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Journal of Bacteriology, April 2008, p. 3018-3025, Vol. 190, No. 8
0021-9193/08/$08.00+0     doi:10.1128/JB.01831-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Basis of Arginine Sensitivity of Microbial N-Acetyl-L-Glutamate Kinases: Mutagenesis and Protein Engineering Study with the Pseudomonas aeruginosa and Escherichia coli Enzymes{triangledown}

M. Leonor Fernández-Murga1 and Vicente Rubio1,2*

Instituto de Biomedicina de Valencia (IBV-CSIC),1 Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER-ISCIII), C/Jaime Roig 11, 46010 Valencia, Spain2

Received 20 November 2007/ Accepted 2 February 2008


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ABSTRACT
 
N-Acetylglutamate kinase (NAGK) catalyzes the second step of arginine biosynthesis. In Pseudomonas aeruginosa, but not in Escherichia coli, this step is rate limiting and feedback and sigmoidally inhibited by arginine. Crystal structures revealed that arginine-insensitive E. coli NAGK (EcNAGK) is homodimeric, whereas arginine-inhibitable NAGKs, including P. aeruginosa NAGK (PaNAGK), are hexamers in which an extra N-terminal kinked helix (N-helix) interlinks three dimers. By introducing single amino acid replacements in PaNAGK, we prove the functionality of the structurally identified arginine site, as arginine site mutations selectively decreased the apparent affinity for arginine. N-helix mutations affecting R24 and E17 increased and decreased, respectively, the apparent affinity of PaNAGK for arginine, as predicted from enzyme structures that revealed the respective formation by these residues of bonds favoring inaccessible and accessible arginine site conformations. N-helix N-terminal deletions spanning ≥16 residues dissociated PaNAGK to active dimers, those of ≤20 residues decreased the apparent affinity for arginine, and complete N-helix deletion (26 residues) abolished arginine inhibition. Upon attachment of the PaNAGK N-terminal extension to the EcNAGK N terminus, EcNAGK remained dimeric and arginine insensitive. We concluded that the N-helix and its C-terminal portion after the kink are essential but not sufficient for hexamer formation and arginine inhibition, respectively; that the N-helix modulates NAGK affinity for arginine and mediates signal transmission between arginine sites, thus establishing sigmoidal arginine inhibition kinetics; that the mobile {alpha}H-β16 loop of the arginine site is the modulatory signal receiver; and that the hexameric architecture is not essential for arginine inhibition but is functionally essential for physiologically relevant arginine control of NAGK.


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INTRODUCTION
 
Microorganisms and plants make arginine from glutamate in eight steps (6), among which the first five (glutamate->N-acetylglutamate->N-acetylglutamyl-{gamma}-phosphate->N-acetylglutamate-{gamma}- semialdehyde->N-acetylornithine->ornithine) involve N-acetylated intermediates and generate the arginine precursor ornithine (5, 21, 22). The absence of steps 2 to 5 in animals (2, 14) renders the enzymes catalyzing these steps potential candidates for developing antibacterials and biocides. In some microorganisms, such as Escherichia coli, the arginine biosynthetic route is linear because ornithine is produced by the hydrolysis of N-acetylornithine (5). In such cases, the first enzyme of the route, N-acetylglutamate synthase (NAGS), catalyzes the controlling step and is feedback inhibited by the final product, arginine. Many more organisms, including yeasts, algae, plants, and many bacteria, such as Pseudomonas aeruginosa, have a more evolved, cyclic variant of this route in which the acetyl group is recycled by its transfer from acetylornithine to glutamate (5, 6, 22). In this case, NAGS plays a purely anaplerotic role, and the main controlling step that is feedback inhibited by arginine is catalyzed by the second enzyme of the route, N-acetylglutamate kinase (NAGK). Therefore, the latter enzyme exists in different organisms as arginine-insensitive and arginine-inhibitable forms (6). The importance of the controlling function of the arginine-inhibitable form is highlighted by the observation that in photosynthetic organisms, NAGK is a key target of the nitrogen signaling protein PII: when nitrogen is abundant, PII binds to NAGK, relieving arginine inhibition and allowing fast arginine synthesis (4, 13, 16, 23). Further controlling functions for arginine-sensitive NAGKs have been unveiled in Saccharomyces cerevisiae, where NAGK is involved in gene regulation (12) and in the formation of a complex with NAGS having the characteristics of a true metabolon, in which the regulatory properties of NAGS and NAGK are influenced mutually (1, 17, 18).

