Acyl Carrier Protein Synthases from Gram-Negative, Gram-Positive, and Atypical Bacterial Species: Biochemical and Structural Properties and Physiological Implications

Materials.Unless specified otherwise, fine chemicals were purchased from Sigma Chemical Company (St. Louis, Mo.). Fast-protein liquid chromatography resins and columns used for purification were purchased from Amersham Biosciences (Piscataway, NJ). Expression vectors and expression strains were purchased from Novagen (Madison, WI). Luria-Bertani (LB) broth medium was purchased from Bio 101, Inc. (Vista, CA). Polyacrylamide gels and reagents were purchased from Invitrogen (Carlsbad, CA). The Bradford protein assay reagent was purchased from Bio-Rad (Hercules, CA), and ethylene glycolbis (succinimidylsuccinate) (sulfo-EGS) was purchased from Pierce (Rockford, IL).

Cloning and expression of the acpS and acpP genes.Cloning and expression of the S. pneumoniae acpS and acpP genes have been described (32). The cloning and expression of the acpP and acpS genes from M. pneumoniae were carried out as described before (47). All reagents, plasmids, and cell lines used for cloning and expression were the same as those described before (47). To clone the acpS gene, the following PCR primers were designed and used to amplify the acpS gene for cloning into E. coli expression systems. The 5′ PCR primer (5′-CGCGGATCCCATATGATTCTA GGCATAGGGATTGATTTAGTC-3′) was designed at the ATG start codon of acpS and contains BamHI and NdeI sites for cloning purposes. The 3′ PCR primer (5′-CGCGGATCCTCATGGTGTTTGTTGTGCCAAACAGATGGC-3′) was designed at the stop codon of acpS and contains a BamHI site after the stop codon. Using these primers, acpS was PCR amplified from M. pneumoniae for 25 cycles under the conditions as described before (47). Five PCR products were combined, and a portion of the pooled PCR products was digested with BamHI. The BamHI-digested PCR fragment was cloned into pCZA342, a low-copy-number plasmid (2) that had been digested with BamHI and dephosphorylated with calf intestinal alkaline phosphatase. The acpS gene from several pCZA342 clones was sequenced, and a clone containing the consensus acpS gene sequence was used for constructing expression systems. This pCZA342 clone was digested with NdeI and BamHI. The NdeI-BamHI DNA fragment containing acpS was subcloned into pET-1la (Novagen). The resulting plasmid was designated pLY270.

To clone the acpP gene from M. pneumoniae, the following PCR primers were used for amplification: the 5′ PCR primer (5′-CGCGGATCCCATATGCAAGAGCGTGACATTC-3′) and the 3′ PCR primer (5′-CGCGGATCCCTATACCCCTTTTTGACTTA TTA-3′). Using these primers, acpP was PCR amplified from M. pneumoniae as described above. The PCR products were digested and cloned into pCZA342 and finally pET-11a (Novagen) exactly as described above. The resulting plasmid was designated pLY368.

To clone the acpS gene from E. coli, the following PCR primers were used for amplification: the 5′ PCR primer (5′-CGCGGATCCCATATGAGCACCATCGAAGAACGTGTGAAAAAA-3′) and the 3′ PCR primer (5′-CGCGGATCCTTACGCCTGGTTT CCGTTAATATAGACAAT-3′). Using these primers, acpP was PCR amplified from E. coli as described above. The PCR products were digested and cloned into pCZA342 and finally pET-11a (Novagen) exactly as described above. The resulting plasmid was designated pLY296.

For the expression of M. pneumoniae ACP and AcpS and E. coli AcpS, the expression plasmids were transformed into E. coli BL21 pLysS as described before (47), and the resulting E. coli expression strains were designated LY368, LY270, and LY296, respectively.

For the expression of E. coli ACP, DK554, an E. coli ACP overexpresser strain, was used as described previously (29).

Purification of the AcpS and apo-ACP proteins of E. coli, M. pneumoniae, and S. pneumoniae.The S. pneumoniae AcpS and apo-ACP proteins were expressed and purified as described previously (32).

