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J Bacteriol. Jun 2007; 189(12): 4456–4464.
Published online Apr 6, 2007. doi:  10.1128/JB.00099-07
PMCID: PMC1913372

Identification of Essential Residues in Apolipoprotein N-Acyl Transferase, a Member of the CN Hydrolase Family[down-pointing small open triangle]

Abstract

Apolipoprotein N-acyl transferase (Lnt) is an essential membrane-bound protein involved in lipid modification of all lipoproteins in gram-negative bacteria. Essential residues in Lnt of Escherichia coli were identified by using site-directed mutagenesis and an in vivo complementation assay. Based on sequence conservation and known protein structures, we predict a model for Lnt, which is a member of the CN hydrolase family. Besides the potential catalytic triad E267-K335-C387, four residues that directly affect the modification of Braun's lipoprotein Lpp are absolutely required for Lnt function. Residues Y388 and E389 are part of the hydrophobic pocket that constitutes the active site. Residues W237 and E343 are located on two flexible arms that face away from the active site and are expected to open and close upon the binding and release of phospholipid and/or apolipoprotein. Substitutions causing temperature-dependent effects were located at different positions in the structural model. These mutants were not affected in protein stability. Lnt proteins from other proteobacteria, but not from actinomycetes, were functional in vivo, and the essential residues identified in Lnt of E. coli are conserved in these proteins.

Lipoproteins are unique combinations of a polypeptide and a water-insoluble lipid. Bacterial lipoproteins are involved in a wide variety of biological processes such as virulence, maintenance of cell wall integrity, insertion and stabilization of outer membrane proteins, bacteriocin release, adherence, protein secretion, antibiotic production, and solute uptake.

Lipoproteins are synthesized as prolipoproteins and are translocated across the inner membrane via the Sec machinery (5). Subsequent posttranslational modifications take place on the outer leaflet of the inner membrane (34). The cysteine residue targeted for lipid modification is located in a motif known as the lipobox [Leu(Ala,Val)−4-Leu−3-Ala(Ser)−2-Gly(Ala)−1-Cys+1]. First, the cysteine that will become the N-terminal amino acid of the mature lipoprotein receives an sn-1,2-diacylglyceryl group from phosphatidylglycerol on its sulfhydryl group through the action of phosphatidylglycerol::apolipoprotein diacylglyceryl transferase (Lgt) (7, 8, 28). The amino-terminal signal peptide is then processed by prolipoprotein signal peptidase (LspA), producing an apolipoprotein (12, 31). The third and last step is the acylation of the N-terminal glyceride-cysteine residue by apolipoprotein N-acyl transferase (Lnt), resulting in mature lipoprotein (11). Biochemical analyses showed that Escherichia coli Lnt (LntEc) can use all available phospholipids, i.e., phosphatidylglycerol, phosphatidylethanolamine, and cardiolipin, as acyl donors, but phosphatidylglycerol is the preferred substrate (10). All three lipoprotein-processing enzymes are essential for growth and viability and are located in the cytoplasmic membrane (20, 27, 33).

N acylation is required for the correct sorting of lipoproteins to the outer membrane by the Lol machinery (6). LolCDE is an ABC transporter that causes the ATP-dependent release of outer membrane lipoproteins (13), which are then captured by the specific periplasmic carrier protein LolA (17). LolA interacts with the lipid moiety and transfers it to the outer membrane lipoprotein receptor LolB, which is itself a lipoprotein (18). The amino acid residues at positions +2, +3, and +4 in the mature lipoprotein determine its final destination (29, 35). The general nature of this so-called inner membrane retention or Lol avoidance signal is an aspartic acid residue at position +2 in E. coli or lysine and serine residues at positions +3 and +4, respectively, in Pseudomonas aeruginosa (22; S. Lewenza, unpublished data). Lipoproteins that harbor this signal are not recognized by LolCDE and therefore remain in the inner membrane (30). The sorting rules are conserved in Enterobacteriaceae (16).

