![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Department of Microbiology,1 Center for Biofilm Engineering, Montana State University, Bozeman, Montana 59717,4 Department of Microbiology and Immunology, Medical College of Virginia Campus of Virginia Commonwealth University, Richmond, Virginia,2 McGuire Veterans Affairs Medical Center, Richmond, Virginia3
Received 21 November 2004/ Accepted 30 March 2005
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-L-guluronic acid (G) linked by ß1-4 glycosidic bonds. In bacterial but not in algal alginates, the M residues are modified by the addition of O-acetyl groups at the O-2 and/or O-3 position (70). In the opportunistic pathogen Pseudomonas aeruginosa, alginate is an important virulence factor, particularly in patients with the genetic disorder cystic fibrosis, where conversion of strains to the alginate-overproducing (mucoid) phenotype often results in chronic pulmonary P. aeruginosa infections (48). Alginate acts as a virulence factor in these infections by contributing to the matrix material of the mucoid P. aeruginosa biofilms (45) and by protecting the bacteria from opsonic phagocytosis (55). Alginate also neutralizes oxygen radicals produced by inflammatory immune cells (38) and stimulates the production of inflammatory cytokines indicative of a Th2-type immune response (47, 49).
The structure of alginate contributes to its activity as a virulence factor. For example, the M/G block structure is important for the chemical and physical properties of the polymer, including its viscosity and its interaction with divalent cations (70, 71). The presence of O-acetyl substitutions on the O-2 and O-3 positions of the D-mannuronate residues (70) is essential for the formation of microcolonies and for the avoidance of antibody-independent and antibody-mediated opsonic phagocytosis (45, 55).
The block structures of alginate vary depending on the organism producing the polymer. Algal and Azotobacter vinelandii alginates contain continuous stretches of G residues (G-blocks), while alginates from pseudomonads contain primarily M residues randomly interspersed with G residues (21, 66, 70). The G-blocks of A. vinelandii alginate chelate calcium and give the polymer its gelling properties (19, 21, 34, 70, 71), important for this organism to build capsules of cysts (60). In P. aeruginosa, G-blocks are not found, most likely because the polymer-level epimerase of pseudomonads, AlgG, is not capable of introducing G residues adjacent to other G residues (66, 70). In A. vinelandii, G-blocks are introduced into the alginate polymer by a series of extracellular mannuronan epimerases, AlgE1 to AlgE7, that are encoded by tandem gene repeats (11, 76). Genes for these extracellular epimerases are not found in P. aeruginosa (75).
The bacterial alginate biosynthetic pathway occurs in four steps: (i) synthesis of the alginate precursor GDP-mannuronic acid, (ii) polymerization of GDP-mannuronic acid into ß1-4-linked polymannuronate, (iii) modification of the polymannuronate into its final alginate structure, and (iv) polymer secretion. Precursor synthesis, carried out by AlgA, AlgC, and AlgD, has been well characterized, and the crystal structures of AlgD and AlgC have been solved (56, 72). Less is known about the polymerization step, which may be carried out by Alg8 and Alg44, putative inner membrane proteins (39). The periplasmic proteins AlgX and AlgK, whose functions have not yet been determined, may also play a role in polymerization (26, 43). Enzymes that localize primarily to the periplasm carry out modifications of polymannuronic acid to its structurally mature and functionally active form. These enzymes include AlgI, AlgJ, and AlgF, forming the alginate O-acetylation complex; AlgG, the periplasmic mannuronan epimerase; and AlgL, an alginate lyase (14-16, 43, 64). Polymer secretion is likely mediated by AlgE (6, 57).