For the purification of E. coli AcpS, an E. coli expression strain (LY296) carrying the acpS gene on an expression plasmid as described above was inoculated from an overnight culture into LB with 100 μg/ml ampicillin and grown at 35°C with shaking at 250 rpm until an optical density of 0.5 to 0.6 at 590 nm was reached. The culture was induced with 1 mM isopropyl-1-thio-β-d-galactopyranoside for 3 h. Cells were harvested by centrifugation at 4,500 × g at 4°C for 8 min, washed twice in phosphate-buffered saline, and frozen at −80°C. The cell pellet was thawed and resuspended in 50 mM Tris-HCl, pH 7.0, and 100 mM KCl (buffer A) and disrupted by passing it twice through a French press cell. The resulting cell lysate was centrifuged at 160,000 × g for 40 min at 4°C. The supernatant fraction was collected and applied to a Source S column (15S, 2.5 by 8 cm) that had been equilibrated with buffer A. The column was washed with buffer A and eluted with a linear gradient of 0.1 to 1.0 M KCl in buffer A. Fractions (7.0 ml each) were collected, and the presence of AcpS in the fractions was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis (16% Tricine gels). The fractions containing AcpS were pooled, adjusted to final concentrations of 1 mM dithiothreitol (DTT) and 15% glycerol (vol/vol), and stored in small aliquots at −80°C. Protein concentrations were determined using a Bradford protein assay kit (Bio-Rad) with bovine serum albumin as a standard.

For the purification of E. coli ACP, an E. coli ACP overexpression strain, DK554, was grown in LB containing glucose (50 mM), kanamycin (50 μg/ml), and pantothenate (25 mM) at 30°C until an optical density of 0.6 at 590 nm was reached (29). The cells were induced using 1 mM isopropyl-1-thio-β-d-galactopyranoside for 3 h, harvested by centrifugation, and washed twice in phosphate-buffered saline prior to storage at −80°C. Cell pellets were thawed and resuspended in 50 mM Tris-HCl, pH 7.0, and 50 mM KCl (buffer B), and disrupted by passage through a French pressure cell as described above. The resulting cell lysate was centrifuged as described above, and the supernatant fraction collected was applied to a Source Q column (30 ml) equilibrated with buffer B. Fractions (7 ml each) containing apo-ACP, as judged by SDS-PAGE (4 to 20% Tris-glycine) analysis, were pooled and applied to a Sepharose (900 ml, S-100) size exclusion column preequilibrated with 50 mM Tris-HCl, pH 8.0, and 100 mM KCl. Fractions containing apo-ACP were pooled, adjusted to a final concentration of 15% glycerol, and stored frozen at −80°C. Protein concentration was determined as described above.

For the purification of M. pneumoniae AcpS and apo-ACP, E. coli cells (LY270 and LY368 carrying M. pneumoniae AcpS and apo-ACP genes on expression plasmids, respectively) were grown and induced as described above. Cells were harvested and disrupted in buffer B as described above. The supernatant fraction containing M. pneumoniae AcpS was applied to a Source S column (15S, 2.5 by 8 cm), whereas the supernatant fraction containing M. pneumoniae apo-ACP was applied to a Source Q column (15S, 2.5 by 8 cm), both of which had been equilibrated in buffer B. The columns were washed with buffer B and eluted with a linear gradient of 0.1 to 1.0 M KCl in buffer B. Fractions (7 ml each) were collected, and the presence of AcpS and apo-ACP was determined by SDS-PAGE (16% Tricine gels) analysis. The fractions containing AcpS were collected, adjusted to a final concentration of 15% glycerol (vol/vol), and frozen at −80°C. The fractions containing M. pneumoniae apo-ACP protein were further purified by using a Sepharose gel filtration column (S-100, 5 by 60 cm) equilibrated in 50 mM Tris-HCl, pH 8.0, and 50 mM KCl. The column was eluted with the same buffer, and fractions (10 ml each) were collected. Fractions containing apo-ACP were pooled, adjusted to a final concentration of 15% glycerol (vol/vol), and frozen at −80°C. Protein concentrations were determined as described above.