In an effort to ascertain the bases for the arginine sensitivity of NAGKs, we determined the crystal structures of four NAGKs, with one of them, that from E. coli (EcNAGK) (9, 19), being arginine insensitive, and the other three, from P. aeruginosa (PaNAGK), Thermotoga maritima (TmNAGK) (20), and Synechococcus elongatus (bound to PII) (16), being inhibited by arginine. These studies have revealed that EcNAGK is homodimeric and that arginine-sensitive NAGKs are hexameric and composed of three homodimers, with each one of them resembling EcNAGK (20). The three dimers forming the hexamer are linked by an N-terminal extension of 16 to 25 residues that is found exclusively in arginine-sensitive NAGKs and which forms a mobile, kinked {alpha} helix (the N-helix) (20) that interlaces with the N-helix attached to the adjacent dimer. Despite the absence of added arginine, we found this inhibitor in the crystal structure of TmNAGK, binding to each subunit in a crevice near the interdimeric junction, flanking the N-helix, and interacting with the C-terminal portion of it (20). This location next to the interconnecting helix appeared to lend structural support to the strong cooperative character (Hill coefficient of ~4) of the inhibition by arginine (6, 11). This cooperativity is a crucial control feature, since it makes NAGK a true switch that is turned off quite abruptly when a certain threshold arginine concentration is exceeded (6, 11). These studies also suggested that the interlaced kinked N-helix is the structural basis for hexamer formation and for the cooperative nature of the inhibition, mediating the cross talk between the arginine sites of different dimers (20).

In the present study, we tested these structure-based inferences experimentally. We used site-directed mutagenesis to introduce single amino acid changes in the putative arginine site of PaNAGK (schematized in Fig. 1), demonstrating that this site is indeed functional. We also introduced single amino acid substitutions and deletions of increasing length in the N-helix, showing that this helix is essential for hexamer formation and for arginine inhibition. Nevertheless, a chimera prepared by incorporating the N-terminal 25 residues of PaNAGK at the N terminus of EcNAGK remained arginine insensitive and dimeric. Although the hexameric organization was not found to be absolutely essential for arginine inhibition, it appears to be functionally essential, since both the hexameric organization and the N-helix were proven to be crucial for the cooperative kinetics and the relatively high apparent affinity for arginine exhibited by the enzyme.


Figure 1
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  		FIG. 1. Schematic representation of the proposed arginine site and the interlaced N-helices in PaNAGK. The N-helix of one subunit (in gray) is shown clamped between the N-helix and the body of the other subunit (in white). The inhibitor is shown with a thick trace and is labeled Arg. The side chains of the residues that have been mutated here and the side chain of D288 are illustrated and labeled. The dotted lines represent polar interactions between enzyme residues and the inhibitor. The curved lines and scissors illustrate the protein starting points in the deletion mutants prepared here.


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MATERIALS AND METHODS
 
Preparation of wild-type and mutant NAGK forms. Wild-type PaNAGK was produced as described previously (7) from the pET-22b (Novagen)-derived plasmid pNAGK-PA25, which carries the P. aeruginosa argB gene (argB encodes PaNAGK). Single-nucleotide substitutions causing missense mutations were introduced into the argB gene carried within pNAGK-PA25 by using a QuikChange kit (Stratagene) and the appropriate primer pairs listed in Table 1. Both wild-type and mutant forms of the enzyme were purified according to the reported procedure for wild-type PaNAGK (7), except for the utilization in the ion-exchange step of a 1-ml HiTrap Q HP column mounted on an ÁKTA fast-performance liquid chromatography system (both from GE Healthcare) and a 20-ml linear gradient of 0 to 0.5 M NaCl in 20 mM sodium phosphate, pH 8, containing 1 mM dithiothreitol for elution.


