Báo cáo khoa học: Functional dissection of Escherichia coli phosphotransacetylase structural domains and analysis of key compounds involved in activity regulation potx

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Báo cáo khoa học: Functional dissection of Escherichia coli phosphotransacetylase structural domains and analysis of key compounds involved in activity regulation potx

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Functional dissection of Escherichia coli phosphotransacetylase 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 differentially 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. FEBS Journal 277 (2010) 1957–1966 ª 2010 The Authors Journal compilation ª 2010 FEBS 1957 producing 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 deletions 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-AMPAcetyl-P Ack Acs Acs P i CoA ADP ATP AMP CoA PP i ATP 2P i PPasa Glucose PEP Pyruvate Acetyl-CoA Acetate CoA NADH NAD + CO 2 Acetyl-P Pta P i CoA P i CoA ADP ATP + - Acetate excretion Acetate assimilatio n AB 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. Escherichia coli phosphotransacetylase V. A. Campos-Bermudez et al. 1958 FEBS Journal 277 (2010) 1957–1966 ª 2010 The Authors Journal compilation ª 2010 FEBS Results Expression 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 Ni 2+ –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. 1234 MM 51- 38- 36- 77- -116 -66 -45 -35 kDa -25 -18 1234 MM -660 -440 -232 -140 -66 kDa AB 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. V. A. Campos-Bermudez et al. Escherichia coli phosphotransacetylase FEBS Journal 277 (2010) 1957–1966 ª 2010 The Authors Journal compilation ª 2010 FEBS 1959 Kinetic 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 k cat 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 k cat 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 k cat 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 k cat value 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 k cat values were measured, from 1.5% to 0.1% of the estimated k cat 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 (mM) K m, CoA (lM) Hill constant for CoA V max (UI ⁄ mg) k cat (s )1 ) Pta 0.9 ± 0.1 67.2 ± 5.3 a 1.7 ± 0.2 177.4 ± 6.2 227.6 ± 9.3 Pta–F1 1.7 ± 0.2 59.6 ± 3.5 NA 2.6 ± 0.4 1.56 ± 0.5 Pta–F2 2.4 ± 0.4 62.3 ± 2.5 NA 0.24 ± 0.05 0.15 ± 0.03 Pta–F3 1.1 ± 0.1 65.8 ± 2.2 a 1.6 ± 0.1 2.6 ± 0.2 2.16 ± 0.3 Acetyl phosphate-forming reaction S 0.5, Ac-CoA (lM) Hill constant K m , phosphate (m M) V max (UI ⁄ mg) K cat (s )1 ) Pta 44.9 ± 4.1 1.3 ± 0.3 2.1 ± 0.2 23.1 ± 2.1 29.6 ± 2.3 Pta–F1 28.5 ± 5.2 2.1 ± 0.4 1.5 ± 0.1 0.38 ± 0.1 0.23 ± 0.1 Pta–F2 39.1 ± 5.5 1.3 ± 0.2 1.9 ± 0.2 0.05 ± 0.02 0.029 ± 0.01 Pta–F3 58.3 ± 6.1 1.8 ± 0.1 3.0 ± 0.3 0.51 ± 0.2 0.43 ± 0.2 a Kinetics for these reactions are sigmoidal, and the reported values are S 0.5 values, not true K m values. Escherichia coli phosphotransacetylase V. A. Campos-Bermudez et al. 1960 FEBS Journal 277 (2010) 1957–1966 ª 2010 The Authors Journal compilation ª 2010 FEBS not 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 Ac-P synthesis activity (%) No addition NADH Pyr ATP PEP [PEP] (mM) 100 105 110 115 120 0 0.5 1.0 1.5 2.0 [Pyruvate] (m M ) 0 5 10 15 20 25 30 Ac-P synthesis activity (%) Ac-P synthesis activity (%) 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 Ac-P synthesis activity (%) No addition NADH Pyr ATP PEP Pyr + 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 mM 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. V. A. Campos-Bermudez et al. Escherichia coli phosphotransacetylase FEBS Journal 277 (2010) 1957–1966 ª 2010 The Authors Journal compilation ª 2010 FEBS 1961 Oligomeric 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 37 °C. 