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Originally published In Press as doi:10.1074/jbc.M402466200 on May 10, 2004

J. Biol. Chem., Vol. 279, Issue 30, 31863-31872, July 23, 2004
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Structure and Function of a Hypothetical Pseudomonas aeruginosa Protein PA1167 Classified into Family PL-7

A NOVEL ALGINATE LYASE WITH A {beta}-SANDWICH FOLD*

Masayuki Yamasaki{ddagger}, Satoko Moriwaki§, Osamu Miyake§, Wataru Hashimoto§, Kousaku Murata§||, and Bunzo Mikami{ddagger}

From the {ddagger}Division of Agronomy and Horticultural Science, Graduate School of Agriculture, and §Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan

Received for publication, March 4, 2004 , and in revised form, April 28, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Structural and functional analyses of alginate lyases are important in the clarification of the biofilm-dependent ecosystem in Pseudomonas aeruginosa and in the development of therapeutic agents for bacterial disease. Most alginate lyases are classified into polysaccharide lyase (PL) family-5 and -7 based on their primary structures. Family PL-7 enzymes are still poorly characterized especially in structural properties. Among family PL-7, a gene coding for a hypothetical protein (PA1167) homologous to Sphingomonas alginate lyase A1-II was found to be present in the P. aeruginosa genome. PA1167 overexpressed in Escherichia coli cleaved glycosidic bonds in alginate and released unsaturated saccharides, indicating that PA1167 is an alginate lyase catalyzing a {beta}-elimination reaction. The enzyme acted preferably on heteropolymeric regions endolytically and worked most efficiently at pH 8.5 and 40 °C. The specific activity of PA1167, however, was much weaker than that of the known alginate lyase AlgL, suggesting that AlgL plays a main role in alginate depolymerization in P. aeruginosa. In addition to this specific activity, differences were found between PA1167 and AlgL in enzyme properties such as molecular mass, optimum pH, salt effect, and substrate specificity. The first crystal structure of the family PL-7 alginate lyase was determined at 2.0 Å resolution. PA1167 was found to form a glove-like {beta}-sandwich composed of 15 {beta}-strands and 3 {alpha}-helices. The structural difference between the {beta}-sandwich PA1167 of family PL-7 and {alpha}/{alpha}-barrel AlgL of family PL-5 may be responsible for the enzyme characteristics. Crystal structures of polysaccharide lyases determined so far indicate that they can be assigned to three folding groups having parallel {beta}-helix, {alpha}/{alpha}-barrel, and {alpha}/{alpha}-barrel + antiparallel {beta}-sheet structures as basic frames. PA1167 is the fourth novel folding structure found among polysaccharide lyases.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Polysaccharide lyases recognize uronic acid residues in polysaccharides and catalyze a {beta}-elimination reaction by releasing unsaturated saccharides with C=C double bonds at nonreducing terminal uronate residues. These characteristics of lyases indicate that they share common structural features determining their uronate-recognition sites and reaction modes ({beta}-elimination reaction). Based on their primary structures, polysaccharide lyases are classified into 14 families (PL1-1–14), each of which also includes various function-unknown proteins that have sequence homology in the Carbohydrate-Active enZYme (CAZY) data base (afmb.cnrs-mrs.fr/~cazy/CAZY/index.html). The crystal structures of lyases for pectate (families PL-1, -3, -9, and -10) (1–4), alginate (family PL-5) (5), hyaluronate (family PL-8) (6), chondroitin (families PL-6 and -8) (7, 8), and xanthan (family PL-8) (9) have been determined and assigned to three folding groups having parallel {beta}-helix, {alpha}/{alpha}-barrel, and {alpha}/{alpha}-barrel + antiparallel {beta}-sheet structures as basic frames. No three-dimensional structures for family PL-2, -4, -7, and -11–14 lyases have been solved, however, and little information exists on structural rules common to polysaccharide lyases.

Alginate is a linear polysaccharide consisting of {alpha}-L-guluronate and its C5 epimer, {beta}-D-mannuronate, arranged in three different ways: poly-{alpha}-L-guluronate (poly(G)), poly-{beta}-D-mannuronate (poly(M)), and heteropolymeric (poly(MG)) regions (Fig. 1, A–C) (10). Alginate produced by brown seaweed is widely used in the food and pharmaceutical industries due to its ability to chelate metal ions and to form a highly viscous solution, whereas some pathogenic bacteria such as Pseudomonas aeruginosa produce alginate, which is involved in pathogenicity (11).



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  		FIG. 1.
Block sites of alginate polymer and alginate lyase reactions. A, MM; B, GG; C, MG block sites. M and G represent {beta}-D-mannuronate and {alpha}-L-guluronate. Vertical arrows indicate cleavage sites for alginate lyase reactions and horizontal arrows indicate those for reactions. D, reaction scheme for the TBA method.