Jain et al. (25) proposed a model for alginate biosynthesis in which the alginate biosynthetic proteins form a multicomponent scaffold for polymerization, modification, and export. Evidence supporting this model includes deletion mutation of algG, algK, and algX (25, 26, 59). Strains with these deletions secrete depolymerized alginate, suggesting that removal of any of these components from the biosynthetic scaffold results in access to and degradation of the alginate by the periplasmic alginate lyase AlgL. Further support for the scaffold model is the work of Gimmestad et al. (18), who demonstrated that expression of both a wild-type and epimerase-defective mutant form of AlgG in Pseudomonas fluorescens results in two distinct polymer types, a wild-type M-G alginate and a poly(M) alginate. Those results suggest that one epimerase is required for each alginate molecule synthesized, rather than multiple epimerases acting at random sites on the polymer. Therefore, alginate is likely shuttled across the periplasm through an alginate biosynthetic complex, with the mannuronan epimerase, AlgG, involved in both epimerization and the transfer process.
To further characterize AlgG, its interaction with polymannuronate, and its role in the alginate biosynthetic scaffold, we performed sequence analysis and structural modeling of AlgG as well as phenotypic characterization of AlgG mutant proteins. The results show that AlgG contains a repeating sequence in its C terminus that is predicted to fold into a right-handed ß-helix (RHßH), similar to proteins with a carbohydrate-binding and sugar hydrolase (CASH) domain (7). The model of the AlgG RHßH contains a shallow groove that may accommodate the linear polysaccharide, similar to other CASH domain proteins. This groove contains amino acids that are necessary for mannuronan epimerization and transfer of alginate through the periplasm. The structure of AlgG suggests that it may play a protective role as part of the alginate biosynthetic scaffold against alginate degradation by AlgL while performing enzymatic modification of polymannuronic acid.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
algG::aacCI contains a nonpolar deletion of algG (25). This strain secretes depolymerized alginate, presumably due to disruption of the alginate biosynthetic scaffold and degradation of alginate by AlgL (25). However, it can be complemented in trans by algG, indicating the that the algG::aacCI insertion is nonpolar on expression of the downstream alg biosynthetic genes (25). P. aeruginosa and Escherichia coli were cultured on L broth (LB) (10 g tryptone, 5 g yeast extract, and 5 g NaCl per liter). Triparental matings were used to transfer plasmids from E. coli to P. aeruginosa using the helper plasmid pRK2013 (13) and Pseudomonas isolation agar (Difco) as the selective medium. Antibiotics, when used, were used at the following concentrations: carbenicillin, 300 µg/ml; ampicillin, 100 µg/ml; tetracycline, 20 µg/ml; and kanamycin, 60 µg/ml.
|
-helical region while retaining the native AlgG signal peptide. The primers used for the AlgG 37-137 deletion were 5'-CGACTGCACGGTGCACCAATGCTTC-3' and 5'-GAAGATCGCCTGGGGCGCCGCCCACGCCTGGCCGTGCAG-3' to amplify the upstream algG fragment and 5'-CAGGCGTGGGCGGCGCCCCAGGCGATCTTCATCGAAGGC-3' and 5'-GGGCCATCTAGAGGCCGGCGGCC-3' to amplify the downstream algG fragment. The primers used for the AlgG 37 to 161 deletion were 5'-CGACTGCACGGTGCACCAATGCTTC-3' and 5'-CTCGACCTGTTCCAGCGCCGCCCACGCCTGGCCGTGCAG-3' for the upstream fragment and 5'-CAGGCGTGGGCGGCGCTGGAACAGGTCGAGCCGGGGGTG-3' and 5'-TGGGCCATCTAGAGGCCGGCGGCC-3' for the downstream fragment. The reverse primers introduced XbaI restriction sites into the 3' end of the PCR products to facilitate ligation of the NcoI-XbaI fragments containing the mutant algG into pMF54. The resulting plasmids were labeled pSAD149 for the AlgG 37 to 137 deletion and pSAD151 for the AlgG 37 to 161 deletion. Plasmids containing algG point mutations and N-terminal deletion mutations were introduced into P. aeruginosa and tested for complementation of the epimerization defect of FRD462 and complementation of the secretion and epimerization defects of FRD1200.