The identity of each purified ACP or AcpS protein preparation was confirmed by N-terminal sequencing analysis as described before (47). The identity of each purified ACP preparation was also confirmed by electrospray mass spectrophotometry (ESMS) analysis as described before (32). In addition, each purified ACP preparation was also subjected to ESMS analysis to verify their primary presence in the apo form.

Determination of the native structures of purified AcpS proteins.For the determination of the native structure of each purified AcpS protein, analytical gel filtration column chromatography was carried out. A sample of 100 μl of a purified AcpS preparation (≈300 μg/ml) was applied to a Superdex gel filtration column (S-75, HR 1.0 by 30 cm) that was equilibrated with 50 mM Tris-HCl, pH 7.5, 50 mM KCl, and 1 mM DTT. The column was calibrated with protein molecular weight standards (Sigma). On the basis of the elution profiles of the molecular weight standards, a standard curve was generated, which was used to calculate the molecular weight of each protein according to its elution profiles.

To further verify the native oligomerization state of AcpS, each purified AcpS preparation was subjected to a cross-linking analysis as described below. Each AcpS protein preparation was first dialyzed against 4 liters of 20 mM HEPES, pH 7.5, and 1 mM DTT at 4°C for 18 h. Then, 100 μl of each protein preparation (50 μM) was mixed with 0.5, 2.5, or 5.0 mM of sulfo-EGS and the reaction mixtures were incubated at room temperature for 30 min. The reactions were terminated by the addition of 50 mM Tris-HCl, pH 7.5, at room temperature for 30 min. The sulfo-EGS-treated and untreated AcpS preparations (10 μl each) were analyzed by SDS-PAGE (10 μl each gel) and stained with Coomassie blue R-250. On the basis of the relative mobilities of the molecular weight markers (Invitrogen) versus their molecular weights, a standard curve was generated, which was then used to calculate the molecular weight of sulfo-EGS-treated and untreated AcpS protein preparations (Invitrogen).

Biochemical characterization of purified AcpS proteins.The ability of the purified AcpS enzymes to convert their native apo-ACP substrates to holo-ACP was assessed by ESMS analysis. The reaction mixtures containing 50 mM Tris-HCl buffer, 10 mM MgCl₂, 1 mM DTT, 300 μM CoA, 5 μM AcpS, and apo-ACP (100 μM of S. pneumoniae ACP, 47 μM of E. coli apo-ACP, or 120 μM of M. pneumoniae apo-ACP) were incubated at room temperature for 1 h before ESMS analysis as described previously (32).

The conversion of apo-ACP to acyl-ACP in the presence of different CoA derivatives was monitored by a high-pressure liquid chromatography (HPLC)-based assay (32) that was used for subsequent enzyme characterization and kinetic analysis. Reaction mixtures (100 μl each) were injected onto a reverse-phase column (Vydac Selectapore 300M) and separated by a 25 to 100% acetonitrile gradient containing 0.1% trifluoroacetic acid (31). Acyl-ACP products were detected by their absorption at 220 nm, and the product formation was determined by comparing the peak areas of the acyl-ACP (products) to those of the substrate (apo-ACP) peak and the product peak.

For the determination of the kinetic parameters (K_m and k _cat) of AcpS, the activity of each enzyme was assayed using an HPLC method (32) at a fixed concentration of one substrate and various concentrations of the other as described below. For the determination of the kinetic parameters of AcpS with respect to CoA, each enzyme was assayed at a fixed concentration of the apo-ACP (1 μM) and various concentrations of CoA (0.5 to 100 μM). For the determination of the kinetic parameters of AcpS with respect to apo-ACP, each enzyme was assayed at a fixed concentration of the CoA (100, 40, and 20 μM for E. coli, M. pneumoniae, and S. pneumoniae AcpS, respectively) and various concentrations of apo-ACP (0 to 20 μM). The concentrations of apo-ACP used for each AcpS enzyme were determined on the basis of their lack of significant inhibition of each enzyme activity. In all cases, the reaction mixtures containing 50 mM Tris-HCl at different optimal pHs (8.0, 7.2, and 7.0 for E. coli, M. pneumoniae, S. pneumoniae AcpS enzymes, respectively), 10 mM MgCl₂, and 1 mM DTT were incubated at 37°C for 1 h, in quadruplicate, and the reactions were terminated by the addition of 50 mM EDTA.