On the basis of sequence similarity, Lnt is classified as a member of the nitrilase superfamily (24). These nitrilases, amidases, and carbamylases are multimeric proteins with an α-β-β-α fold that hydrolyze carbon-nitrogen bonds and have a common Glu-Lys-Cys catalytic triad. The nitrilase mechanism indicates that Lnt will probably function by a nucleophilic attack of the activated thiol on the sn-1-glycerolphospholipid carbonyl group to generate a lysophospholipid by-product and an acyl enzyme intermediate, which is then resolved by the apolipoprotein alpha-amino group.

How Lnt interacts with phospholipids and/or with apolipoprotein substrates remains unknown. We have identified essential residues in LntEc by site-directed mutagenesis as a first step towards an understanding of its function. Lnt homologues from various Proteobacteria and Actinomycetes were tested for Lnt activity, and conserved residues were identified. These findings are correlated with a predicted structural model for Lnt based on known structures of CN hydrolases.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

E. coli strains used in this study are listed in Table Table1.1. Luria broth (LB) and agar were prepared as described previously (19). When appropriate, media were supplemented with l-arabinose (0.2%), l-fucose (0.2%), or d-glucose (0.2%). Ampicillin was used at 100 μg/ml.

TABLE 1.
Bacterial strains and plasmids

Plasmid construction.

For immunodetection of Lnt, the protein was tagged at the carboxy terminus with a double c-myc epitope. An oligonucleotide sequence composed of primers upperMYC2 and lowerMYC2 (Table (Table2),2), encoding a double c-myc tag, was inserted directly between the XbaI and HindIII sites in pBAD18, resulting in plasmid pCHAP7521. The lnt gene was amplified by PCR using primers 5′-cutE (27) and lntXbaIrev and inserted between the EcoRI and XbaI sites in pCHAP7521, resulting in plasmid pCHAP7526; the 3′ end of lnt is in frame with the c-myc tag. The lnt-myc2 gene was recloned into pUC18 as an EcoRI-HindIII fragment, resulting in pCHAP7530. The mutated lnt genes were recloned into pCHAP7530 as ScaI-AscI fragments, except L436A (mutant 33), which was recloned as an HpaI fragment and verified by sequencing.

TABLE 2.
Cloning primers

lnt homologues were PCR amplified with a C-terminal His6 tag and cloned in frame with the 5′ end of lacZ in pUC18 under the control of the plac promoter. PCR amplification was performed in 30 cycles of 94°C for 30s, 52°C for 30s, and 72°C for 2 min using 0.4 μM primers and the JumpStart REDTaq ReadyMix kit (Sigma). Ten percent dimethyl sulfoxide was added to PCRs using template DNA from Streptomyces coelicolor. PCR products were column purified, digested, and cloned as EcoRI-BamHI fragments with lnt from P. aeruginosa (lntPa) and lnt from Neisseria meningitidis (lntNm). Vibrio cholerae lnt (lntVc) was cloned as an XmaI-BamHI fragment, Yersinia pseudotuberculosis lnt (lntYp) was cloned as a SacI-PstI fragment, S. coelicolor lnt (lntSc) was cloned as an EcoRI-XbaI fragment, and Corynebacterium glutamicum lnt (lntCg) was cloned as a SacI-BamHI fragment. All plasmid inserts were confirmed by DNA sequencing. Primers for amplification are listed in Table Table22.

Sequence alignments and structural models.

Conserved residues in Lnt were identified by an alignment of 27 Lnt proteins of the family Gammaproteobacteriaceae (see Fig. S1 in the supplemental material). The CN hydrolase domain in Lnt was identified by an NCBI BLAST search. Residues conserved between Lnt and CN hydrolases were identified using MultAlin (3). The Protein Homology/Analogy Recognition Engine (PHYRE) server (http://www.sbg.bio.ic.ac.uk/phyre) was used to predict a structural model for Lnt, and Swiss-Pdb Viewer v3.9b1 (http://www.expasy.org/spdbv) and PyMol (http://www.pymol.org) were used for modeling and analysis of the mutants.

Site-directed mutagenesis of lnt of E. coli.