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 K m 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 K m , indicating that Pta is operating at the maximum rate when catalyzing acetyl phosphate Time (h) 0 20406080 D 600 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. Escherichia coli phosphotransacetylase V. A. Campos-Bermudez et al. 1962 FEBS Journal 277 (2010) 1957–1966 ª 2010 The Authors Journal compilation ª 2010 FEBS formation for acetate excretion (Fig. 1B). However, for the reverse reaction, the estimated K m for 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 K m values for CoA and acetyl-CoA are 2.4-fold and 7.3-fold lower than the K m 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, containing 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 located 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. coli Pta; 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 V. A. Campos-Bermudez et al. Escherichia coli phosphotransacetylase FEBS Journal 277 (2010) 1957–1966 ª 2010 The Authors Journal compilation ª 2010 FEBS 1963 domain 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 E. coli DH5a was used as a general cloning host. E. coli 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: E. coli K-12 AG1 for pCA24N–Pta; E. coli BL21(DE3) for 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 Datsenko and Wanner [17]. The cat + cassette in plasmid 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 CAAACACACCA TTGTG TAGGC TGGAGCT GCTTC G-3¢; and delacs P2 (reverse), 5¢-GGCAATTGTGGGTTAC GATGGCATCGCGATAGCCTGCTTCATATGAATATC CTCCTTA-3¢. The presence of the acs pta deletion was confirmed by sequencing. The mutated acs pta E. coli strain, called FB22, was transformed with plasmids 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 the 3¢-end: Pta-F1 (F1 Fw_NcoI, 5¢-CCATGGTCC GTTATCAGCTGACTGAACT-3¢; and F1 Rv_XhoI, 5¢-C TCGAGCTGCTGCTGTGCAGACTGAAT-3¢); Pta-F2 (F2 Fw_NcoI, 5¢-CCATGGCTAACTACATCAACGCT 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, 0.2 pmolÆlL )1 each primer, 100 ng of DNA template, 5 lL 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 2 min at 94 °C; 30 cycles of 1 min at 94 ° C, 30 s at 55 °C, and 1 min 30 s at 72 °C; and one cycle of 5 min at 72 °C. 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 Pta-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 E. coli Pta and Pta-F1, Pta-F2 and Pta-F3 were produced 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 Escherichia coli phosphotransacetylase V. A. Campos-Bermudez et al. 1964 FEBS Journal 277 (2010) 1957–1966 ª 2010 The Authors Journal compilation ª 2010 FEBS steps 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 induc- ing each E. coli culture at D 600 0.4–0.6 with 0.5 mm IPTG, and growing for 4 h at 30 °C. In a typical protein preparation, a 500 mL culture of E. coli transformed with the corresponding expression 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 min, and resuspended in 50 mm Tris ⁄ HCl (pH 8.0), 1 mm phenylmethanesulfonyl fluoride, 0.01 mg ⁄ mL DNase, and 5mm MgCl 2 . Sonication was performed four times for 30 s, and this was followed by centrifugation for 10 min at 7000 g at 4 °C. The bacterial lysate was applied to a col- umn of Ni 2+ –agarose (Qiagen). After washing with 50 mm Tris ⁄ HCl (pH 8.0), 300 mm NaCl, and 20 mm imidazole, 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, USA) and stored in buffer 50 mm Tris ⁄ HCl (pH 8.0). 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 (forward reaction; Fig. 1B) was assayed at 30 °Cby monitoring the thioester bond formation of acetyl-CoA at 233 nm (e 233 nm = 5.55 mm )1 Æcm )1 ). The assay mixture contained 50 mm Tris ⁄ HCl (pH 8.0), 20 mm KCl, 10 mm 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 412 nm (e 412 nm = 13 600 m )1 Æcm )1 ). The assay mixture 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 5mm KH 2 PO 4 . 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 concen- trations were maintained at the K m for each enzyme (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 SDS ⁄ PAGE was performed in 10% (w ⁄ v) or 12% (w ⁄ v) 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 and 10 °C. The gels were analyzed by Coomassie Blue 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 volume of 50 lL at a constant flow rate of 1 mLÆmin )1 . All 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. References 1 Wolfe AJ (2005) The acetate switch. Microbiol Mol Biol Rev 69, 12–50. V. A. Campos-Bermudez et al. 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Functional dissection of Escherichia coli phosphotransacetylase structural domains and analysis of key compounds involved in activity regulation Valeria. FEBS Results Expression 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,

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