Determination of mannuronan epimerase activity. P. aeruginosa FRD1, FRD462, and FRD1200 with plasmids containing algG mutations were incubated for 24 h in 10 ml of LB supplemented with 300 µg/ml carbenicillin and 1.0 mM isopropyl-ß-D-thiogalactopyranoside (IPTG). Alginates were purified from the culture supernatants as described previously (14), using one precipitation with 2% (wt/vol) cetyl pyridinium chloride and one precipitation with isopropanol.
Alginate pellets were resuspended in 4 ml of saline. Alginate concentrations were assayed by a modification of the Knutson and Jeanes (35) protocols, using Macrocystis pyrifera alginate as a standard. Briefly, partially purified alginates, 20 µl, was mixed with 1.0 ml of borate-sulfuric acid reagent (10 mM H3BO3 in concentrated H2SO4) and 30 µl of carbazole reagent (0.1% in ethanol). The mixtures were heated to 50°C for 30 min, and the concentrations were determined spectrophotometrically at 535 nm.
Alginate epimerization was determined using the alginate lyase assay (4, 14). Alginate lyase of Klebsiella aerogenes cleaves L-guluronate at MG or GG blocks (3). Therefore, the relative number of G residues per gram of alginate can be determining by assaying the abundance of cleaved, unsaturated residues produced by alginate lyase. For this assay, 65 µg of alginate was diluted to 150 µl in deionized water. The alginates were deacetylated with 50 µl of 1 M NaOH for 15 min at 65°C and then neutralized to pH 7.2 with 50 µl of 1 M HCl and 100 µl of lyase buffer (50 mM Tris-HCl, pH 8.0, 10 mM MgCl2 · 6H2O, 5.0 mM CaCl2). Alginate lyase, 10 µl, was added, and the mixture was incubated for 1 h at 25°C. The following were then added to the reaction mix: periodic acid (250 µl of 0.2 M, incubation for 40 min at 25°C), sodium arsenite (100 µl of 2% in 0.5N HCl, incubation for 1 min at 25°C), and thiobarbituric acid (1 ml of 0.6% solution, pH 2.0, incubation for 30 min at 65°C). The samples were cooled for 1 h and centrifuged at 12,000 rpm for 5 min to remove the precipitate. G-residue abundances were measured spectrophotometrically at 535 nm. Results were normalized to one optical density unit at 535 nm/ng uronic acid and compared to FRD1 alginate as a percentage of wild-type activity. Student's t tests were performed for the AlgG mutants and wild-type alginate, with P < 0.05 considered significantly different.
Assays for mannuronan epimerization in vitro. The in vitro assays for alginate epimerization were similar to those described previously (14). Briefly, 1-liter cultures of E. coli expressing wild-type and mutant forms of AlgG were incubated for 24 h in LB medium supplemented with ampicillin and IPTG. The cultures were centrifuged, and cell pellets were resuspended in 1 ml of lyase buffer. Cells were then subjected to freeze-thawing followed by sonication to lyse the cells. Cell debris was removed by centrifugation, and the supernatants containing AlgG (50 µl) were mixed with 100 µl of polymannuronic acid purified from the supernatants of P. aeruginosa FRD462 algG4. The mixtures were incubated for 2 to 18 h at 25°C. Introduction of L-guluronate residues into the mannuronan was determined by the G-specific alginate lyase assay as described above.
Immunoblot analysis. Proteins from P. aeruginosa containing plasmids with algG mutations were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) (36) using 12% resolving gels. Proteins were electroblotted onto nitrocellulose membrane and then incubated with AlgG antibodies (14). Horseradish peroxidase-conjugated anti-rabbit immunoglobulin G was used as the secondary antibody. Immunoblots were developed using a chemiluminescence protocol as described previously (2).
Preparation of alginate lyases. The recombinant guluronate-specific alginate lyase of K. aerogenes was expressed in E. coli and prepared from culture supernatants as described previously (3, 14).