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  		TABLE 1. Synthetic oligonucleotides used for this study

PaNAGK forms carrying deletions spanning residues 2 to 13, 2 to 16, 2 to 20, and 2 to 26 (abbreviated from here on as {Delta}2-13, {Delta}2-16, {Delta}2-20, and {Delta}2-26, respectively) were prepared by a PCR approach using a high-fidelity thermostable DNA polymerase (Deep-Vent; New England Biolabs), P. aeruginosa PAO1 (11) genomic DNA as the template, and the appropriate forward and reverse primers (Table 1), which included NdeI and HindIII sites, respectively, for cloning. The reverse primer was the same for all the deletion mutants and contained the translation stop codon. The engineered genes were cloned into the NdeI and HindIII sites of pET-22b (Novagen) and were expressed in E. coli BL21(DE3) cells (Novagen) grown at 37°C in Luria-Bertani (LB) medium supplemented with 0.1 mg ml–1 ampicillin. When the cultures attained an optical density at 600 nm of {approx}0.5, they were left at 4°C for 1 h, and then 2% (vol/vol) ethanol and 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) were added and the incubation was continued overnight, with shaking, at 15°C. In the case of the {Delta}2-26 mutation, the BL21(DE3) cells were also cotransformed with pGroESL (a pACYC184-derived expression plasmid [provided by A. E. Gatenby, DuPont de Nemours, Wilmington, DE] encoding the E. coli chaperonins GroES and GroEL) (10) and were grown to an optical density at 600 nm of ~0.5 in ampicillin- and chloramphenicol-containing (both at 0.1 mg ml–1) LB medium before induction with 0.01 mM IPTG for 6 h at 30°C. The deletion mutant forms were used without purification, as centrifuged cell extracts prepared by sonication in 15 ml g–1 cells of 0.1 M sodium phosphate, pH 7.0, containing 0.2 mM dithioerythritol.

EcNAGK (8) was provided by F. Gil-Ortiz, from our laboratory. Chimeric EcNAGK with residues 1 to 25 of PaNAGK fused to its N terminus was prepared by first carrying out a PCR amplification of E. coli wild-type argB, using primers 28 and 29 (Table 1). In addition, the region of pNAGK-PA25 comprising base –371 (relative to the first argB codon) to base 75 of P. aeruginosa argB was PCR amplified in the same way, using primers 26 and 27 (Table 1). Primer 27 is complementary to codons 21 to 25 of P. aeruginosa argB and to codons 1 to 7 of E. coli argB (in that order; the E. coli sequence is shown in italics in Table 1), and it contains a unique AseI site (shown in bold). After AseI digestion of both amplified DNA regions, the fragments were ligated. The product was digested with NdeI and EcoRI and inserted directionally at the NdeI and EcoRI sites of pET-22b, generating plasmid pNAGK/PaEco. The latter plasmid was transformed into E. coli BL21(DE3) cells (Novagen), and the chimeric protein was overexpressed and purified exactly as previously described for wild-type EcNAGK (8).

The correctness of all the constructs and mutants prepared here was confirmed by DNA sequencing.

Other methods. NAGK activity was determined at 37°C at pH 7.5 as described previously (6), using the hydroxylamine-containing colorimetric assay of Haas and Leisinger (11). The data shown are the results of at least duplicate assays. One enzyme unit is the amount of enzyme that generates 1 µmol of product in 1 min. Analytical gel filtration chromatography was carried out, also as reported previously (6), using a Superdex 200HR (10/30) column mounted on an Akta fast protein liquid chromatography system (Amersham Biosciences). The column was equilibrated and run at 24°C with a solution of 50 mM Tris-HCl, pH 7.5, and 0.15 M NaCl. For analysis of crude extracts, 1 mg of protein was applied to the column, and 0.5-ml fractions were collected over ice and used for the assay of enzyme activity and protein content (3) and for immunodetection of NAGK protein in Western blots. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (15) was carried out in 15% polyacrylamide gels. For Western blotting, proteins were transferred to nitrocellulose membranes, followed by chemiluminescence immunodetection (ECL Plus Western blotting detection system; GH Healthcare) using a 10–3 dilution of a polyclonal rabbit antiserum (prepared by Genosphere-Biotech, Paris, France) against PaNAGK as primary antibody.