Mutagenic oligonucleotides (Sigma) were designed to contain the desired codon change and a new restriction site (Table (Table3).3). Site-directed mutagenesis of 34 conserved residues was performed by PCR based on the QuikChange site-directed mutagenesis protocol (Stratagene) using lnt in pCHAP6571 (27) as the template DNA and Pfu high-fidelity DNA polymerase (Invitrogen) for DNA synthesis. PCR cycles included 18 cycles of a 5-min extension step to amplify the template plasmid fully. Template DNA was digested with DpnI, and part of the PCR mixture was transformed into competent PAP105 cells. The mutant lnt genes were amplified by colony PCR from transformants using universal primers M13for and M13rev. The fragments were verified by digestion with restriction enzymes for which sites were introduced by the mutagenic primers. All constructs were sequenced to confirm the introduction of the desired mutation.

TABLE 3.
Mutagenic lnt primers

Complementation assay of conditional lnt mutant.

Plasmids carrying lnt mutations were introduced into PAP8504 or PAP8508 and selected on LB agar plates containing ampicillin and arabinose. Single colonies were restreaked onto plates containing arabinose or fucose/glucose with or without 1 mM IPTG (isopropyl-β-d-thiogalactopyranoside) and grown at different temperatures to test for complementation of the conditional lnt mutant.

Modification of Lpp by Lnt mutants.

The modification of Lpp by Lnt variants was analyzed in cell lysates from cells depleted for wild-type lnt in PAP8504 expressing nonfunctional lnt. Strains were grown overnight at 37°C in LB medium with 0.2% arabinose and washed in LB medium before diluting 1:100 into fresh LB medium with 0.2% arabinose or 0.2% fucose and 1 mM IPTG and ampicillin. Cells were grown at 37°C with agitation to an A600 0.8 and rediluted 1:10. Samples were taken shortly before growth arrest was observed.

SDS-PAGE and immunoblotting.

Total cell lysates were solubilized in loading buffer with 4 mM dithiothreitol, heated at 100°C for 5 min, and separated by 10%- or 8%-acrylamide sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or 16%-acrylamide Tricine SDS-PAGE gels. After transfer onto nitrocellulose membranes, proteins were detected by incubating the membranes with primary polyclonal rabbit antibodies against c-Myc, Lpp (27), or His6 (Sigma) and horseradish peroxidase-conjugated secondary antibodies to rabbit immunoglobulin G (Amersham). Secondary antibodies were detected by enhanced chemiluminescence (Pierce).

RESULTS AND DISCUSSION

Identification of conserved residues and site-directed mutagenesis of lnt.

Conserved residues in LntEc were identified by aligning 27 Lnt sequences of γ-proteobacteria using MultAlin (3). A total of 63 conserved residues were identified among 512 amino acids (Fig. (Fig.1;1; see the supplemental material). We previously determined the membrane topology of Lnt (27). Conserved residues are found in transmembrane segment 1 (TM1), TM2, and TM3 and in two domains, one located in the cytoplasm between TM4 and TM5 and the other in the periplasm between TM5 and TM6. Of the 63 conserved residues, 34 were chosen for mutagenesis based on their conservation among Lnt proteins or on their potential role in enzymatic activity as predicted from CN hydrolases. One alanine residue was converted into aspartate, and the other 33 conserved amino acids were changed into alanine. Glycine residues and most proline residues were not targeted for mutagenesis. All mutations could be readily introduced into LntEc, suggesting that none of them resulted in a dominant negative phenotype in wild-type E. coli.

FIG. 1.
Comparison of protein levels and migrations of 34 Lnt protein variants. Numbers correspond to the replaced residues listed in Table Table4.4. The proteins were produced in wild-type E. coli strain PAP105 in the absence (−) or presence ...

Functional analysis of Lnt mutants in a conditional lnt mutant strain.