Computational analysis.
Initial searches using the SMART database (at http://smart.embl-heidelberg.de/) (65) revealed that AlgG contains the CASH domain (7). The amino acid sequence of AlgG was subsequently submitted to the 3D-PSSM server (at http://www.sbg.bio.ic.ac.uk/
3dpssm/) (33). This server compares unknown sequences to proteins from the Protein Data Bank (PDB) whose crystal structures have been solved. Unknown proteins are scored on sequence similarity, secondary structural predictions using Psi-Pred (at http://bioinf.cs.ucl.ac.uk/psipred/) (32) and solvent accessibility. The best-fitting proteins are then used as models to thread the unknown protein and create a coordinate files. Support for the predicted right-handed ß-helical structure of AlgG was also generated using FFAS (at http://ffas.ljcrf.edu/ffas-cgi/cgi/ffas.pl) and BETAWRAP (at http://betawrap.lcs.mit.edu/) (8).
Comparative modeling was performed by satisfying spatial restraints as implemented in MODELLER (http://salilab.org/modeller/modeller.html) (61). Since the alignment returned by 3D-PSSM had low target-template sequence identity (
13%), it was necessary to edit the alignments to account for secondary structure predictions (32). Given that the evaluation of three-dimensional models are more reliable than alignment evaluation (40), all models were independently assessed and rebuilt by adjusting the initial alignment. The resulting alignment had 15% identity. Models were evaluated by Prosa II empirical mean-force potentials devised to detect problematic areas of structural models (69). The Z-score produced by Prosa II was used to estimate the probability (pG) on a 0 to 1 scale that the model is reliable (http://sanchezlab.org/servers/pg/) (62). High pG values mean that the model is likely to be based on a correct alignment with an appropriate template, although models with pG values very close to 1 may not be accurate in all of the spatial details when the models target-template identities are below 30% (40). The model building and evaluation procedures employed in this work are described in detail at http://www.homepage.montana.edu/mdlakic/modeling/methods.html.
The sequences of ß-helical repeats were aligned using the secondary structural predictions of AlgG and secondary structures of the known RHßH proteins. Residues that stack in the RHßH fold were aligned. Alignments with the A modules of AlgE1 to AlgE7 C-5 epimerases of Azotobacter vinelandii, coat protein GP1 of Ectocarpus siliculosis virus, and AlgG homologs from P. fluorescens and A. vinelandii were performed using ClustalX multiple sequence alignment (23, 27).
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-helical-rich region at its N terminus and a ß-rich region in its C terminus. The C terminus of AlgG has a tandem repeating sequence, approximately from amino acids F237 to D462, containing a 24-mer amino acid repeat (Fig. 1). The repeating sequence has the consensus IXZX+XSX+XZZZXNXXXXNXXXG, where Z represents an aliphatic residue, + represents a charged residue, and X represents any amino acid. AlgG contains up to nine of these 24-mer repeats, with six repeats easily identified and three repeats that are more difficult to align due to extended loops in the variable regions (described below). This repeating sequence is characteristic of proteins with carbohydrate-binding and sugar hydrolase (CASH) domains, often found in carbohydrate lyases such as polygalacturonases and pectate lyases (7, 52, 54, 82). This domain is also proposed to exist in surface layer proteins of Archaea and the extracellular mannuronan alginate epimerases of A. vinelandii (7). These proteins are classified in the pectin-lyase superfamily (44). The structures of several of these CASH domain proteins have been solved by X-ray crystallography and shown to form parallel right-handed ß-helices (RHßHs).