 
P. aeruginosa is a Gram-negative bacterium that thrives in diverse environments such as soil, plant, and animal tissues due to its ability to assimilate a large number of organic compounds (12). The bacterium is also known to cause opportunistic infections in humans (13). In the lungs of patients with cystic fibrosis, it is the most predominant bacterium (60–90%). Many antimicrobial agents have been used to repress bacterial growth in cystic fibrosis (14), but the appearance of drug-resistant bacteria and the toxicity of agents, especially in older people, have decreased the usefulness of such treatment. Mucoid cells of P. aeruginosa produce an alginate biofilm, a capsule-like exopolysaccharide containing no poly(G) region, which is responsible for both chronic pulmonary infection and respiratory difficulties in cystic fibrosis (15–17). Bacterial alginate plays an important role in the colonization of target cells by P. aeruginosa, so the biosynthesis of alginate in the bacterium has been well characterized and documented (18, 19). The importance of alginate metabolism has drawn attention because transfer of the bacterium from one colonization site to another, which is induced by a change in environmental or nutritional conditions, requires depolymerization of the alginate biofilm (20).

Alginate lyase cleaves glycosidic bonds in alginate through a {beta}-elimination reaction and produces unsaturated saccharides with a double bond between C4 and C5 sites in the nonreducing terminal sugar (Fig. 1, A–C). The enzyme is thus applicable to useful biochemicals for the analysis of biofilm-dependent ecosystems in P. aeruginosa and for establishment of an effective therapy for bacterial disease. In a study to substantiate our proposal that the use of Sphingomonas alginate lyases (A1-I (65 kDa), A1-II (25 kDa), and A1-III (40 kDa)) as biofilm-degrading enzymes together with antimicrobial agents may be feasible for therapy for P. aeruginosa infectious disease (21, 22), the gene coding for a hypothetical protein (PA1167) of P. aeruginosa PAO1 was found to be homologous to that for alginate lyase A1-II. PA1167 is one of nine function-unknown proteins categorized into family PL-7.

Based on their primary structures, alginate lyases are grouped into three families, PL-5, -7, and -14. Most of the family PL-5 and -7 alginate lyases specifically depolymerize poly(M) and poly(G), respectively, although family PL-14 contains enzymes specific for poly(M) or poly(G). P. aeruginosa alginate lyase (AlgL, PA3547) categorized into family PL-5 has been characterized functionally (23, 24), and we have clarified the structure/function relationship of family PL-5 alginate lyase A1-III (5, 25). Structural and functional analyses of family PL-7 alginate lyases together with family PL-5 enzymes are thought to be important in clarifying the biofilm-dependent ecosystem in P. aeruginosa, for the development of therapeutic agents for bacterial disease, and for the establishment of common structural features and catalytic mechanisms of alginate lyases. Family PL-7 alginate lyases show no homology with other family lyases, suggesting that family PL-7 enzymes exhibit a novel folding distinct from the three types of lyase thus far analyzed.

This article identifies and characterizes the novel alginate lyase PA1167 (family PL-7) from P. aeruginosa PAO1 and determines the structure of the enzyme by x-ray crystallography.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Sodium alginate from Eisenia bicyclis (average molecular weight, 25,700; viscosity, 1,000 centipoises) was purchased from Nacalai Tesque, Kyoto, Japan. Poly(M) (M, 95.1%, G, 4.9%), poly(G) (M, 6.1%; G, 93.9%), and poly(MG) (M, 53.2%; G, 46.8%) were prepared from brown seaweed alginate as described elsewhere (26). CM-Toyopearl 650M, DEAE-Toyopearl 650M, butyl-Toyopearl 650M, and SuperQ-Toyopearl 650S were purchased from Tosoh, Tokyo, Japan. Sephacryl S-200HR was purchased from Amersham Biosciences. Restriction endonucleases were purchased from Takara Shuzo Co., Kyoto, Japan, and DNA-modifying enzymes were from Toyobo Co., Tokyo, Japan.

Alignment of Amino Acid Sequences of Alginate Lyases Specific for Poly(G)
To find P. aeruginosa proteins homologous to alginate lyases specific for poly(G), the FASTA program, assisted by GenomeNet on the internet, was used for a sequence similarity search, and the ClustalW program was used for multiple sequence alignment (www.genome.ad.jp/).

Microorganisms and Culture Conditions
P. aeruginosa PAO1 and 8830 were gifts from Dr. A. Markaryan (University of Illinois College of Medicine); Escherichia coli [BL21(DE3)pLysS] (Novagen, Madison, WI) was used as the host for overexpression of PA1167 and AlgL. For purification of proteins expressed in E. coli, cells were aerobically precultured in Luria-Bertani (LB) medium (27) supplemented with ampicillin (0.1 mg/ml) at 37 °C. When turbidity reached 0.4 at 600 nm, isopropyl-{beta}-D-thiogalactopyranoside was added to the culture at a concentration of 0.1 mM, and the culture was continued at 16 °C for 42 h.

Construction of Plasmids for Overexpression of PA1167 and AlgL
To introduce PA1167 and AlgL genes into an expression vector, pET3a (Novagen, Madison, WI), a PCR was conducted using KOD polymerase (Toyobo Co, Tokyo, Japan), genomic DNA isolated from P. aeruginosa PAO1 as a template, and two synthetic oligonucleotides as primers. Oligonucleotides for PA1167 were 5'-CCCATATGCCTGACCTGAGTACCTGGAAC-3' and 5'-CCGGATCCTCATTGATGGCTAACGCGCAGC-3' with NdeI and BamHI sites added to their 5' regions, and those for AlgL had 5'-GCCATATGGCCGACCTGGTACCCCCGCCC-3' and 5'-CCGGATCCTCAACTTCCCCCTTCGCGGCT-3' with NdeI and BamHI sites added to their 5' regions. PCR conditions recommended by the KOD polymerase manufacturer (Toyobo Co., Tokyo, Japan) were used. Fragments amplified through PCR were digested with NdeI and BamHI and then ligated with NdeI- and BamHI-digested pET3a. Resultant plasmids containing PA1167 genes were designated pET3a-PA1167 and those containing AlgL were designated pET3a-AlgL.

