Native electrophoresis of recombinant E coli Pta was
also performed (Fig 3B) In this case, the estimated
molecular mass obtained (nearly 475 kDa) was similar
to that obtained by size exclusion chromatography,
validating the use of this technique for estimating the
native assembly of this protein
On the other hand, in order to evaluate the
contri-bution of the N-terminal end to the formation of the
final oligomeric state of Pta, the native conformational
state of the truncated polypeptides was analyzed By
size exclusion chromatography, several different
pro-tein peaks were obtained (not shown), indicating that
Pta-F1, Pta-F2 and Pta-F3 displayed different
aggre-gates, ranging from dimers to hexamers, in similar
pro-portions The results with native electrophoresis were
same as those obtained by exclusion chromatography,
and the truncated Ptas presented a mixture of different
oligomers (Fig 3B) Thus, the profiles obtained suggest
the existence of dimers and hexamers in equilibrium
for the truncated Ptas Therefore, the absence of the
N-terminal domain is unfavorable for the formation of
the native hexameric structure of Pta
Complementation experiments on the E coli
acs pta mutant growing on acetate
The E coli acs pta double mutant (FB22) is not able
to grow on a minimal medium with acetate as a sole
carbon source (Fig 6) In order to evaluate the ability
of Pta-F3 to complement FB22 when growing on a
high acetate concentration, complete E coli Pta or
Pta-F3 were introduced into this mutant strain, and
the growth on acetate was evaluated
The results obtained indicate that the introduction
of complete E coli Pta or Pta-F3 was able to
comple-ment FB22 growth on acetate (Fig 6), giving similar
final attenuance values after 90 h of incubation at
37C
Discussion
Biochemical properties of E coli Pta in relation to
its physiological role
In the present work, detailed biochemical
characteriza-tion of E coli Pta was performed The enzyme was
nearly eight-fold more active in the direction of acetyl-CoA synthesis than in the direction of acetyl phosphate formation (Table 1) However, these two activities are differentially regulated by pyruvate and PEP, which both act as positive effectors of the acetyl phosphate-forming reaction and as negative effectors of the opposite reaction (Fig 5) Thus, these compounds highly favor E coli Pta acetyl phosphate synthesis activity This differential Pta activity regulation by pyruvate and PEP may be important in vivo, as this enzyme is involved in balancing pyruvate flux when
E coli grows on rich medium, by opting for acetate excretion [13] Thus, high levels of pyruvate and⁄ or PEP activate acetate excretion by favoring the Pta acetyl phosphate reaction (Fig 1B) On the other hand, E coli Pta was negatively modified by NADH and ATP in both directions of the reaction, which is in accord with the fact that when the tricarboxylic acid cycle is operating, acetate excretion by the Pta–AckA pathway is reduced However, in the presence of pyruvate or PEP, the inhibitory effect of NADH or ATP is partially or totally reversed (Fig 5A), indicating the relevance of these compounds in the activation of acetate excretion (Fig 1B)
E coli Pta Km values for the substrates (Table 1) were compared with the absolute metabolite concentra-tions in E coli growing on glucose or acetate [14], as these concentrations are critical for understanding the
in vivo rate of the Pta reaction In this regard, the acetyl-CoA concentration in E coli is far higher than the estimated Pta Km, indicating that Pta is operating
at the maximum rate when catalyzing acetyl phosphate
Time (h)
0 20 40 60 80
D600 nm
0.6
0.4
0.2
0
Fig 6 Growth on acetate of the E coli acs pta double mutant (•) transformed with E coli Pta (s) or Pta-F3 (.) The culture medium contained M9 salts supplemented with 15 m M acetate Results are the mean of at least three independent studies with no more than 5% standard deviation.