To examine the substrate specificity of AcpS enzymes, the activity of each enzyme was assayed in the presence of a fixed concentration of their native apo-ACP and different concentrations of CoA derivatives: CoA, acetyl-CoA, malonyl-CoA, acetoacetyl-CoA, butyryl-CoA, crotonyl-CoA, decanoyl-CoA, myristoyl-CoA, and palmitoleoyl-CoA. The ability of S. pneumoniae AcpS to utilize myristoleoyl-CoA and palmitoyl-CoA was also tested. For the determination of optimal activity of each enzyme for CoA derivatives, the activity of each AcpS was measured in the presence of CoA derivatives and different enzyme concentrations as follows: 0.001 to 1 μM of E. coli AcpS, 0.1 to 1.0 μM of M. pneumoniae AcpS, and 1.0 to 20 nM of S. pneumoniae AcpS. On the basis of these results, the appropriate enzyme concentrations for the assay were determined for the assessment of the kinetic parameters of each enzyme for the CoA derivatives. For the assessment of E. coli AcpS substrate specificity, the enzyme activity was assayed in the presence of 1 μM of E. coli apo-ACP and 0.5 to 50 μM of CoA derivatives. For the assessment of M. pneumoniae AcpS substrate specificity, the enzyme activity was assayed in the presence of 1 μM of M. pneumoniae apo-ACP and the following concentrations of different CoA derivatives: 0 to 100 μM (acetyl-CoA, crotonyl-CoA, butyryl-CoA), 0 to 30 μM (decanoyl-CoA, myristoleoyl-CoA, palmitoleoyl-CoA), and 0 to 250 μM (malonyl-CoA, acetoacetyl-CoA). For the assessment of S. pneumoniae AcpS substrate specificity, the enzyme activity was assayed in the presence of 1 μM S. pneumoniae apo-ACP and the following concentrations of different CoA derivatives: 0 to 50 μM except for malonyl-CoA, of which the enzyme required much higher concentrations (0 to 400 μM) for activity. From the progress curves generated, kinetic parameters (K_m and k _cat) were calculated by fitting the curves obtained to the Michaelis-Menten equation using Sigma-Plot.

Cloning and expression of the acpS and acpP genes of E. coli and M. pneumoniae.To further understand the physiological role of AcpS in gram-negative (E. coli), gram-positive (S. pneumoniae), and also atypical (M. pneumoniae) bacteria and to examine their structure and activity relationship, we wanted to characterize the AcpS enzymes from these phylogenetically well-spaced and evolutionarily diverse organisms with regard to their native structures and substrate specificities. Since the acpS gene from M. pneumoniae has not been identified and characterized, we first searched the acpS and also acpP genes from the genome of M. pneumoniae by using the S. pneumoniae and E. coli acpS and acpP gene sequences as queries in the BLAST program (1). We identified a gene consisting of 360 base pairs, which encodes a protein with a predicted molecular mass of 13,774 Da (accession number NP_109986). As shown next, we have confirmed the identity of this gene as acpS. The M. pneumoniae acpS gene appears to be clustered with the genes in the order of unknown-fmt-acpS, since rnc and rpsT, located downstream and upstream to the acpS cluster, respectively, are transcribed from the opposite directions with respect to the acpS cluster (18). Thus, the acpS cluster appears to consist of fmt, involved in protein synthesis (methionyl-tRNA formyltransferase), and a gene of unknown function. In this regard, the genomic organization of acpS in M. pneumoniae is quite different from that of acpS in E. coli and S. pneumoniae (27, 32). The acpS gene in E. coli consists of an operon with its upstream pdxJ gene that is required for vitamin B6 biosynthesis (27, 41). It is also interesting to note that in M. pneumoniae, rnc, a gene encoding RNase III, is located immediately downstream of the acpS cluster and is transcribed from an opposite direction (18), whereas in E. coli, rnc is located in an operon immediately upstream to the acpS operon (27, 41). Thus, rnc and acpS appear to be located in the same vicinity on the chromosomes of both organisms. The acpS cluster in S. pneumoniae, aroG-aroF-acpS-alr-recG, consists of the genes that are required for aromatic amino acid biosynthesis, cell wall biosynthesis, and DNA recombination (32).