|
The most distinctive characteristic of the RHßH fold is that it forms a spring-like structure with a shallow groove on one face that accommodates long-chain linear polysaccharides (54, 82). One rung of the spring is made of one repeating sequence, as in Fig. 1. The length of the helix depends on the number of rungs in the protein and can range from 7 to 11 repeats (7). The components of one rung of a RHßH are three ß-sheets (PB1, PB2, and PB3) separated by three turn regions (T1, T2, and T3) (Fig. 2). PB1, PB2, and PB3 of one rung stack with their respective PB1, PB2, and PB3 of the next rung (28, 83). PB1 is associated with a shallow groove. Predictive secondary-structure modeling shows that the AlgG repeats contains these three ß-sheets separated by three turn regions and that this secondary structure of the tandem repeats aligns to the structure of several of the crystallized RHßH proteins, including pectate lyase C of Erwinia chrysanthemi, as shown in Fig. 2.
|
Structural modeling of AlgG. The alignment provided by 3D-PSSM was used as a starting point for protein structural modeling. Several iterations of model building were performed by manually adjusting the alignments and model evaluation (61, 69). The final model has good stereochemistry and can be considered reliable (pG = 0.99). Figure 3A shows the transparent surface of the space-filling model around the schematic secondary-structure elements and indicates the boundaries of the shallow groove of the RHßH (top side of the molecule in Fig. 3A). Two views in Fig. 3B and 3C are related by 180o rotation around the y axis. Shown using the ball and stick representation are the conserved asparagine residues on the opposite side of the shallow groove (Fig. 3B) and the 324-DPHD motif within the groove (Fig. 3C). The models in Fig. 3B and C are colored by sequence conservation (50) between AlgG homologs, with the most conserved amino acids shown in red and the least conserved shown in blue.
|
|
Mutations that affect mannuronan epimerization and alginate secretion.
To investigate the importance of these conserved motifs in mannuronan epimerization and in alginate secretion, we performed site-directed mutagenesis on algG for key amino acids in domains predicted to be important for AlgG activity. For these experiments, AlgG point mutants were assayed for their ability to complement two algG mutant strains. In strain P. aeruginosa FRD462 algG4, the ability of the mutant proteins to complement the epimerization defect was determined, and in strain P. aeruginosa FRD1200 algG::aacCI, the ability of the mutant proteins to complement both the epimerization and the alginate secretion defects was determined. The mutations described here fall into several phenotypic classes, including mutations that cause a defect in mannuronan epimerization, mutations that fail to complement the defective alginate biosynthetic scaffold, and mutations that showed a dominant negative phenotype for epimerization.
AlgG proteins with mutations in the 324-DPHD motif of the proposed epimerase catalytic domain, D324A, P325A, H326A, and D327A, were all functional in restoring the alginate secretion defect of FRD1200 (Fig. 5A), suggesting that these mutant proteins fold correctly and that they are properly inserted into the alginate biosynthetic scaffold. However, none of these mutant proteins was functional in restoring the epimerization defect of FRD1200. These proteins also failed to complement the epimerase defect of FRD462 (Fig. 5A). Immunoblots demonstrated that full-length AlgG proteins with these mutations were produced in FRD1200 (Fig. 6). Therefore, these mutations likely do not disrupt protein folding but are located in a portion of the protein necessary for mannuronan epimerization and likely the epimerase catalytic center, as predicted by the structural modeling.
|
|
Mutations in the 361-NNRSYE-365 and 381-NLVAY-385 motifs that do not affect epimerase activity. Since 361-NNRSYE and 381-NLVAY show sequence conservation in AlgG, the AlgE proteins of A. vinelandii, and the P. aeruginosa alginate lyase AlgL, we tested the ability of AlgG with mutations in other conserved amino acids to restore the mucoid phenotype in FRD1200 and the epimerase defect of FRD462. With the exception of N362A (described above) and V383, which is involved in hydrophobic stacking, none of the other mutants tested here affected mannuronan epimerization. AlgG with mutations S364A, Y365F, and E366A of the 361-NNRSYE motif and Y385F of the 381-NLVAY motif fully complemented FRD1200 to wild-type levels of both alginate production and epimerization and complemented FRD462 to wild-type levels of epimerization (data not shown). Figure 6 (lanes 11 to 12) shows examples of mutant proteins that did not affect mannuronan epimerization, demonstrating that full-length proteins were produced. Therefore, these mutant proteins act as functional epimerases. The results suggest that, although conserved, amino acids in these positions of AlgG do not play a functional role in mannuronan epimerization or in the alginate biosynthetic scaffold.