Trang 7formation for acetate excretion (Fig 1B) However, for
the reverse reaction, the estimated Kmfor acetyl
phos-phate is almost equal to the absolute concentration of
this compound in E coli growing on glucose [14]
Thus, although E coli Pta is more active in the
direc-tion of acetyl-CoA synthesis (Table 1), the in vivo
con-centrations of Pta substrates and products when
E coli grows on glucose favor acetyl phosphate
syn-thesis (Fig 1B) On the other hand, the acetyl
phos-phate concentration significantly increases when E coli
grows on acetate [14], allowing Pta to operate at the
maximum rate for acetate assimilation (Fig 1B)
By size exclusion chromatography, E coli Pta was
found to assemble as a hexamer Practically the same
native molecular mass was estimated for S enterica
Pta [5] In this way, the positive cooperative effect
found in CoA and acetyl-CoA binding (Table 1) would
be due to interactions among the active sites in the
oligomeric Pta
Recently, a detailed biochemical characterization of
S enterica Pta was performed [5] When the kinetic
performance of the enzymes is compared, although
the maximum activities in both directions of the
reaction are in the same order of magnitude, there is
a notably higher affinity of E coli Pta for both CoA
and acetyl-CoA Thus, E coli Pta Km values for CoA
and acetyl-CoA are 2.4-fold and 7.3-fold lower than
the Km values for S enterica Pta, respectively
(Table 1 [5]) Thus, although the two proteins share
95% identity, specific changes in amino acids may be
involved in the affinity differences With regard to
metabolic regulation, acetyl phosphate synthesis by
S enterica Pta is also activated by pyruvate and
inhibited by NADH [5], as in E coli (Fig 5),
although these compounds were not tested in the
acetyl-CoA synthesis direction
Pta-F3 is able to complement E coli acs pta
growth on acetate
E coli employs two different mechanisms for the
incorporation of acetate into the cell, either directly
through the activity of Acs (high-affinity pathway), or
in a way involving AckA and Pta enzymes (low-affinity
pathway) (Fig 1A) Therefore, an acs pta double
mutant strain is unable to grow on minimal medium
with acetate as a sole carbon source (Fig 6) In the
present work, we have found that this deficiency can
be corrected not only by complementation with the
complete E coli Pta, but also by complementation
with Pta-F3 (Fig 6), which displays very low activity
and is not regulated by metabolites at all It is thus
possible that the metabolic regulation of E coli Pta is
relevant for E coli metabolic fitness when growing on glucose
The E coli Pta N-terminal end is involved in native protein stabilization and metabolic regulation
In the present work, three truncated Ptas with deletions
in the N-terminal region were constructed: Pta-F1, con-taining only the PTA_PTB domain; Pta-F3, concon-taining the DRTGG and the PTA_PTB domains; and Pta-F2, which is 30 amino acids longer than Pta-F1 (Fig 2) The three truncated Ptas were successfully purified to homo-geneity (Fig 3), and conserved the secondary structure
of the complete Pta, as assessed by CD spectroscopy (Fig 4) Moreover, the truncated Ptas displayed Pta activity in both reaction directions, with comparable affinity for the substrates relative to the complete Pta (Table 1) However, they displayed notably lower maxi-mum activity (Table 1) Consequently, although the binding sites for the substrates are conserved in the trun-cated Ptas and are thus lotrun-cated in the PTA_PTB domain, residues from the N-terminal domain, specifi-cally from the P-loop NTPase domain (Fig 2), are needed for maximal catalytic activity, participating either directly in the catalytic mechanism, or indirectly
in the conformation of the catalytic site
The oligomeric state of the truncated Ptas was eval-uated by gel filtration chromatography and native gel electrophoresis (Fig 3B) The results indicate that the N-terminal domain is important for stabilization of hexameric native Pta, as none of the truncated Ptas was able to assemble as a hexamer (Fig 3B) Specifi-cally, the P-loop NTPase domain is important for native hexameric stabilization, as Pta-F3 did not dis-play a stable native conformation (Figs 2 and 3B) Therefore, another possible explanation for the low activity displayed by the truncated Ptas is that the for-mation of a hexameric protein is critical for maximal catalytic activity