The mutated lnt alleles were tested for functionality in a conditional lnt mutant (27). In this strain, the chromosomal lnt gene is under the exclusive control of an arabinose promoter, paraB. The addition of arabinose to the medium allows growth, whereas glucose or fucose represses the expression of lnt from paraB, resulting in Lnt depletion. Cell lysis occurs after approximately eight generations due to the mislocalization of (apo)lipoproteins (27). The functionality of the mutated lnt alleles was tested by the growth of the conditional lnt mutant on LB agar plates containing fucose and expressing the lnt mutants from a plac promoter located on a high-copy-number plasmid. The mutants were classified according to their complementation phenotypes (Table (Table4).4). Class I mutants were unable to restore the growth of the conditional lnt mutant at any growth temperature, even in the presence of the inducer IPTG, and, therefore, were unable to produce nonfunctional Lnt proteins. These include mutations affecting the potential active-site residues E267, K335, and C387 and residues Y388, E389, W237, and E343. Both class II and class III mutants were temperature sensitive; class II mutants restored the growth of the conditional lnt mutant only at 30°C, and class III mutants were nonfunctional at 42°C. Two mutants belong to class IV, which complemented the conditional lnt mutant only in the presence of IPTG. Class V mutants could complement the growth of the conditional lnt mutant under all conditions tested, indicating that the amino acids affected are not essential for Lnt function.

TABLE 4.
Complementation of PAP8508 by Lnt variantsa

Analysis of Lnt protein levels and mobility behavior by gel electrophoresis.

The inability of mutated lnt alleles to complement a conditional lnt mutant might be caused by the instability or misfolding of the protein. To verify the production of the Lnt mutant proteins, the constructs encoding the 34 Lnt variants were expressed in wild-type E. coli, and the proteins were analyzed by immunoblotting. To facilitate the detection of Lnt, a double c-myc tag was fused in frame with the C terminus of Lnt and the panel of single-amino-acid mutants. The c-myc2 tag does not interfere with Lnt function, since Lnt-myc2 restored the growth of a conditional lnt mutant like wild-type Lnt (data not shown). All class I nonfunctional mutants were produced, indicating that protein stability was not affected (Fig. (Fig.1).1). Higher amounts of Lnt were observed in samples induced with IPTG, but protein levels were essentially similar in the different mutants. Two Lnt variants, W237A (mutant 11) and E267A (mutant 14), migrated more slowly than wild-type Lnt upon SDS-PAGE and appeared as double bands (Fig. (Fig.22 and data not shown), suggesting that they are modified by the interaction with an acyl group. The introduction of C387A in W237A or E267A resulted in the disappearance of the double bands, indicating that C387 plays a role in the aberrant migration of the W237A and E267A mutants (Fig. (Fig.22 and data not shown). The proteins encoded by mutated alleles in classes II and III were also produced. For all temperature-sensitive variants except A433D (mutant 30) (Fig. (Fig.1),1), the protein level increased at elevated growth temperatures (Fig. (Fig.3).3). This was not due to the mutations, since wild-type Lnt shows a similar production pattern, but might be caused by an increased plasmid copy number. In any case, elevated temperatures did not destabilize these Lnt variants. In vitro Lnt activity is destroyed only at 80°C (12), suggesting that the Lnt variants have altered conformations at 42°C. Lnt(A433D) was less abundant than wild-type Lnt at 37°C, indicating that this is the only replacement that causes instability (or reduced production at elevated temperatures). The two class IV mutants that are dependent on IPTG for complementation produced Lnt at levels comparable to those of wild-type Lnt. These proteins might have an altered conformation that results in lower affinity for phospholipids or apolipoprotein and that is overcome by producing more enzyme. The functional Lnt variants (class V) were also stably produced, as expected (Fig. (Fig.11).

FIG. 2.
Role of active-site cysteine in mobility behavior of Lnt(W237A). C387A (mutant 24) was introduced into Lnt(W237A) (mutant 11) and is indicated as 11 24 (lane 4). Lnt variants were produced in wild-type E. coli strain PAP105, separated by 8% SDS-PAGE, ...
FIG. 3.
Protein stability of temperature-sensitive Lnt variants. E. coli PAP105 producing two Lnt variants representative of class IV (mutants 13 and 29) or three variants representative of class III (mutants 8, 17, and 27) were grown at a permissive temperature ...

The model lipoprotein Lpp was analyzed for modification by nonfunctional Lnt variants. All class I mutants, except W148A (mutant 6), caused an accumulation of the apo form of Lpp (Fig. (Fig.4),4), indicating that the N acylation activity of these Lnt variants is affected. Residue W148A, which is located outside the CN hydrolase domain, does not affect lipidation of apo-Lpp and may be involved in a different step in the mechanism catalyzed by Lnt.