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RESULTS
 
The arginine site identified in structural studies is functional. None of the point mutations of residues lining or close to the arginine site (Y21A, R24E, K213A, H271N, E284D, and G290A) significantly altered solubility or gross stability or affected purification of the enzyme, carried out as described for wild-type PaNAGK (Fig. 2A). Gel filtration assays showed that these mutant enzymes remained hexameric (data not shown). The kinetic parameters for both substrates of the reaction in the absence of arginine were little changed in the mutants (Table 2). However, in all of the mutants, arginine inhibition was affected (Fig. 3).


Figure 2
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  		FIG. 2. Coomassie-stained SDS-PAGE of the wild-type and mutant forms of PaNAGK. St, protein markers with masses, in kDa, indicated at the side. (A) Enzyme forms having single amino acid substitutions, after purification. (B) Centrifuged crude extracts of E. coli cells expressing the indicated PaNAGK deletion mutants. As negative and positive controls, similar extracts of cells transformed with the parental pET22b plasmid without an insert and of cells expressing wild-type PaNAGK, respectively, are shown. The arrow points to the NAGK band. The enzyme activities in the different extracts (expressed per mg of protein in each extract) are given below the tracks. The specific activity of the pure enzyme in each extract is also shown. This value was calculated by estimating the amount of enzyme in each extract on the basis of densitometric estimation (Sigmagel program; Jandel Corporation) of the Coomassie staining in the band corresponding to NAGK. Because of the low intensity of the band, this estimate was unreliable for the {Delta}2-20 mutant, and therefore the specific activity for this mutant is not given. (C) Purified wild-type EcNAGK and the chimeric enzyme in which residues 1 to 25 of PaNAGK are glued to the EcNAGK N terminus. In the case of the chimeric enzyme, SDS-PAGE of the centrifuged extract is also shown.


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  		TABLE 2. Influence of single amino acid changes affecting the putative arginine site and the N-helix on the kinetic parameters of PaNAGK for substrates in the absence of argininea


Figure 3
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  		FIG. 3. Influence of point mutations affecting residues of the putative arginine site on arginine inhibition of PaNAGK. The standard error for each point does not exceed 15% of the value given. Curves fitting the data and extrapolating to 100% inhibition at an infinite concentration of arginine were drawn using the equation v = v[Arg]=0 x [1 – [Arg]H/(I0.5H + [Arg]H)] (11), where v[Arg]=0 is the activity in the absence of arginine, I0.5 is the concentration of arginine giving 50% inhibition, and H is the Hill coefficient. The curves drawn for the data for wild-type (WT) PaNAGK, for the R24E, E284D, and H271N mutants, and for the remaining three mutants are those for respective I0.5 values of 0.77, 0.11, 2.9, 7.7, and 57 mM and for respective H values of 4, 2, 2, 1, and 1. The framed inset details the inhibition at lower concentrations of arginine to illustrate the important increase in apparent affinity for the inhibitor observed with the R24E mutant.

Mutations Y21A and K213A replace arginine-interacting residues, and the G290A mutation affects a glycine of the {alpha}H-β16 loop that confers this loop with the appropriate flexibility (20) for encircling the arginine (Fig. 1). These mutations increased the concentration of arginine needed for 50% inhibition (I0.5) >50-fold. The H271N mutation affects a residue that lines the arginine site but that does not contact the inhibitor. This mutation increased the I0.5 for arginine ~10-fold. The E284D mutation, affecting a residue that interacts with the guanidinium group of the arginine and which, given the mildness of the change, may be expected to have only a small effect, gave a lesser increase in the I0.5 value (~4-fold).