Sequencing and Manipulation of DNA
Nucleotide sequences of PA1167 and AlgL genes amplified by PCR were determined by dideoxy chain termination using an automated DNA sequencer (model 377, Applied Biosystems Division, PerkinElmer Life Sciences) (28). Subcloning, transformation, and gel electrophoresis were conducted as described elsewhere (27).

Assays for Enzyme Activity and Protein Concentration
The assay for alginate lyase was conducted as follows. In each assay, reaction products were confirmed to increase proportionally with time and enzyme concentration in the reaction mixture. The enzyme was incubated at 37 °C in a reaction mixture (100 µl) consisting of 0.025% sodium alginate and 50 mM Tris-HCl buffer (pH 7.5). The reaction was terminated by immersing the test tube in boiling water for 5 min, and then a 50-µl portion of the mixture was used for the thiobarbituric acid (TBA) method (29). Enzyme activity was determined by monitoring the increase in absorbance at 548 nm arising from the condensation of {beta}-formyl-pyruvic acid and TBA (molar absorption coefficient {epsilon} = 29.0 x 103 M-1 cm-1) (Fig. 1D). One unit of enzyme activity was defined as the amount of enzyme required to liberate 1 µmol of {beta}-formyl-pyruvic acid per min at 37 °C. Protein concentrations were determined by the method of Bradford (30), with bovine serum albumin as the standard, or by measuring the absorbance at 280 nm using a 1-cm path length cuvette, assuming that A280 = 1.64 (PA1167) and 1.74 (AlgL) correspond to 1 mg/ml.

Purification of PA1167 and AlgL from E. coli Cells
Unless otherwise specified, all procedures were conducted at 0–4 °C.

Purification of PA1167—E. coli cells harboring pET3a-PA1167 were grown in 6 liters of LB medium (1.5 liters/flask), collected by centrifugation at 6,000 x g and 4 °C for 5 min, washed with 20 mM potassium phosphate buffer (KPB) (pH 6.0), containing 1 mM EDTA, and were then resuspended in the same buffer. Cells were ultrasonically disrupted (Insonator model 201M, Kubota, Tokyo, Japan) at 0 °C and 9 kHz for 20 min, and the clear solution obtained on centrifugation at 15,000 x g and 4 °C for 20 min was used as the cell extract. After supplementation with 1 mM phenylmethylsulfonyl fluoride and 0.1 µM pepstatin A, the cell extract was fractionated with ammonium sulfate. The precipitate (0–30% saturation) containing the enzyme was collected by centrifugation at 15,000 x g and 4 °C for 20 min and then dialyzed against 20 mM KPB (pH 6.0) containing 1 mM EDTA overnight. The enzyme solution was applied to a CM-Toyopearl 650M column (2.6 by 9.5 cm) equilibrated with 20 mM KPB (pH 6.0) containing 1 mM EDTA. The enzyme was eluted with a linear gradient of NaCl (0–0.7 M) in 20 mM KPB (pH 6.0) containing 1 mM EDTA (150 ml), with a 3-ml fraction being collected every 3 min. Active fractions, which were eluted at about 0.4 M NaCl, were combined and concentrated to about 6 ml by ultrafiltration using Centriprep (molecular mass cut-off, 10 kDa; Millipore). The concentrate was applied to a Sephacryl S-200HR column (2.6 by 65 cm) equilibrated previously with 20 mM KPB (pH 6.0) containing 1 mM EDTA and 0.15 M NaCl. The enzyme was eluted with the same buffer, with a 3-ml fraction being collected every 5 min. The enzyme was eluted in fraction numbers 79–88. These fractions were combined and dialyzed overnight against 20 mM KPB (pH 6.0) containing 1 mM EDTA. The dialysate was used as purified PA1167.