FIG. 4.
Modification of Lpp by nonfunctional Lnt variants. The lnt depletion strain PAP8504 expressing nonfunctional lnt variants was grown in the presence of fucose and IPTG. Numbers correspond to the replaced residues listed in Table Table4.4. Lpp was ...

A structural model for Lnt, a member of the CN hydrolase family.

We used the PHYRE server to predict a structural model for LntEc based on primary sequence conservation and known protein structures. Four structures of carbon-nitrogen hydrolases are known: Agrobacterium sp. (1UF5A) (21, 32), Saccharomyces cerevisiae (1F89A) (15), Caenorhabditis elegans (1EMSA) (25), and Pyrococcus horikoshii (1J31A). LntEc shows low overall sequence conservation with these proteins (less than 20% identity), but a structure could be predicted for LntEc based on known structures. Like CN hydrolases, Lnt is predicted to have an α-β-β-α fold (Fig. (Fig.5).5). Based on the CN hydrolase structures, helix 2 (H2) and H3 are probably solvent exposed. Helices H7 and H8 might be involved in α-α-subunit interactions, but the multimeric state of Lnt is currently unknown. The inside of the structure is surrounded by several β-strands. H1, H4, and H5 face outward and are likely to be flexible, making the potential active-site pocket readily accessible to substrates. This is different from CN hydrolases, in which the active site, though solvent exposed, is located in a deep cleft. H1, H4, and H5 in the Lnt model might fulfill a lid function, allowing the enzyme to bind phospholipid, cleave the acyl group, release the lysophospholipid, and subsequently bind the apolipoprotein substrate to be N acylated.

FIG. 5.
Predicted structural model for Lnt (K220-E468). (A) Secondary-structure motifs are colored red for α-helices and yellow for β-strands. The active site is blue. (B) Superposition of mutagenized residues on the model are colored white, corresponding ...

The site-directed replacements in LntEc were mapped onto the model using Swiss-PdbViewer (9) and PyMol. Among the seven nonfunctional replacements, five are located in the active-site pocket that includes the potential catalytic triad E267-K335-C387 (Fig. (Fig.55 and Table Table4).4). Two residues, W237 and E343, are located in loops that extend from this cleft. Several of the affected residues in class II and class III (temperature-sensitive) mutants, i.e., Y333, Q372, N314, and P346, are also located in the hydrophobic pocket. Residues Q424, R342, A433, E435, and L436 are all located in H8. P266 is located in the loop between H2 and H3 and is probably involved in the correct orientation of E267 towards the reactive cysteine. Q228 and Y249 are located inside the structure in the vicinity of P266, facing away from the catalytic triad. The IPTG-dependent replacement Q233A (class IV) is upstream from H1.

The reactive cysteine has been the main target in many mutational analyses with various CN hydrolases, which always result in enzyme inactivation (1, 4, 14, 26). Mutations affecting the catalytic triad of an aliphatic amidase of P. aeruginosa showed that the replacement of Glu causes a loss of activity and that the stability of variants in which Lys is substituted depends on the amino acid (23). Structural studies with N-carbamoyl-d-amino acid amidohydrolase from Agrobacterium radiobacter indicated that several variants affected in the α-α dimeric interface have a structure that is nearly identical to that of the wild-type enzyme but have higher stability and activity at higher temperatures (2). Furthermore, the enzyme does not change conformation in the presence of a ligand (1). Our ongoing studies are directed at determining whether Lnt shares these characteristics.

The essential residues located in the membrane-bound N-terminal domain (W74, F146, and W148) are not part of the CN hydrolase domain and could therefore not be mapped onto the Lnt model. These residues are not required for protein stability (Fig. (Fig.1)1) but might play a role in the folding and/or positioning of the enzymatic portion of the protein. Coproduction of two parts of Lnt, domain A (M1-P218) and domain B (V188-K512), in the conditional lnt mutant did not restore growth on plates containing fucose (data not shown). Furthermore, an Lnt-PhoA chimera in which the PhoA part was fused in frame with P476 (27), thereby deleting the sixth and last transmembrane segment, did not allow the growth of a conditional lnt mutant (data not shown). This clearly indicates that the CN hydrolase domain of Lnt must be anchored in the cytoplasmic membrane and correctly oriented towards the periplasm.