The acpP gene from M. pneumoniae is 255 base pairs long and encodes a protein consisting of 84 amino acid residues with a predicted molecular mass of 9,838 Da (accession number S73758). The acpP gene appears to be in a cluster with the genes in the order of unknown-unknown-acpP-unknown. There is a long noncoding region (145 bp) upstream of the first gene of unknown function. In addition, the gene with an unknown function located downstream of the acpP cluster is transcribed from an opposite direction. Like the acpS gene, the acpP gene also appears to organize into an operon with genes of unknown functions.

The subunits of M. pneumoniae AcpS and ACP exhibit molecular weights similar to those of their counterparts in S. pneumoniae and E. coli. The two proteins, AcpS and ACP, share 27 and 33% and 25 and 32% identities with their respective counterparts in E. coli and S. pneumoniae. The pI value of M. pneumoniae AcpS is estimated to be 9.98, which is identical to that of E. coli AcpS but much higher than that (6.13) of S. pneumoniae AcpS (32). Thus, M. pneumoniae AcpS, like E. coli AcpS, is significantly more basic than that of S. pneumoniae (27, 29, 32). Like other ACPs (8, 26, 34, 37-39), M. pneumoniae apo-ACP is very acidic, with a pI value of 4.82.

The acpS and acpP genes from M. pneumoniae and E. coli were cloned by PCR methodology and expressed in E. coli by using expression vectors (see Materials and Methods). The acpS and acpP genes from S. pneumoniae were cloned and expressed in E. coli as described previously (32).

Purification and identification of the AcpS and ACP proteins of M. pneumoniae, S. pneumoniae, and E. coli.The purification and identification of S. pneumoniae AcpS and apo-ACP have been described elsewhere (32). To purify E. coli and M. pneumoniae AcpS proteins, we overexpressed both proteins in E. coli and purified them to approximately 85% pure by a single step of anion-exchange column chromatography (data not shown). Using this purification scheme, we obtained approximately 50 and 13 mg AcpS proteins of E. coli and M. pneumoniae, respectively, from one liter of E. coli cells overexpressing the proteins. The purified AcpS proteins of E. coli and M. pneumoniae exhibited molecular masses of 15 kDa, which were very similar to the predicted values of 14.1 and 13.8 kDa, respectively, on the basis of their amino acid sequences (27) (accession number NP_109986). To further confirm the identity of each purified protein, we determined their N-terminal sequences. The N-terminal amino acid sequences obtained for E. coli and M. pneumoniae AcpS proteins were AILGLGTDIV and MILGIGIDLV, respectively, which were identical to the predicted amino acid sequences except that the first Met residue of E. coli AcpS was processed. Thus, these results have confirmed the identities of purified proteins as E. coli and M. pneumoniae AcpS enzymes.

We also overexpressed M. pneumoniae apo-ACP protein in E. coli and purified both E. coli and M. pneumoniae apo-ACP proteins to apparent homogeneity, as judged by SDS-PAGE analysis, using a two-step purification scheme consisting of anion-exchange and gel filtration chromatography (data not shown). Using this scheme, we obtained approximately 23 and 50 mg of E. coli and M. pneumoniae apo-ACP proteins, respectively, from one liter of E. coli cells overexpressing the proteins. To confirm the identities of both proteins, we carried out N-terminal amino acid sequencing and ESMS analyses. The N-terminal amino acid sequences obtained for E. coli and M. pneumoniae apo-ACP proteins were STIEERVKKIxG and MQERDILLKIKE, respectively, which were identical to the predicted amino acid sequences except that the first Met residue of E. coli ACP was processed and the identities of the residue between Lys and Gly could not be established due to a weak signal (data not shown). Furthermore, ESMS analysis showed that the purified ACP proteins of E. coli and M. pneumoniae exhibited molecular masses of 8,508 and 9,838 Da, respectively, which matched exactly the predicted molecular masses of both proteins on the basis of their amino acid sequences (accession numbers AAA24316 and S73758, respectively). Thus, the results of these studies have confirmed the identities of the purified proteins as E. coli and M. pneumoniae ACP proteins.