Purification of AlgL—E. coli cells harboring pET3a-AlgL were grown in 6 liters of LB medium (1.5 liters/flask), collected by centrifugation at 6,000 x g and 4 °C for 5 min, washed with 20 mM KPB (pH 7.0), and then resuspended in the same buffer. Cells were ultrasonically disrupted (Insonator model 201M, Kubota, Tokyo, Japan) at 0 °C and 9 kHz for 20 min, and the clear solution obtained on centrifugation at 15,000 x g and 4 °C for 20 min was used as the cell extract. After supplementation with 1 mM phenylmethylsulfonyl fluoride and 0.1 µM pepstatin A, the cell extract was applied to a DEAE-Toyopearl 650M column (2.6 by 17 cm) equilibrated with 20 mM KPB (pH 7.0). The enzyme was eluted with a linear gradient of NaCl (0–0.7 M) in 20 mM KPB (pH 7.0) (300 ml), with a 3-ml fraction being collected every 5 min. Active fractions, which were eluted at about 0.4 M NaCl, were combined and saturated with ammonium sulfate (30%). The enzyme was applied to a butyl-Toyopearl 650M column (2.6 by 7 cm) equilibrated with 20 mM KPB (pH 7.0), saturated with ammonium sulfate (30%), and eluted with a linear gradient of ammonium sulfate (30 to 0% saturation) in 20 mM KPB (pH 7.0) (140 ml). Two-milliliter fractions were collected every 2 min. Active fractions, which were eluted with ammonium sulfate at less than 3%, were combined and dialyzed overnight against 20 mM Tris-HCl (pH 7.5), and the dialysate was applied to a SuperQ-Toyopearl 650S column (1.6 by 4 cm) equilibrated with 20 mM Tris-HCl (pH 7.5). The enzyme was eluted with 20 mM Tris-HCl (pH 7.5) and concentrated to about 3 ml by ultrafiltration using Centriprep (molecular mass cut-off, 10 kDa; Millipore). The concentrate was applied to a Sephacryl S-200HR column (2.6 x 65 cm) previously equilibrated with 20 mM KPB (pH 7.0) containing 0.15 M NaCl. The enzyme was eluted with the same buffer, with a 3-ml fraction being collected every 5 min. The enzyme was eluted in fraction numbers 66–73. These fractions were combined and dialyzed against 20 mM KPB (pH 7.0) overnight. The dialysate was used as purified AlgL.

Criteria for Purity
SDS-PAGE was conducted to confirm purity (31).

Preparation of Sphingomonas Alginate Lyases A1-II and A1-III
Alginate lyases A1-II and A1-III of Sphingomonas sp. A1 were purified from recombinant E. coli cells as described elsewhere (21).

Thin Layer Chromatography
Products derived from alginate through the reaction of PA1167 were separated by TLC using a solvent system of 1-butanol/acetic acid/water (3:2:2, v/v). Products were visualized by heating TLC plates at 130 °C for 5 min after spraying with 10% (v/v) sulfuric acid in ethanol. Unsaturated saccharides on TLC plates were stained with TBA as described elsewhere (32). Standard alginate di-, tri-, and tetrasaccharides, with molecular weights of 352, 528, and 704, were prepared as described elsewhere (33).

N-terminal Amino Acid Sequence
The N-terminal amino acid sequence of PA1167 purified from E. coli cells was determined by Edman degradation with a Procise 492 protein sequencing system (Applied Biosystems Division, PerkinElmer Life Sciences).

Crystallization
PA1167 was crystallized at 20 °C by hanging-drop vapor diffusion as described elsewhere (34).

Multiple Isomorphous Replacement Phasing and Initial Model Building
The structure of PA1167 was determined by multiple isomorphous replacement (MIR). PA1167 crystals were soaked in solutions containing several heavy atom compounds such as 5 mM PrCl3, 2 mM Sm2(SO4)3, 5 mM LuCl3, 5 mM CdCl2, 2 mM KAuCl4, and 10 mM K2PtCl4 for 0.5–2 h at 20 °C. All heavy atom solutions were prepared in 2.0 M sodium chloride and 0.1 M Tris-HCl buffer (pH 7.0). Crystals were mounted in a thin walled glass capillary for x-ray analysis. Both ends of the capillary were sealed with wax after one end was filled with the mother liquor. Diffraction data for native crystals up to 2.8 Å and for derivative crystals around 3.5 Å were collected with a Bruker Hi-Star multiwire area detector at 7 °C with FTS air-cooling system, using CuK{alpha} radiation generated by a MAC Science M18XHF rotating anode generator, and were processed, merged, and scaled with the SADIE and SAINT software packages (Bruker, Karlsruhe Germany). The precession image indicated that the crystal belongs to a space group of P21. Two molecules were included in the asymmetric unit. MIR phasing was conducted with a PHASES program package (35). An electron density map was made with the solvent-flattened MIR phase using data from 15.0 to 3.5 Å, and the map was averaged using noncrystallographic symmetry. The initial model built using an averaged MIR map at 3.5 Å resolution was refined by simulated annealing with molecular dynamics using a CNS program package (36). Then several rounds of restrained least squares refinement, followed by manual model building using the program TURBO-FRODO (AFMB-CNRS, France), were conducted to improve the initial model on to the R-factor value of 27.5% at 2.8 Å resolution.

Data Collection and Refinement for the Final Model at 2.0 Å
Diffraction data for the following refinement were collected in the resolution range between 50 and 1.9 Å by using synchrotron radiation of wavelength 0.9 Å at the BL-41XU station of SPring-8, Hyogo, Japan, as described elsewhere (34). Data obtained were processed, merged, and scaled using program package HKL 2000 (DENZO and SCALEPACK) (37), and truncated with the CCP4 program package. The model refined at 2.8 Å was further refined by simulated annealing with molecular dynamics using a CNS program package. Several rounds of restrained least square refinement to a resolution of 2.0 Å, followed by manual modeling, were conducted to improve the model. We then added 298 water molecules having more than 3{sigma} on the Fo - Fc map to the model. The final model was determined with an R-factor value of 18.7% (free R-factor = 24.0%) for 30,777 reflections with F > 2{sigma}(F) at 50–2.0 Å resolution.