In contrast to the mutations described above, the R24E mutation decreased the I0.5 for arginine ~7-fold relative to that of the wild type, and the inhibition remained sigmoidal. The enhancing effect of this mutation on arginine inhibition agrees with the findings on the enzyme structure. R24 forms an ion bridge with D288 that favors the closed, inaccessible conformation of the arginine site (20), and the mutation, by disrupting this bridge, should make the site more accessible to the inhibitor.

The present results therefore confirm the functionality of the arginine site identified in our structural studies (20).

Key role of the N-helix in hexamer formation. We tested the role of the N-helix in hexamer formation by deleting all or parts of this helix (Fig. 1). Except for the {Delta}2-20 mutant, which was expressed poorly, the deletion mutants were present in the extracts in soluble form and in substantial amounts (Fig. 2B) and were enzymatically active (although apparently less active than the wild-type enzyme) (Fig. 2B). However, the deletion mutants tended to aggregate and could not be purified. Gel filtration of the crude extracts through a Superdex-HR200 column showed (Fig. 4A) that the enzyme activity of the {Delta}2-13 mutant was eluted essentially like that of the wild-type enzyme, emerging as a large peak at the position corresponding to the hexamer (Fig. 4A, inset). Western blots of the wild-type and the {Delta}2-13 mutant also revealed (Fig. 4A) a strong parallelism between immunoreactive material and enzyme activity, although a small fraction of the NAGK protein emerged early from the column, possibly as large aggregates. With the other two mutants tested ({Delta}2-16 and {Delta}2-26), the majority of the activity eluted late, as a large peak corresponding to dimers (Fig. 4A and inset therein). Again, particularly with the {Delta}2-26 deletion, some activity emerged early, as a broad peak that decreased slowly, suggesting the existence of large aggregates that dissociate during chromatography, but little activity emerged at the position expected for hexamers. This was also shown by immunodetection of the protein for the {Delta}2-16 mutant (Fig. 4A). There was enzyme protein eluting early, corresponding to the large aggregates and the peak of the dimer, but not at the position expected for the hexamer. The Western blots for the {Delta}2-26 mutant exhibited a pattern similar to that for {Delta}2-16, although they were somewhat blurred because of a greater presence of large aggregates (Fig. 4A). Overall, these results indicate that the deletion of ≥16 residues from the N terminus of the N-terminal extension of PaNAGK leads to dissociation of the hexamer to the basic dimers, which are active. In contrast, the deletion of residues 2 to 13 does not cause dissociation.


Figure 4
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  		FIG. 4. Influence of the N-helix on the oligomeric state of NAGK, monitored by gel-exclusion chromatography through a Superdex 200HR (10/30) column. (A) Elution of wild-type PaNAGK and of deletion mutants of this enzyme, applied separately to the column as crude extracts. The peaks illustrate the enzyme activity in the 0.5-ml fractions collected from the column effluent. The panels at the top illustrate the results of immunodetection of NAGK in Western blots of the fractions. (Inset) Semilogarithmic plot of molecular masses of protein standards versus elution volumes. The peaks of activity for the wild-type enzyme and for the {Delta}2-13 mutant are represented in the plot at the sites corresponding to the sequence-deduced masses for their hexamers, whereas the {Delta}2-16 and {Delta}2-26 mutants are represented at the sites corresponding to the sequence-deduced masses of their dimers. The open circles correspond to the following protein standards: cytochrome c (12.3 kDa), lactalbumin (14.2 kDa), carbonic anhydrase (29.0 kDa), ovalbumin (42.7 kDa), wild-type EcNAGK (54.3 kDa), bovine serum albumin (66.4 kDa), alcohol dehydrogenase (146.8 kDa), aldolase (156.8 kDa), amylase (223.8 kDa), catalase (230.3 kDa), ferritin (440 kDa), and thyroglobulin (669 kDa). (B) Superimposed patterns of elution (as optical absorption at 280 nm [O.D.280]) of pure wild-type EcNAGK, wild-type PaNAGK, and chimeric EcNAGK having residues 1 to 25 of PaNAGK glued to its N terminus. The positions of elution of three marker proteins of the indicated masses (from higher to lower, amylase, alcohol dehydrogenase, and bovine serum albumin) are shown with arrows.