On the other hand, the activity of the truncated Ptas was not regulated by any of the metabolites that were able to modify the activity of the complete Pta (Fig 5A) Thus, the N-terminal domain, specifically the P-loop NTPase domain (Fig 1), is involved in the metabolic regulation of E coli Pta Two explanations may account for this result: the first is that the binding site of the effectors is located at the N-terminal end of
E coliPta; and the second is that the native hexameric structure of E coli Pta is important for the metabolic regulation
Recently, analysis of several S enterica Pta mutants with single amino acid changes in the N-terminal
Trang 8domain revealed specific amino acids involved in
meta-bolic regulation and stabilization of this Pta [5] On
the other hand, crystal structure analysis of several
PtaIs, which lack the N-terminal end of PtaII, revealed
that these enzymes form homodimers [8] Moreover,
the activity of these shorter Ptas is not modulated at
all by the metabolite effectors of larger Ptas [5] These
results are in agreement with the characterization of
the truncated E coli Pta performed in the present
work, indicating that the N-terminal end is involved in
metabolic regulation and the hexameric conformation
The full characterization of more PtaIIs, as well as
three-dimensional structure analysis, would reveal
spe-cific N-terminal residues involved in the particular
properties of this enzyme and also the function of the
DRTGG domain Taking into account the important
role of Ptas, the results obtained in the present work,
dissecting the different domains forming E coli Pta,
will help in the future manipulation of these enzymes
by protein engineering in order to obtain Ptas better
suited for particular metabolic purposes
Experimental procedures
Bacterial strains and growth media
K-12 AG1, containing the plasmid pCA24N–Pta (ASKA
clone JW2294), was obtained from the ASKA library [15]
Strains were routinely cultured aerobically in LB broth with
appropriate antibiotics Alternatively, the different E coli
strains were grown on minimal medium M9 containing
15 mm acetate For expression and purification, different
strains, depending on the expression vector, were used:
pET28–F1 and pET28–F2; and E coli M15 for pQE30–F3
Construction of the E coli acs pta deletion strain
The E coli acs pta deletion strain (FB22) was constructed
using the pta single-gene deletion mutant JW2294, obtained
from the NIG Collection [16], as recipient strain The acs
deletion in JW2294 was performed as described by
pKD3 was amplified using primers with 60 bp of perfect
identity for the 5¢-end and 3¢-end of acs: delacs P1
(forward), 5¢-GAGAACAAAAGCATGAGCCAAATTCA
CAAACACACCATTGTGTAGGCTGGAGCTGCTTCG-3¢;
GATGGCATCGCGATAGCCTGCTTCATATGAATATC
CTCCTTA-3¢ The presence of the acs pta deletion was
confirmed by sequencing The mutated acs pta E coli
pCA24N–Pta or pQE–F3 for complementation analysis Induction of the introduced plasmids was performed by the addition of 0.5 mm isopropyl thio-b-d-galactoside (IPTG)
Gene amplification and cloning of the truncated Ptas
Pta fragments were amplified by PCR from plasmid pCA24N–Pta, hereafter called pPta, containing the entire coding sequence of pta from E coli Different sets of primers were used to amplify different E coli Pta fragments from
GTTATCAGCTGACTGAACT-3¢; and F1 Rv_XhoI, 5¢-C
GAC-3¢; and F2 Rv_XhoI, 5¢-CTCGAGCTGCTGCTGTG CAGACTGAAT-3¢); and Pta-F3 (F3 Fw_SacI, 5¢-CCGA GCTCCGCGTTAAATCCGTCAC-3¢; and F3 Rv_HindIII, 5¢-GGGAAGCTTACTGCTGTGCAGACTGAA-3¢) Each primer includes the restriction sites at the 5¢-end and 3¢-end
of the fragment, as indicated The primers were designed in order to generate three different truncated Ptas, containing the last 326 amino acids in the case of Pta-F1, the last 352 amino acids in the case of Pta-F2, and the last 470 amino acids in the case of Pta-F3 (Fig 2)
PCR reactions performed in a final volume of 25 lL, and using the following components: 0.2 mm each dNTP,
of 5· GoTaq DNA polymerase buffer, and 0.6 U of GoTaq DNA polymerase (Promega, Madison, WI, USA) The amplification protocol was as follows: one cycle of