The stereo quality of the model was assessed using the programs PROCHECK (38) and WHAT-CHECK (39). Ribbon plots were prepared using the programs MOLSCRIPT (40) and RASTER3D (41).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
P. aeruginosa Protein Homologous with Alginate Lyase A1-II of Sphingomonas sp. A1
P. aeruginosa produces the periplasmic alginate lyase AlgL, which is specific for poly(M) (42) and belongs to family PL-5. Gene coding for the alginate lyase responsible for depolymerization of other blocks (poly(G) and/or poly(MG)) in alginate was screened in the genome of P. aeruginosa. In a homology analysis with the FASTA program, a hypothetical protein (PA1167) of P. aeruginosa PAO1 showed slight homology with a family PL-7 alginate lyase (A1-II) of Sphingomonas sp. A1 specific for poly(G) (36.1% identity in a 233-aa overlap; GenBankTM accession number AB011415 [GenBank] ) (21). In the Pseudomonas genome project data base, the PA1167 gene is categorized into a family composed of function-unknown genes, although PA1167 belongs to family PL-7 in the CAZY data base. Since no report has been made on the presence of a family PL-7 alginate lyase in P. aeruginosa, and the structure/function relationship of family PL-7 alginate lyases remains to be clarified, PA1167 was analyzed functionally and structurally.

Overexpression in E. coli Cells and Purification of PA1167 and AlgL
After the construction of plasmids containing genes coding for PA1167 (pET3a-PA1167) and AlgL (pET3a-AlgL), the accuracy of nucleotide sequences of genes was confirmed by DNA sequencing (data not shown). Transformants with plasmids (pET3a-PA1167 and pET3a-AlgL) of BL21(DE3)pLysS with no alginate lyase activity were grown at 16 °C in LB broth in the presence of isopropyl-{beta}-D-thiogalactopyranoside at 0.1 mM for the induction of gene expression. The cell extract of the E. coli transformant with pET3a-PA1167 exhibited alginate lyase activity, suggesting that PA1167 is an alginate lyase.

PA1167 and AlgL were purified 14.0- and 10.4-fold from cell extracts of the E. coli transformant with activity yields of 3.7 and 15.3% (Table I), although the specific activity of PA1167 was about 34-fold lower than that of AlgL. Purified enzymes were confirmed to be homogeneous by SDS-PAGE (Fig. 2).


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  		TABLE I
Purification of PA1167 and AlgL from E. coli cells

 



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  		FIG. 2.
Electrophoretic profiles of PA1167 and AlgL. A, PA1167 purified from E. coli cells was subjected to SDS-PAGE. Lane 1, molecular weight standards (from top): synthetic polypeptides with molecular weights of 250,000, 150,000, 100,000, 75,000, 50,000, 37,000, 25,000, and 15,000; lane 2, E. coli cell extract (3 µg of protein); lane 3, ammonium sulfate fraction (0–30% saturation) (3 µg of protein); lane 4, active fraction from CM-Toyopearl 650M column (3 µg of protein); lane 5, purified PA1167 (3 µg). B, AlgL purified from E. coli cells was subjected to SDS-PAGE. Lane 1, molecular weight standards; lane 2, purified AlgL (10 µg). Arrows indicate positions of PA1167 and AlgL.

 
Characterization of PA1167 Purified from E. coli Cells
Because AlgL has already been well characterized (23, 24, 42–46), only the properties of the purified PA1167 are given, as follows.

Molecular Weight—The molecular mass of PA1167 was determined to be 25 kDa by SDS-PAGE (Fig. 2A). This was comparable with the theoretical value (25,120 Da) deduced from the predicted amino acid sequence of the enzyme. On permeation chromatography on Sephacryl S-200HR, the enzyme was eluted as a protein with a molecular mass of about 25 kDa (data not shown), indicating the enzyme is monomeric.

pH and Temperature—PA1167 was most active at pH 8.5 in 50 mM Tris-HCl buffer (Fig. 3A) and at 40 °C (Fig. 3B); 80% of enzyme activity was lost on preincubation at 45 °C for 10 min in 50 mM Tris-HCl (pH 7.5) (Fig. 3C).



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  		FIG. 3.
Effects of pH and temperature on PA1167 activity. Experiments were conducted at 37 °C using sodium alginate (0.025%) as a substrate and the enzyme (5 µg) purified from E. coli cells. A, effect of pH. Reactions were conducted at 37 °C for 30 min in the following 50 mM buffers; sodium acetate (closed rhombuses), potassium phosphate (closed squares), Tris-HCl (closed triangles), and glycine-NaOH (closed circles). Activity at pH 8.5 in Tris-HCl was taken as 100%. B, optimal temperature. Reactions were conducted for 30 min at different temperatures in 50 mM Tris-HCl (pH 7.5). Activity at 40 °C was taken as 100%. C, thermal stability. After preincubation of the enzyme at different temperatures for 10 min, the remaining activity was measured under conditions specified under "Experimental Procedures." Enzyme activity at 30 °C was taken as 100%.

 
Effect of Salts—Although the production and enzyme activity of AlgL are significantly enhanced in the presence of salts such as 0.2 M NaCl (23), the enzyme activity of PA1167 was not affected by this salt (0.2 M NaCl) (data not shown).