C terminus of AlgG is not sufficient for complementation of the secretion defect of FRD1200.
The structures obtained from 3D-PSSM did not model the first 120 amino acids of AlgG that follow the signal peptide cleavage site. This region is dominated by -helices (25). The domain architecture for RHßH proteins has been suggested to exist in the glycosidic hydrolase families 55 and 87 (58) and in the surface layer protein B of Methanosarcina mazei (31). Two crystallized RHßH proteins, the tail spike protein of phage P22 (67) and dextranase from Penicillium minioluteum (37), have been shown to contain multiple domains in addition to the CASH domain.
The -helical region predicted in the amino terminus of AlgG suggests that it is a separate domain from the RHßH. To test this, we constructed AlgG proteins with deletions in the N-terminal region. An AlgG deletion mutant missing amino acids 89 to 102 fully complemented the epimerase and secretion defects of algG deletion strain FRD1200, suggesting that this region of AlgG is not important for epimerase activity (Fig. 5C). AlgG proteins missing amino acids 37 to 137 and 37 to 161 were unable to complement FRD1200 to the mucoid phenotype (Fig. 5C). By using an in vitro assay with cell extracts of strains with AlgG missing amino acids 37 to 137 and 37 to 161, no epimerase activity was observed compared to the wild-type control. The results indicate that a portion of the N-terminal region is required for epimerase activity and/or for proper folding and insertion of AlgG into the alginate biosynthetic scaffold.
Dominant negative mutations.
Within the 361-NNRSY motif, N362 is predicted to be involved in the asparagine stacking of T2. However, it is possible that the adjacent asparagine, N361, stacks at T2. Therefore, we tested an AlgG N361A mutation for its ability to complement FRD1200 and FRD462. Polymer production and epimerase activity were restored in FRD1200, and full-length AlgG was seen in immunoblots (Fig. 6, lane 5), suggesting that this mutant protein folds correctly. Interestingly, epimerase activity, although restored in FRD1200, was not recovered in FRD462 (Fig. 7A). The results suggest that the chromosomally encoded AlgG S272N protein of FRD462 is dominant negative over the plasmid-encoded AlgG N361A mutant protein and therefore may outcompete the plasmid-encoded AlgG N361A for binding sites on mannuronan or within the proposed alginate scaffold. An AlgG R316A mutation was similar in phenotype and by immunoblot to AlgG N361A (Fig. 7A; Fig. 6, lane 4). The S272N mutation of FRD462 was also dominate negative over AlgG 89-102 (Fig. 5C).
|
We also determined if AlgG S272N was dominant negative in the wild-type background. Interestingly, expression of AlgG S272N did not affect epimerization in the wild-type strain. The results suggest that the S272N mutation does not have an enhanced capacity for binding the alginate polymer or the alginate biosynthetic scaffold compared to the wild-type protein. However, AlgG S272N may outcompete AlgG N361A and AlgG R316A for binding sites on alginate or on the alginate biosynthetic scaffold, even though AlgG N361A and AlgG R316A are functional epimerases.