The amplified PCR fragments were cloned using pGEM T-Easy (Promega), and digested with the corresponding restriction enzymes The resulting fragments were purified from a 1% agarose gel using a Qiaex band purification kit (Qiagen, Hilden, Germany), and cloned between the corresponding restriction sites in pET28 (Novagen, EMD Chemicals Inc., Gibbstown, NJ, USA) for F1 and Pta-F2, or in pQE30 (Qiagen) for Pta-F3 The plasmids were finally introduced into E coli DH5a cells by electropora-tion using a Bio-Rad apparatus, following the manufac-turer’s recommendations
Protein expression and purification
in E coli K-12 AG1, E coli BL21(DE3) or E coli M15 containing the corresponding expression vectors (p–Pta, pET28–F1, pET28–F2, and pQE30–F3) The systems used yield high-level expression of the recombinant proteins fused to a His-tag sequence at the N-terminal end codified
by the pET and pQE vectors used All chromatographic
Trang 9steps were performed on an A¨KTA purifier (GE
Health-care, Uppsala, Sweden)
Optimal induction conditions for the expression of each
protein were achieved using IPTG as an induction agent,
and different induction temperatures were tried Optimal
overexpression of the fusion proteins was achieved by
In a typical protein preparation, a 500 mL culture of
vec-tor (p–Pta, pET28–F1, pET28–F2, or pQE30–F3) was
grown in LB medium and induced as described above The
bacteria were harvested by centrifugation at 5000 g for 15
30 s, and this was followed by centrifugation for 10 min at
the fusion protein was eluted with 50 mm Tris⁄ HCl
(pH 8.0), 300 mm NaCl, and 250 mm imidazole The fusion
protein was diafiltrated in a concentrator (Millipore, MA,
Protein concentration
The protein concentration was determined by the method
of Sedmak and Grossberg [18], using BSA as standard
Steady-state kinetics
Pta activity in the direction of acetyl-CoA synthesis
monitoring the thioester bond formation of acetyl-CoA at
lithium acetyl phosphate, 0.2 mm lithium-CoA, and 2 mm
dithiothreitol
The reverse Pta activity (Fig 1B) was monitored by
mea-suring the phosphate-dependent CoA release from
acetyl-CoA with Ellman’s thiol reagent,
5¢,5-dithiobis(2-nitroben-zoic acid), as the formation of thiophenolate anion at
contained 50 mm Tris⁄ HCl (pH 8.0), 20 mm KCl, 0.1 mm
5¢,5-dithiobis(2-nitrobenzoic acid), 0.1 mm acetyl-CoA, and
Steady-state kinetic parameters were determined for both
the forward and the reverse reactions by measuring the
initial rates of acetyl-CoA or CoA formation, respectively
The measurements were performed at least in triplicate
Kinetic constants were determined by fitting the data of
initial rates to the Michaelis–Menten equation by nonlinear
regression [19] When sigmoidal curves were observed,
initial rates were fitted to the Hill equation [19]
Different compounds were tested as potential inhibitors
or activators of Pta Pta activity was measured in the absence or presence of 0.8 mm each effector (NADH, ATP, and PEP) or 15 mm pyruvate, while the substrate
(Table 1) The results are presented as the percentages of activity in the presence of the effectors relative to the activ-ity measured in the absence of the metabolites
Gel electrophoresis
polyacrylamide gels, according to the method of Laemmli [20] Proteins were visualized with Coomassie Blue stain-ing Native PAGE was performed according to the method of Davis [21], employing a 6% or 8% acrylamide separating gel Electrophoresis was performed at 150 V
staining
Gel filtration chromatography The molecular masses of recombinant native Pta variants were evaluated by gel filtration chromatography on an FPLC system with a Biosep-Sec S3000 (Phenomenex, CA, USA) The column was equilibrated with 100 mm phos-phate buffer at pH 7.4, and calibrated using molecular mass standards (Sigma-Aldrich, St Louis, MO, USA) The sample and the standards were applied separately in a final
chromatographic steps were performed on an A¨KTA purifier (GE Healthcare)
CD
CD spectra of purified Pta variants were obtained with a Jasco J-810 spectropolarimeter, using a 0.2 cm pathlength cell and averaging five repetitive scans between 260 nm and
200 nm Typically, 10 lm protein in 10 mm Tris (pH 8.0) was used for each assay
Acknowledgements
This work was funded by grants from CONICET and Agencia Nacional de Promocio´n Cientı´fica y Tecnolo´g-ica M F Drincovich and C S Andreo are mem-bers of the Researcher Career of CONICET, and
V A Campos-Bermu´dez and F P Bologna are fellows of the same institution
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