Complementation of the conditional lnt mutant by Lnt homologues.

Lnt is conserved in the entire family of proteobacteria and in some actinomycetes but not in low-GC-content gram-positive bacteria. Our initial studies on conserved residues and domains in Lnt were based on the alignment of Lnt from 27 γ-proteobacteria. We tested three Lnt proteins from other γ-proteobacteria, one from β-proteobacteria, and two from actinomycetes for complementation of the conditional lnt mutant to determine the degree of functional conservation between these species and to get more insight in the overall protein structure. Lnt from Y. pseudotuberculosis, V. cholerae, N. meningitidis, and P. aeruginosa restored the growth of the conditional lnt mutant under restrictive conditions (Table (Table5).5). Expression of lntPa caused growth inhibition in the presence of arabinose but not in the presence of glucose at 37°C, suggesting that it is either toxic or dominant negative over lntEc expressed from paraB. A similar inhibitory effect was observed when LntEc(E435K) was produced in wild-type Salmonella enterica at 42°C (27). Overproduction of LntEc in wild-type E. coli did not inhibit growth (data not shown). These observations could be explained if Lnt is multimeric. The formation of a heterodimer/multimer between LntPa and LntEc could inhibit the acylation of the former if it is slightly different in structure or has a reduced affinity for its substrates. In the absence of LntEc, the LntPa homodimer/multimer might catalyze acylation although less efficiently than LntEc. It is unclear why the other functional Lnt homologues do not inhibit growth in the presence of LntEc. Membrane topology predictions suggest a similar orientation for LntPa, LntVc, LntNm, and LntYp compared to LntEc (data not shown) (27).

TABLE 5.
Restoration of growth of PAP8504 by Lnt homologues in the absence of arabinose

lnt genes from S. coelicolor and C. glutamicum were not able to complement the conditional lnt mutant (Table (Table5)5) but were expressed in wild-type E. coli, as judged by immunoblotting with anti-His antibodies (data not shown). The alignments of the primary Lnt sequences indicated a relative high percentage of similarity (77%) between LntEc and LntYp but low similarity with other Lnt homologues (between 10% for LntSc and 50% for LntVc). A closer inspection of the conservation of important residues identified in LntEc showed that all essential residues (class I mutants) are conserved in the Lnt homologues, with the exception of W237 in LntSc and LntCg, where it is replaced by serine and aspartate, respectively (Table (Table6).6). Y388 in LntSc is replaced by another hydrophobic residue, phenylalanine. All other residues implicated in Lnt activity (class II, III, and IV mutants) are conserved only in the Lnt homologues that restore Lnt function in the conditional lnt mutant, except in LntPa, in which L436 is a serine (Table (Table6).6). This illustrates that the essential residues identified in LntEc are also important for Lnt's of other proteobacteria. The differences observed between proteobacteria and actinomycetes might reflect differences in the acyl donor and/or substrate specificity of Lnt.

TABLE 6.
Conservation of essential residues in Lnt homologuesa

We have presented data on essential residues in Lnt, their positions in a structural Lnt model, their potential roles in Lnt activity, and their conservation among other bacterial species. These findings will be beneficial for studying the mechanisms by which Lnt hydrolyzes phospholipids and transfers the acyl group onto apolipoprotein substrates.

Supplementary Material

[Supplemental material]

Acknowledgments

We thank Tony Pugsley for constructive advice and corrections of the manuscript, members of the Pugsley laboratory for support and helpful discussions, Michael Nilges for help with protein modeling, and Hajime Tokuda for antibodies against Lpp.

N.B. was supported by the Fondation Recherche Médicale, and S.L. was supported by a postdoctoral fellowship from the Canadian Louis Pasteur Foundation. The work was supported in part by a grant from the Programme de Microbiologie Fondamentale of the French Ministère Délégué de la Recherche et aux Nouvelles Technologies.

Footnotes

[down-pointing small open triangle]Published ahead of print on 6 April 2007.

Supplemental material for this article may be found at http://jb.asm.org/.

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