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-carrageenase of Alteromonas sp. (42)., methylesterase of Erwinia chrysanthemi (29), the tail spike protein of phage P22 (73), and the P69 pertactin of Bordetella pertussis (10). This structure is found in proteins of all three domains of life, although as yet it has not been found in higher eukaryotes. Most of these proteins cleave polysaccharides that compose the extracellular matrix of plants and animals. The high charge densities of the nonmethylated component of plant pectins, polyglacturonates, and -carrageen is similar to the negatively charged alginate. Therefore, this protein fold appears to be a general carbohydrate-binding structure that accommodates high-molecular-weight linear polysaccharides and occurs in several glycosidic hydrolases and also, as shown here, in the P. aeruginosa AlgG epimerase. Evidence presented here supports the structure of the AlgG C terminus as an RHßH. First, the scores obtained from hidden Markov model searches of the SMART database support this model. 3D-PSSM modeling, BETAWRAP analysis, and FFAS analysis indicate that AlgG has a high probability of folding as an RHßH. Second, the tandem repeats in AlgG are similar to those of other RHßH proteins. Third, each repeat of AlgG is similar in secondary structures to other RHßH proteins, including three ß-sheets and three turn regions. Fourth, similar amino acids in each repeat align, including the aliphatic residues in the ß-sheets, the asparagines in the turn regions, and the charged residues in PB1. Fifth, AlgG is biologically similar to other members of the RHßH family in that it associate with a linear, negatively charged polymer. Finally, model evaluation confirmed that AlgG is likely to have the RHßH fold. A recent study using hydrophobic cluster analysis showed that an epimerase from the marine brown alga Laminaria digitata has a secondary structure similar to that of P. aeruginosa AlgG (46). Our modeling studies also show a similar fold for the A-module of the extracellular alginate epimerases from A. vinelandii (data not shown). Therefore, this structure appears to be conserved among mannuronan epimerases.
Site-directed mutagenesis of AlgG 324-DPHD indicates that this motif is important for catalytic function and supports the results of Svanem et al. (77) that suggest that the amino acid equivalent of D324 (D152 in A. vinelandii AlgE7) is important for catalytic activity. In the RHßH model for AlgG, this motif lies in the center of the grooved face on PB1 (Fig. 3A). This position corresponds to the location of the catalytic centers for all the known RHßHs, where the reactive residues are either on PB1 or adjacent to it. An alternative explanation of the role of the DPHD motif is that these amino acids (as well as D375 and R422) are important in protein/polymer interactions.
Several RHßH proteins have been cocrystallized with their respective substrates (1, 9, 37, 63, 68). The results of those studies demonstrated that amino acids, often charged or aromatic, are located along the PB1 groove and are in contact with the substrate. It has also been demonstrated in the cellobiohydrolase I of Trichoderma reesei that at least seven sugar residues are in contact with the protein along its binding face, suggesting that many amino acids in the shallow groove are required for polysaccharide/protein interactions (9). Our docking studies indicate that the spacing between helical turns of the AlgG RHßH is such that each repeat could bind one modified sugar residue (data not shown). Therefore, as many as nine uronic acid residues may reside in the AlgG groove at one time. Interestingly, a summary of mutations that affect mannuronan epimerization shown here and those described for equivalent positions of P. fluorescens AlgG (18) all lie along the PB1 groove (shown in Fig. 2). Therefore, this entire groove is required for substrate interaction and/or epimerization.
An interesting observation in these studies is that AlgG with the D324A mutation is dominant negative over wild-type AlgG, suggesting that the mutant protein displaces the wild-type protein from the mannuronan precursor molecule or from the alginate biosynthetic scaffold. Since the mutant gene is plasmid encoded, it may have a higher abundance in the cell periplasm than the chromosomally encoded wild-type protein. If this is the case, the mutant protein may occupy a limited number of sites on the alginate biosynthetic scaffold, thereby displacing the wild-type protein from the complex. When expression of the mutant gene is not induced, we see a return of some epimerase activity, although not a complete restoration of wild-type levels of epimerization (Fig. 7B).
The results suggest that the increased abundance of the mutant AlgG may cause the observed dominant negative phenotype. However, the dominant negative effect is not fully explained by increased abundance, since a dominant negative phenotype was not observed for other mutants of the proposed catalytic domain (AlgG P325A, H326A, and D227A) (Fig. 7B). An alternative explanation is that AlgG D324A may bind to the alginate polymer with greater affinity than the wild-type protein and not allow access of the alginate polymer to the wild-type AlgG. This hypothesis could explain the lack of a dominant negative phenotype of the other catalytic domain mutants if they are defective in polymer binding. Further studies addressing the binding efficiency of these mutant proteins to the substrate will provide insight into the nature of this dominant negative mutation.