To establish that the purified E. coli and M. pneumoniae AcpS proteins were enzymatically active, we performed ESMS analysis. We found that the molecular mass of E. coli apo-ACP was increased by 340 Da (from 8,508 to 8,848.6 Da) after the incubation with E. coli AcpS (data not shown). This increase in the molecular mass of ACP indicates that the transfer of the phosphopantetheine group from CoA to apo-ACP occurred, since the phosphopantetheine group is known to have a molecular mass of approximately 340 Da. Similarly, we found that the molecular mass of M. pneumoniae apo-ACP was increased by approximately 340 Da (from 9,838 to 10,178 Da) after the incubation with M. pneumoniae AcpS (data not shown). This increase in molecular mass again indicates a transfer of the phosphopantetheine group from CoA to apo-ACP. Finally, we have shown previously that S. pneumoniae apo-ACP exhibited a molecular mass increase of approximately 340 Da (from 8,834 to 9,174 Da) after the incubation with S. pneumoniae AcpS (32). Together, these results have clearly established that the purified AcpS enzymes are enzymatically active.

Determination of native molecular structures of E. coli and M. pneumoniae AcpS enzymes.To better understand the structure and activity relationship of AcpS enzymes, we wanted to determine the native structures of each purified enzyme. We have previously shown that the AcpS enzyme from S. pneumoniae is a homotrimeric enzyme (5, 32). Interestingly, E. coli AcpS appeared to be a dimeric enzyme as indicated by gel filtration column chromatography (29). To further confirm this finding and to determine the native structure of M. pneumoniae AcpS, we subjected both purified proteins to analytical gel filtration column chromatography (Materials and Methods). Since we found that DTT was important for E. coli AcpS activity, we included it in all the subsequent experiments (32). As a control, we also subjected S. pneumoniae AcpS to gel filtration column chromatography (Materials and Methods). Under this condition, we found that S. pneumoniae AcpS was eluted in the fractions corresponding to a molecular mass of 39 kDa (data not shown), consistent with our previous finding that this enzyme is homotrimeric (i.e., 13-kDa/monomer). However, we found that both the E. coli and M. pneumoniae AcpS proteins were eluted in the fractions corresponding to a molecular mass of 29 kDa (data not shown) when subjected to analysis under identical conditions. These results indicate a homodimeric structure (13.8 to 14.1 kDa/monomer). To further confirm these results, we performed cross-linking analyses using sulfo-EGS as a cross-linker (Materials and Methods). We found that a higher-molecular-mass protein species (34 kDa) was present in the S. pneumoniae AcpS preparation after the treatment with sulfo-EGS (Fig. 1). This 2.6-fold (from 13.3 to 34 kDa) increase in the molecular mass of S. pneumoniae AcpS indicates a trimeric structure. Similarly, we found that higher-molecular-mass protein species (32 and 27 kDa) were present in the E. coli and M. pneumoniae AcpS preparation, respectively, after the treatment with the cross-linker (Fig. 1). This increase in the molecular mass of E. coli AcpS (15 to 32 kDa) or M. pneumoniae AcpS (15 to 27 kDa) indicates a dimeric structure. Together, the results of these studies have confirmed the previous findings regarding the native structures of E. coli and S. pneumoniae AcpS enzymes and have also suggested that M. pneumoniae AcpS is a dimeric enzyme.

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FIG. 1.