Substrate Specificity—Because alginate has three block structures (poly(M), poly(G), and poly(MG)), substrate specificities of PA1167 and AlgL were investigated using poly(M), poly(G), and poly(MG). Alginate lyases A1-II and A1-III of Sphingomonas sp. A1 specific for poly(G) and poly(M), respectively, were used as positive controls (21). The reaction was conducted at 37 °C for 60 min in a mixture consisting of 50 mM Tris-HCl buffer (pH 7.5), various substrates (0.025%), and a purified enzyme (137 microunits). A1-II and A1-III were specific for poly(G) and poly(M), respectively, whereas PA1167 preferably depolymerized poly(MG) rather than poly(M) or poly(G) (Table II). AlgL was specific for poly(M) rather than poly(MG) or poly(G), as reported elsewhere (42), and the specific activity of AlgL toward each block was stronger than that of PA1167 (Table II). In addition to algal alginate, PA1167 also acted on alginate biofilm produced by P. aeruginosa 8830 (data not shown).


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  		TABLE II
Substrate specificities of PA1167, AlgL, A1-II, and A1-III

 
Kinetics—kcat values of PA1167 and A1-II for alginate were determined to be 2.42 ± 0.10 and 748 ± 13.0 (s-1), respectively, although the Km value of PA1167 (0.0096 ± 0.0005%) was comparable with that of A1-II (0.0139 ± 0.0010%). The activity of PA1167 was also low toward alginate-oligosaccharides such as the tetrasaccharides produced from alginate by alginate lyase A1-III from Sphingomonas sp. A1 (data not shown).

Mode of Action—From the results of TLC analysis, products derived from alginate through the reaction of PA1167 were considered to be alginate oligosaccharides with different degrees of polymerization, which were positive in the TBA reaction (Fig. 4, lane 2). The enzyme was thus found to act on alginate endolytically and to catalyze a {beta}-elimination reaction.



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  		FIG. 4.
Mode of action of PA1167. The reaction was conducted at 30 °C for 24 h in a mixture of purified PA1167 (137 microunits) and 0.25% alginate (lanes 1 and 2). Reaction products were analyzed on TLC plates. A, reaction products were stained with sulfuric acid. B, reaction products were stained with TBA. Reaction times: lane 1, 0 h; lane 2, 24 h; lane 3, alginate-tetrasaccharide; lane 4, alginate-trisaccharide; and lane 5, alginate-disaccharide.

 
N-terminal Amino Acid Sequence—The N-terminal amino acid sequence of PA1167 expressed in E. coli cells was determined to be NH2-PDLST, which corresponds to 2PDLST6 of the PA1167 amino acid sequence predicted from the nucleotide sequence. This shows that the N-terminal methionine residue of the enzyme was removed by E. coli aminopeptidase.

Crystallization and Structural Determination of PA1167
PA1167 was crystallized, and the structure was determined at 3.5 Å resolution by MIR, using Pr3+, Sm3+, Lu3+, Cd2+, Au3+, and Pt2+ derivatives. The final structure was refined at 2.0 Å resolution using the native data collected at SPring-8. Refinement statistics for MIR phasing and the final model are given in Tables III and IV.


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  		TABLE III
Statistics for MIR phasing of PA1167

 


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  		TABLE IV
Data collection and refinement statistics for PA1167

 
Quality of Refined Model
The refined model consisted of 444 amino acid and 298 water molecules for two molecules in the asymmetric unit. The N- and C-terminal amino acid residues of the mature form produced from the proform through post-translational processing were confirmed to be Pro2 and Gln223, respectively, by electron density mapping and N-terminal amino acid analysis. All polypeptide chain sequences could be traced well, and the electron density of the main and side chains was generally very well defined in the 2Fo - Fc map. The final overall R-factor for the refined model was 18.7% with 24,766 unique reflections within the 50.0–2.0-Å resolution range. The final free R-factor calculated with randomly selected 10% data was 24.0%. Final root mean square deviations from standard geometry were 0.005 Å for bond lengths and 1.44° for bond angles. Based on theoretical curves in the plot calculated according to Luzzati (47), absolute positional error was estimated to be close to 0.22 Å at 5.0–2.0 Å resolution. Judging from the results of the Ramachandran plot (48) analysis, in which the stereochemical correctness of the backbone structure is indicated by the ({phi}, {Psi}) torsion angles (49), most of the nonglycine residues (89.4%) lie within the most favored region, and other residues (10.1%) fell in the additional and generously allowed regions, except for the Leu125 residue. This residue has a well defined density in type I {beta}-turns. There is one cis-peptide between the Ile11 and Pro12 residues.

Overall Structure of PA1167
Fig. 5A displays a ribbon model of the overall structure of PA1167 that forms a glove-like {beta}-sandwich. As shown in Fig. 5B, PA1167 consists of three short {alpha}-helices (H1, amino acid residues 22–26; H2, 76–79; and H3, 185–189) and two antiparallel {beta}-sheets; sheet A consists of 8 {beta}-strands (SA1, 19–20; SA2, 7–10; SA3, 59–64; SA4, 192–200; SA5, 97–105; SA6, 115–124; SA7, 127–134; SA8, 144–152) and sheet B, 7 {beta}-strands (SB1, 34–37; SB2, 40–46; SB3, 210–222; SB4, 81–91; SB5, 158–164; SB6, 169–174; SB7, 177–182).