Although these results confirm that the N-helix is essential for hexamer formation, results obtained with a PaNAGK- EcNAGK chimera in which residues 1 to 25 of PaNAGK were attached to the N terminus of EcNAGK suggest that the presence of the N-helix is not enough for hexamer formation. This chimeric enzyme was soluble, could be purified easily (Fig. 2C), had the expected size (determined by SDS-PAGE [Fig. 2C] and by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, yielding a mass of 29,875 Da [sequence-expected mass, 29,861 Da]), and exhibited the same specific activity, within experimental error, as wild-type EcNAGK (65 U mg–1, compared with 67 U mg–1 for wild-type EcNAGK). As shown in Fig. 4B, the chimeric E. coli enzyme behaved in gel exclusion chromatography essentially like wild-type dimeric EcNAGK. These data also exclude the possibility that a partially stable hexamer that dissociates slowly during chromatography is formed, since the peak was symmetrical, with a very similar shape to the peak observed with the wild-type enzyme. Thus, the attachment of the N-terminal extension of PaNAGK to EcNAGK does not lead to hexamer formation.

Key role of the N-helix in the inhibition of PaNAGK by arginine. Figure 5 shows that the elimination of the entire N-helix ({Delta}2-26) renders PaNAGK arginine insensitive, whereas the attachment of the N-terminal extension of PaNAGK to the N terminus of EcNAGK does not render EcNAGK arginine sensitive. Thus, the N-helix is necessary but appears not to be sufficient for arginine inhibition (Fig. 5). Since the wild-type and the chimeric EcNAGK proteins, as well as the {Delta}2-26 mutant of PaNAGK, are dimeric (Fig. 4), it might be that a hexameric organization is essential for arginine inhibition. This possibility was excluded by the finding (Fig. 5) that arginine inhibits the {Delta}2-16 and {Delta}2-20 mutants, although with decreased apparent affinity and without sigmoidal inhibition kinetics. The {Delta}2-16 mutant has been shown to be dimeric (Fig. 4A), and the {Delta}2-20 mutant may be expected to be dimeric, given the longer deletion. Furthermore, we have confirmed that the dimers of the {Delta}2-16 mutant, which were separated from larger aggregates by gel filtration, are as sensitive to arginine as the activity in the crude extract (data not shown). Thus, arginine can inhibit the dimeric form of the enzyme. Nevertheless, the concentrations of arginine required for inhibition are much larger for the deletion mutants than for the wild-type enzyme, with estimated I0.5 values that increase with the length of the deletion (~60-, 170-, and 280-fold increases, relative to the value for the wild type, for the {Delta}2-13, {Delta}2-16, and {Delta}2-20 mutants, respectively). Therefore, even the portion of the N-helix that is not directly involved in binding arginine has a large impact on the affinity of the enzyme for the inhibitor, indicating that the N-helix plays a very important modulatory role. Furthermore, the deletions rendered the inhibition nonsigmoidal. This was expected for the {Delta}2-16 and {Delta}2-20 deletions, since the enzyme loses its hexameric architecture, which appears to be a prerequisite for the cooperative inhibition kinetics with these deletions. However, even with the hexameric {Delta}2-13 deletion mutant, the cooperativity for arginine was lost, possibly indicating that the cross talk between the arginine sites of adjacent dimers is mediated by the N-helix through the region that is deleted in the {Delta}2-13 mutant.


Figure 5
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  		FIG. 5. Influence of deletions affecting the N-helix or of the incorporation of residues 1 to 25 of PaNAGK into the N terminus of EcNAGK (chimeric EcNAGK) on the arginine inhibition kinetics of NAGK. The results with wild-type PaNAGK and EcNAGK are also shown for comparison. Standard errors did not exceed 15% of the mean values given. The curves through the points for wild-type PaNAGK and for the {Delta}2-13, {Delta}2-16, and {Delta}2-20 deletion mutants were drawn as indicated in the legend to Fig. 3, for respective I0.5 values of 0.77, 47, 130, and 219 mM and for H values of 4 for wild-type PaNAGK and of 1 for the deletion mutants.