Analysis of AcpS native structures by chemical cross-linking. Purified AcpS (50 μM) was treated with 0.5, 2.5, or 5 mM of sulfo-EGS or left untreated, and the resulting AcpS preparations were analyzed by SDS-PAGE as described (see Materials and Methods). Lane M, protein molecular mass markers; lanes 1 to 3, purified S. pneumoniae AcpS protein treated with 0.5, 2.5, or 5 mM sulfo-EGS, respectively; lanes 4 to 6, purified E. coli AcpS treated with 0.5, 2.5, or 5 mM sulfo-EGS, respectively; lanes 7 to 9, purified M. pneumoniae AcpS treated with 0.5, 2.5, or 5 mM sulfo-EGS, respectively. The arrows point to the cross-linked AcpS species.

Utilization of CoA and CoA derivatives as substrates by E. coli, M. pneumoniae, and S. pneumoniae AcpS enzymes.To further assess the physiological role of AcpS in different bacterial species, we wanted to examine the abilities of AcpS enzymes to utilize CoA and its derivatives as physiological substrates for fatty acid and lipid biosyntheses in the cell (Fig. 2). To this end, we measured the activity of each AcpS enzyme in the presence of CoA and its acyl derivatives using an HPLC assay previously developed for the S. pneumoniae AcpS enzyme (32) and determined the kinetic properties of each enzyme with respect to the utilization of CoA and its derivatives. To utilize this HPLC assay for the assessment of a variety of CoA derivatives as substrates for each AcpS enzyme, we optimized the assay, which resulted in enhanced separations of the longer-chain acyl-ACPs for each enzyme and also apo-ACPs from acyl-ACPs for all CoA derivatives tested except hexanoyl-CoA and octanoyl-CoA, whose products could not be separated from the apo-ACP substrates (data not shown). As a result, these two CoA derivatives could not be analyzed. For all AcpS enzymes tested, their activities appeared to increase in a dose-dependent manner within a certain range of apo-ACP concentrations but then appeared inhibited at higher apo-ACP concentrations. Similar to previously reported results (32), the activity of S. pneumoniae AcpS increased with the increase of apo-ACP concentrations until 7.5 μM but then decreased to some degree at 7.5 to 10 μM (Fig. 3A). At >10 μM of apo-ACP, the activity appeared to increase gradually again (Fig. 3A). A similar result was obtained for M. pneumoniae AcpS (Fig. 3C). The activity of this enzyme was inhibited to some degree at 3 μM of apo-ACP but then increased with a further increase in apo-ACP concentrations (Fig. 3C). In contrast to the results obtained for M. pneumoniae and S. pneumoniae enzymes, the activity of the E. coli AcpS enzyme was inhibited to some degree at 3 to 10 μM of apo-ACP and then completely inhibited at 20 μM of apo-ACP (Fig. 3B). Due to the inhibition of AcpS activities by apo-ACP at higher concentrations, the kinetic studies were carried out for each enzyme at the concentrations of apo-ACP which did not significantly inhibit AcpS activity (Materials and Methods).

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FIG. 2.

The structures of CoA and its derivatives used as substrates of S. pneumoniae, E. coli, and M. pneumoniae AcpS enzymes.

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FIG. 3.

Effects of apo-ACP concentrations on activities of S. pneumoniae, E. coli, and M. pneumoniae AcpS enzymes. The activity of each AcpS enzyme was measured using an HPLC method in the presence of a fixed CoA and various apo-ACP concentrations (see Materials and Methods). Panels A, B, and C represent the substrate saturation curves obtained for S. pneumoniae, E. coli, and M. pneumoniae AcpS enzymes, respectively.

All the AcpS enzymes tested appeared to exhibit the Michaelis-Menten kinetics when assayed at different concentrations of CoA or its derivatives and fixed concentrations of apo-ACP at which the enzyme activity was not inhibited (Fig. 4B, D, and F; Fig. 5). The K_m values of the AcpS enzymes of S. pneumoniae, E. coli, and M. pneumoniae for CoA were determined to be 4.0, 9.3, and 40 μM, respectively (Table 1). These results indicate that the affinity of M. pneumoniae AcpS for CoA is much lower than those of the E. coli and S. pneumoniae enzymes. The K_m values of the AcpS enzymes of S. pneumoniae, E. coli, and M. pneumoniae for apo-ACP were determined to be 1.8, 1.3, and 0.8 μM, respectively (Table 1). Thus, the affinities of these enzymes for apo-ACP are similar. The k _cat values obtained for S. pneumoniae and E. coli AcpS enzymes with respect to CoA and apo-ACP were similar but were 150- to 500-fold higher than that obtained for M. pneumoniae AcpS (Table 1). Taken together, these results indicate that the AcpS enzyme of M. pneumoniae is kinetically a sluggish enzyme compared with those of S. pneumoniae and E. coli with regard to the utilization of their native apo-ACP and CoA substrates.