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  		FIG. 5.
Crystal structure of PA1167. A, overall structure of PA1167 (stereo diagram). B, topology diagram. {beta}-Sheets in A are shown in yellow and B in orange.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
To understand comprehensively the environmental versatility and pathogenicity of P. aeruginosa, its complete genome sequence was determined in 2000 (50). Although 5,570 genes are predicted to be present in the genome, the functions of more than 40% of these genes remain unclear. Genes in the genome are grouped into four classes based on the confidence level (50) as follows: (i) class 1,372 genes with a demonstrated function; (ii) class 2, highly homologous genes (1,059) with a demonstrated function from other organisms; (iii) class 3, genes (1,590) with a function proposed through motif and limited homology analyses; and class 4, function-unknown genes (2,549). It is now important to clarify the function of class 4 genes. Although a PA1167 gene is categorized in class 4, PA1167 is assigned as a member of family PL-7 in the CAZY data base. Thus, this article aimed to clarify experimentally the structure and function of PA1167.

The functional and structural evidence shown here demonstrates that PA1167 of P. aeruginosa is an alginate lyase with a preference for poly(MG) and that PA1167, as a family PL-7 protein, forms a {beta}-sandwich differing greatly from that of other family PL proteins. To the best of our knowledge, this is the first report about the crystal structure of a family PL-7 protein.

Almost all family PL-7 lyases from Sphingomonas sp. A1 (21), Klebsiella pneumoniae subsp. aerogenes (33), Corynebacterium sp. ALY1 (51), and Vibrio halioticoli IAM14596T (52) have been shown to be specific for poly(G), the exception being the enzyme from Photobacterium sp. specific for poly(M) (53). PA1167 is a unique enzyme in family PL-7 due to its preference for poly(MG) to poly(M) or poly(G). Family PL-7 thus contains alginate lyases with different substrate specificities. On compositional analysis of alginates from mucoid strains of P. aeruginosa, bacterial alginates are shown to have a high content of mannuronate (67–76%) (54) and to contain no poly(G) region (55). The low enzyme activity of PA1167 and AlgL toward poly(G) is probably reflected by the absence of poly(G) in alginates from P. aeruginosa (Table II). However, between PA1167 and AlgL, differences exist in enzyme properties such as molecular mass (PA1167, 25 kDa; AlgL, 41 kDa (precursor with signal peptide)), specific activity (PA1167, 27.4 milliunits/mg; AlgL, 926 milliunits/mg), optimum pH (PA1167, 8.5; AlgL, 6.2 (20)), salt effect (PA1167, none; AlgL, activation), and substrate specificity (PA1167, poly(MG); AlgL, poly(M)). In contrast to P. aeruginosa,in Sphingomonas sp. A1, family PL-7 A1-II exhibits greater activity than family PL-5 A1-III, thus indicating that the difference in properties between family PL-5 and -7 enzymes are dependent on the enzyme producer. Judging from the significant differences in specific activity and substrate specificity between PA1167 and AlgL, it is thought that periplasmic AlgL with strong activity is the major enzyme for depolymerization of the extracellular alginate biofilm in P. aeruginosa.

One important findings of this study is that the overall structure of the family PL-7 alginate lyase PA1167 clearly differs from that of other PL family proteins such as the parallel {beta}-helix structure of families PL-1, -3, -6, and -9 (1–3, 7), the {alpha}/{alpha}-barrel structure of families PL-5 and -10 (4, 5), and the {alpha}+{beta} structure of family PL-8 (6, 8, 9). Through homology modeling (56), using our coordinates for alginate lyase A1-III of Sphingomonas sp. A1 (5), AlgL was also found to have a {alpha}/{alpha}-barrel structure (Fig. 6). The {beta}-sandwich of family PL-7 is found in sugar-related proteins such as 1,3–1,4-{beta}-glucanase (57), lectin (58), {alpha}-amylase inhibitor (59), and a nonclassified alginate lyase from Alteromonas sp. 272 (Protein Data Bank code 1J1T [PDB] ).2 Although family PL-8 has a {beta}-domain structure in the C-terminal domain, the {beta}-domain of family PL-8 differs completely from that of family PL-7 due to the abundance of 4–5 anti-parallel {beta}-sheets in family 8. The {beta}-domain of family PL-8 makes no direct contribution to the catalytic reaction or substrate binding except for the LB10 loop of xanthan lyase (9), whereas the {alpha}-domain of family PL-8 has a catalytic function similar to family PL-5. These indicate that the {beta}-structure of PL-7 alginate lyases has a unique structural basis working as a catalytic domain.



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  		FIG. 6.
Three-dimensional structure of family PL-5 alginate lyases (stereo diagram). A, AlgL of P. aeruginosa. B, A1-III of Sphingomonas sp. A1 (5). Histidine and tyrosine shown in bonds are putative catalytic residues.