It has been proposed that the lyase reaction by ß-elimination is mechanistically similar to alginate epimerization, since both reactions form a similar intermediate (17). The pectate and pectin lyases use ß-elimination for degrading carbohydrate polymers (24, 41, 63, 84). In those reactions, a proton is abstracted from C-5 by a base and is donated to the glycosidic oxygen at the cleavage site by an acid. This results in the formation of an unsaturated C-4-C-5 bond (20). The base of the reaction is an arginine residue in pectin lyases and a calcium ion in pectate lyases. The acid is often the side chain of an aspartate residue (24, 30, 53, 63). The catalytic residues in AlgG, such as aspartates D324 and D327 and arginines on the catalytic face, would resemble those of the lyases. In the study by Svanem et al. (77), the aspartate corresponding to D324 of AlgG from the A. vinelandii AlgE7 was shown to be important for both epimerase and lyase activity, providing evidence for the similar mechanisms of these two reactions. Those results combined with the studies here indicate why the AlgG epimerase may be structurally similar to a class of proteins involved in polysaccharide cleavage.
The NNRSY motif and the NLVAY motif have been identified previously (18) and appear to be conserved among all of the C-5 epimerases, including the extracellular epimerases of A. vinelandii. Interestingly, a similar motif exists in AlgL and in the alginate lyase A1-III of Sphingomonas sp. (80). Structural analysis of AI-III demonstrated protein-carbohydrate interactions with N191 of this motif and H192 as the catalytic center (84). The results initially suggested that the NNRSY motif of AlgG may play a similar catalytic role, since the lyases and epimerases have similar reaction chemistries. However, the structure of alginate lyase from Sphingomonas sp. forms an 6/5 barrel consisting of many -helices and therefore is structurally distinct from AlgG.
Since N362 and N367 of AlgG lie on the opposite side of the RHßH of the proposed catalytic face, these amino acids are likely involved in asparagine stacking and not in epimerization. As such, mutant proteins AlgG N362A and AlgG N367A were not functional in complementing the secretion defect of FRD1200, suggesting that these mutations caused protein misfolding. In addition, the AlgG S364A and AlgG Y365F mutations of this conserved domain had no effect on AlgG activity in vivo, supporting the hypothesis that this motif is not the epimerase catalytic center.
Three different types of stacking side chains occur in the RHßH: hydrogen bond stacking by asparagine residues, hydrophobic stacking by aliphatic residues, and planar stacking by aromatic residues. Many RHßH structures have asparagine stacks (30). AlgG is unique in that asparagine stacking would occur at all three turn regions. Site-directed mutations of N362 and N367 disrupt alginate secretion and epimerization. Since these amino acids occur in T2 and T3, the mutations likely disrupt the structural stability of the protein. Aromatic stacking may have been disrupted with the Y321F mutation, which would stack with Y254, and Y298 (Fig. 2). The mutation V383A likely disrupts aliphatic stacking of PB2.
The predicted -helices of the N terminus of AlgG suggest that this protein contains multiple domains, similar to several other glycosidic hydrolases. The dextranase of P. minioluteum (37), the tail spike protein of phage P22 (67, 74), and surface layer protein B of M. mazei (31) contain domains in addition to the RHßH. In these examples, the other protein domain is involved in protein-protein interactions. Our results indicate that the N-terminal portion of AlgG may be required for alginate polymerization and/or epimerization. Therefore, the N terminus of AlgG may play a role in protein/protein interactions necessary for the alginate biosynthetic scaffold. Future studies addressing the role of AlgG in the alginate biosynthetic scaffold are necessary to determine the functional role of the N-terminal domain of AlgG.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