Since the {Delta}2-16 and {Delta}2-20 mutants are both dimeric, the higher arginine requirement of the mutant with the longer deletion suggests that among residues 17 to 20, one, at least, may particularly influence the affinity of the enzyme for arginine. E17 appears to be a possible candidate. It is invariant in arginine-sensitive NAGKs (20), and in the structure of TmNAGK, the corresponding residue (E11) makes a hydrogen bond with a main-chain N atom of the {alpha}H-β16 mobile loop of the arginine site. Mutants E17D, E17Q, and E17A and, as a negative control, a Q10A mutant were therefore prepared. None of the mutations impaired enzyme expression or purification (Fig. 2A) or significantly affected the activity or the substrate kinetics of the enzyme in the absence of arginine (Table 2). However, whereas the Q10A mutation had no effect on arginine inhibition, mutations of E17 increased the concentrations of arginine needed for inhibition, consistent with these mutations interfering with hydrogen bond formation with the {alpha}H-β16 loop (Fig. 6).


Figure 6
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  		FIG. 6. Influence of point mutations affecting residues of the N-terminal portion of the N-helix on the arginine inhibition of PaNAGK. The results with wild-type PaNAGK are also shown for comparison. Standard errors did not exceed 15% of the mean values given. The curves through the points were drawn as indicated in the legend to Fig. 3, using a Hill coefficient of 4 and I0.5 values of 0.77, 0.7, 3.0, 5.1, and 19.5 mM for the wild-type and Q10A, E17Q, E17D, and E17A mutant proteins, respectively. (Inset) Inhibition kinetics of the E17A mutant for a larger range of arginine concentrations.


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DISCUSSION
 
The hexameric character and the presence of the N-terminal extension forming a kinked N-helix appear to be constant features of the arginine-sensitive NAGKs that are not found in arginine-insensitive NAGKs (6, 19, 20). The present results provide experimental evidence that the N-helix is essential for hexamer formation. The finding that N-terminal deletions encompassing at least the initial three turns of the N-helix lead to hexamer dissociation is consistent with the enzyme structure. These deletions encompass V11, V14, and L15, three residues forming a hydrophobic patch that mediates the interactions between the N-helices of adjacent subunits. In the {Delta}2-13 mutant, V14 and L15 are preserved, and the N-terminal methionine is brought next to V14. The hydrophobic patch is therefore largely unchanged, providing an explanation for this mutant remaining hexameric. Nevertheless, the dimeric nature of the chimeric EcNAGK incorporating the N-terminal extension of PaNAGK suggests that N-helix-N-helix interactions are not sufficient to generate the hexamer, possibly because of a lack of N-helix clamping against the adjacent subunit body due to the absence of the appropriate hydrophobic patch on the surface of the enzyme (mainly on helices H and A) (Fig. 1) (20). Correspondingly, this hydrophobic patch on the enzyme body of PaNAGK should be exposed to various degrees when all or part of the N-helix is deleted, possibly explaining the tendency of our deletion mutants to aggregate.

Our present structure-function studies with a series of six single-amino-acid mutants have also provided confirmation for the location of the PaNAGK arginine site (schematized in Fig. 1) identified by us on the basis of structural studies on TmNAGK. The C-terminal portion of the N-helix is part of the arginine site, and we show that this portion of the N-helix is also essential for arginine inhibition. Thus, the {Delta}2-26 deletion, which encompasses the C-terminal portion of the N-helix, abolished inhibition, whereas the other three deletions that were prepared did not include this portion of the N-helix and did not abolish arginine inhibition. Furthermore, the single-amino-acid mutation Y21A, affecting a residue of the C-terminal portion of the N-helix that in the crystal structure of TmNAGK accommodates the C-{alpha} of the arginine (20), dramatically increased the arginine concentration needed for inhibition. The requirement of the N-helix for arginine binding agrees with our previous proposal (20) that this helix is a good signature for recognizing arginine-inhibitable NAGKs. Nevertheless, the enzyme structure indicates that the N-helix alone cannot confer sensitivity to arginine, in agreement with the present finding that the attachment of the N-helix of PaNAGK to EcNAGK does not make EcNAGK arginine sensitive.