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FIG. 4.

Kinetic analysis of S. pneumoniae, E. coli, and M. pneumoniae AcpS enzymes. The activity of each AcpS enzyme was measured using an HPLC method in the presence of a fixed apo-ACP and various CoA concentrations (panels A, C, and E) or conversely a fixed CoA and various apo-ACP concentrations (panels B, D, and F) as described in Materials and Methods. Kinetic parameters of each enzyme were generated from the substrate saturation curves obtained as described in Materials and Methods. Panels A and B, C and D, and E and F represent substrate saturation curves obtained for the S. pneumoniae, E. coli, and M. pneumoniae AcpS enzymes, respectively.

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FIG. 5.

Kinetic analysis of S. pneumoniae, E. coli, and M. pneumoniae AcpS enzymes using butyryl-CoA as a substrate. AcpS activity was measured using an HPLC method in the presence of a fixed apo-ACP and various butyryl-CoA concentrations as described in Materials and Methods. Kinetic parameters of each enzyme were generated from the substrate saturation curves obtained as described in Materials and Methods. Panels A, B, and C represent the substrate saturation curves obtained for S. pneumoniae, E. coli, and M. pneumoniae AcpS enzymes, respectively.

To assess the ability of each enzyme to utilize CoA derivatives as their physiological substrates, we determined the K_m and k_cat values of each purified enzyme in the presence of apo-ACP and different CoA derivatives (Table 1). As shown in Table 1, S. pneumoniae AcpS can utilize short- and long-chain CoA derivatives but prefers the long-chain acyl CoA-derivatives (≥C10) as evidenced by the lower K_m values obtained for the long-chain CoA derivatives when tested as substrates (Table 1; Fig. 6, top panel). In contrast, E. coli AcpS can utilize only the short-chain CoA derivatives (≤C4) but not the long-chain CoA-derivatives (>C4) under the conditions tested (Table 1; Fig. 6, middle panel). Interestingly, M. pneumoniae AcpS, like S. pneumoniae AcpS, can utilize both short- and long-chain CoA derivatives but significantly prefers the long-chain CoA derivatives (Table 1; Fig. 6, bottom panel). The affinities of M. pneumoniae AcpS for the long-chain acyl CoA derivatives are significantly higher than those obtained for the short-chain derivatives, although the k_cat values obtained for the enzyme remained similar. Therefore, these results show that the AcpS enzymes from these phylogenetically diverse bacterial species exhibit significantly different substrate specificities with respect to the utilization of CoA and its derivatives.

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FIG. 6.

Catalytic efficiencies of S. pneumoniae, E. coli, and M. pneumoniae AcpS enzymes with respect to the utilization of CoA and its derivatives as substrates. The K_m and k _cat values for each enzyme were obtained by assaying the activity of each enzyme in the presence of fixed concentrations of one substrate and various concentrations of another using an HPLC method (see Materials and Methods). The catalytic efficiencies, k _cat/K_m (M⁻¹ s⁻¹), were calculated on the basis of the K_m and k _cat values obtained for each enzyme. Panels A, B, and C represent the catalytic efficiencies of the S. pneumoniae, E. coli, and M. pneumoniae AcpS enzymes, respectively, for CoA and its derivatives, as follows: malonyl-CoA (Mal), acetyl-CoA (ac), acetoacetyl-CoA (acetac), butyryl-CoA (but), crotonyl-CoA (crot), decanoyl-CoA (dec), myristoyl-CoA (myr), palmitoyl-CoA (palm), myristoleoyl-CoA (myre), and palmitoleoyl-CoA (palme).