 

At present, family PL-7 includes 7 alginate lyases from 6 organisms: PA1167 from P. aeruginosa PAO1, A1-II from Sphingomonas sp. A1, AlyA from K. pneumoniae subsp. aerogenes (GenBankTM accession number L19657 [GenBank] ) (60), AlyVGI and AlyVGII from V. halioticoli IAM14596T (GenBankTM accession numbers AF114039 [GenBank] and AF114037 [GenBank] ) (52), AlyPG from Corynebacterium sp. ALY-1 (GenBankTM accession number AB030481 [GenBank] ) (61), and AlyM from Photobacterium sp. ATCC 43367 (GenBankTM accession number X70036 [GenBank] ) (54). The alignment of these alginate lyases, except for V. halioticoli AlyVGII, which has weak homology with other family-7 enzymes, indicates that most of the conserved residues are located at SA3, SA4, and SA5 in the center of {beta}-sheet A (Fig. 7).



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  		FIG. 7.
Alignment of amino acid sequences of family PL-7 alginate lyases from different sources using the ClustalW program (clustalw.genome.ad.jp/). Pseudomonas, PA1167 (GenBankTM accession number AE004547 [GenBank] ) of P. aeruginosa PAO1; Sphingomonas, alginate lyase A1-II (GenBankTM accession number AB011415 [GenBank] ) of Sphingomonas sp. A1; Klebsiella, alginate lyase AlyA (GenBankTM accession number L19657 [GenBank] ) of K. pneumoniae subsp. aerogenes; Vibrio, alginate lyase AlyVGI (GenBankTM accession number AF114039 [GenBank] ) of V. halioticoli IAM14596T; Corynebacterium, guluronate lyase AlyPG (GenBankTM accession number AB030481 [GenBank] ) of Corynebacterium sp. ALY-1; Photobacterium, alginate lyase AlxM (GenBankTM accession number) of Photobacterium sp. ATCC 43367. Identical and similar amino acid residues in the six kinds of alginate lyases are denoted by asterisks and dots. Identical residues whose side chains are located at the surface of the protein are overlaid with red. Secondary structure elements of PA1167 are shown as above.

 
The side chains of the conserved residues of Phe34, Ser60, Leu62, Phe194, Ala196, and Leu218 are located within the protein. They appear to form hydrophobic blocks along the {beta}-sheets together with the surrounding homologous residues like Trp7, Ile11, Tyr29, Ile89, Ile103, and Phe215. They would be important to the structural stability of family PL-7 enzymes. In contrast, the side chains of residues Arg59, Glu61, and Arg63 on SA3, Gln102 and His104 on SA5, and Tyr193, Lys195, Gly197, Tyr199, and Gln201 on SA4 are located on the surface of the protein. They would make an active cleft and play an important role in the catalytic reaction. In addition to main chain interactions in the anti-parallel {beta}-sheet, there are three hydrogen bonds including two charged ones (Fig. 8) as follows: Arg59 NH1–Glu61 O-{epsilon}1 (2.90 Å), Glu61 O-{epsilon}2–Gln102 N-{epsilon}2 (2.71 Å), and Glu61 O-{epsilon}1–Lys195 N{zeta} (2.74 Å). More characteristic interactions are two sets of stacking-like interactions between Tyr193 and Arg63 and between Tyr199 and Arg59; the benzene ring plane of the Tyr residue and the plane formed by NH1, NH2, C-{zeta}, and N-{epsilon} of the Arg residue exist almost in parallel at 3.5 Å (Fig. 8). The three hydrogen bonds and two stacking-like interactions would retain the rigidity of the active cleft and contribute greatly to substrate binding or catalytic reactions. Considering that tyrosine and histidine residues play a crucial role in the catalytic reaction of the family PL-5 alginate lyase A1-III (Fig. 6B), putative residues responsible for the catalytic reaction are His104, Tyr193, and Tyr199 (Fig. 8).



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  		FIG. 8.
An active cleft of PA1167. Conserved residues located on the surface are shown in bonds. Hydrogen bonds are shown as red lines.

 
Further analysis is required to clarify the structure/function relationship of family PL-7 enzymes. To elucidate the weak specific activity of PA1167 and the broad substrate specificity in family PL-7 alginate lyases, the A1-II and PA1167/substrate complex are now being structurally and mutationally analyzed.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 1VAV [PDB] ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported in part by grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan and by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Both authors contributed equally to this work. Back

|| To whom correspondence should be addressed. Tel.: 81-774-38-3766; Fax: 81-774-38-3767; E-mail: kmurata{at}kais.kyoto-u.ac.jp.

1 The abbreviations used are: PL, polysaccharide lyase; TBA, thiobarbituric acid; MIR, multiple isomorphous replacement. Back

2 H. Motoshima, Y. Iwamoto, K. Watanabe, T. Oda, and T. Muramatsu, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Dr. T. Sawabe, Hokkaido University, for technical guidance in the separation of alginate block structures, and Dr. A. Markaryan, University of Illinois College of Medicine, for providing P. aeruginosa PAO1 and 8830. We also thank Dr. H. Sakai and Dr. M. Kawamoto of Japan Synchrotron Radiation Research Institute (JASRI) for their kind help in data collection. The x-ray data collection at BL41XU of SPring-8 was carried out with approval of the organizing committee of SPring-8 (Proposal 2003A0448-NL1-np, BM).



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HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
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