Our results have also shown a crucial role for the N-helix in the modulation of the sensitivity of the enzyme to arginine. One possible mechanism was perhaps most clearly shown by the effects of mutations affecting the N-helix residues R24 and E17, since these residues do not belong to the arginine site and yet their mutation strongly affects the apparent affinity of the enzyme for arginine. These residues make bonds with D288 in the mobile {alpha}H-β16 loop that is part of the arginine site of the same subunit (20). The bond mediated by R24 (a salt bridge) favors a closed conformation of the loop, rendering the site inaccessible to arginine, whereas the bond mediated by E17 (a hydrogen bond) favors an expanded conformation of the {alpha}H-β16 loop appropriate for the binding of the effector. The respective increasing and decreasing by the R24E and E17A mutations of the apparent affinity of the enzyme for arginine that we also observed are entirely consistent with this interpretation. In the native enzyme, changes in the position of the interlaced N-helices may mediate the breaking or formation of these bonds and may be the basis for the strongly cooperative nature of the inhibition by arginine (6, 11), consistent with our earlier proposal (20) that the N-helix is the mediator for the cross talk between arginine sites. The present work has also shown that deletions of the N-helix not encompassing E17 or R24 ({Delta}2-13 and {Delta}2-16) also dramatically decrease the apparent affinity of the enzyme for arginine. Thus, residues 2 to 16 of the N-helix may also be very important in inducing a high-affinity conformation of the arginine site. The facts that residues 2 to 16 of the N-helix of one subunit interact with the C-terminal part of helix H and the beginning of the {alpha}H-β16 loop of another subunit (schematized in Fig. 1) (see reference 20 for structural detail) and that helix H and the {alpha}H-β16 loop are mobile and make up a large fraction of the arginine site strongly suggest that helix H and the {alpha}H-β16 loop are the receivers in the arginine site of the signals transmitted by the N-helix. This is also supported by the observation that the {Delta}2-13 deletion, which, as already indicated, does not dissociate the hexamer but removes residues that interact with the C-terminal end of helix H of an adjacent subunit, abolishes the cooperative nature of the inhibition.

The finding that the {Delta}2-16 and {Delta}2-20 mutants were inhibited by arginine (although at increased concentrations of the inhibitor) indicates that a hexameric architecture is not essential for inhibition and agrees with the confinement of each individual arginine site within one enzyme subunit. Therefore, the increased separation between the ATP and NAG sites triggered by arginine that appears to be responsible for the inhibition (16, 20) can occur in the absence of a hexameric architecture, suggesting that arginine can induce this separation merely by binding to the subunit. In any case, although mechanistically unessential, the hexameric architecture and the N-terminal portion of the N-helix appear to be functionally essential, since they endow the inhibition with its sigmoidal character and with sensitivity to the appropriate range of arginine concentrations.


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ACKNOWLEDGMENTS
 
This work was supported by grant BFU2004-05159 from the Spanish Ministry of Education and Science. M. L. Fernández-Murga was a fellow of the Consejo Superior de Investigaciones Científicas.

We thank J. J. Calvete (IBV-CSIC, Valencia, Spain) for matrix-assisted laser desorption ionization-time-of-flight mass spectrometer use, S. Masia, E. Laserna (IBV-CSIC), and J. Cervera (CIPF, Valencia) for help with the Western blots, and Hubert G. Britton (Salisbury, United Kingdom) for critical reading of the manuscript.


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FOOTNOTES
 
* Corresponding author. Mailing address: Instituto de Biomedicina de Valencia (IBV-CSIC), C/Jaime Roig 11, 46010 Valencia, Spain. Phone: 34 96 339 17 72. Fax: 34 96 369 08 00. E-mail: rubio{at}ibv.csic.es Back

{triangledown} Published ahead of print on 8 February 2008. Back


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Journal of Bacteriology, April 2008, p. 3018-3025, Vol. 190, No. 8
0021-9193/08/$08.00+0     doi:10.1128/JB.01831-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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