Structural and Mutational Characterization of the Catalytic A-module of the Mannuronan C-5-epimerase AlgE4 from Azotobacter vinelandii
2008; Elsevier BV; Volume: 283; Issue: 35 Linguagem: Inglês
10.1074/jbc.m804119200
ISSN1083-351X
AutoresH.J. Rozeboom, Tonje M. Bjerkan, Kor H. Kalk, Helga Ertesvåg, Synnøve Holtan, Finn L. Aachmann, Svein Valla, Bauke W. Dijkstra,
Tópico(s)Enzyme Structure and Function
ResumoAlginate is a family of linear copolymers of (1→4)-linked β-d-mannuronic acid and its C-5 epimer α-l-guluronic acid. The polymer is first produced as polymannuronic acid and the guluronic acid residues are then introduced at the polymer level by mannuronan C-5-epimerases. The structure of the catalytic A-module of the Azotobacter vinelandii mannuronan C-5-epimerase AlgE4 has been determined by x-ray crystallography at 2.1-Å resolution. AlgE4A folds into a right-handed parallel β-helix structure originally found in pectate lyase C and subsequently in several polysaccharide lyases and hydrolases. The β-helix is composed of four parallel β-sheets, comprising 12 complete turns, and has an amphipathic α-helix near the N terminus. The catalytic site is positioned in a positively charged cleft formed by loops extending from the surface encompassing Asp152, an amino acid previously shown to be important for the reaction. Site-directed mutagenesis further implicates Tyr149, His154, and Asp178 as being essential for activity. Tyr149 probably acts as the proton acceptor, whereas His154 is the proton donor in the epimerization reaction. Alginate is a family of linear copolymers of (1→4)-linked β-d-mannuronic acid and its C-5 epimer α-l-guluronic acid. The polymer is first produced as polymannuronic acid and the guluronic acid residues are then introduced at the polymer level by mannuronan C-5-epimerases. The structure of the catalytic A-module of the Azotobacter vinelandii mannuronan C-5-epimerase AlgE4 has been determined by x-ray crystallography at 2.1-Å resolution. AlgE4A folds into a right-handed parallel β-helix structure originally found in pectate lyase C and subsequently in several polysaccharide lyases and hydrolases. The β-helix is composed of four parallel β-sheets, comprising 12 complete turns, and has an amphipathic α-helix near the N terminus. The catalytic site is positioned in a positively charged cleft formed by loops extending from the surface encompassing Asp152, an amino acid previously shown to be important for the reaction. Site-directed mutagenesis further implicates Tyr149, His154, and Asp178 as being essential for activity. Tyr149 probably acts as the proton acceptor, whereas His154 is the proton donor in the epimerization reaction. Alginate is a family of linear copolymers of (1→4)-linked β-d -mannuronic acid (M) 5The abbreviations used are:Mβ-d -mannuronic acidMG-blocksstretches of contiguous alternating structure ((MG)n)Gα-l -guluronic acidG-blocksstretches of contiguous G-residuesEMPethyl-mercury-phosphatePLpolysaccharide lyaseMOPS3-(N-morpholino)propanesulfonic acid. 5The abbreviations used are:Mβ-d -mannuronic acidMG-blocksstretches of contiguous alternating structure ((MG)n)Gα-l -guluronic acidG-blocksstretches of contiguous G-residuesEMPethyl-mercury-phosphatePLpolysaccharide lyaseMOPS3-(N-morpholino)propanesulfonic acid. and its C-5 epimer α-l-guluronic acid (G) produced by brown algae and some bacteria belonging to the Pseudomonas and Azotobacter genera (1Fischer F.G. Dörfel H. Hoppe-Seyler's Z. Physiol. Chem. 1955; 302: 186-203Crossref PubMed Scopus (57) Google Scholar, 2Drummond D.W. Hirst E.L. Percival E. J. Chem. Soc. 1962; : 1208-1216Crossref Scopus (23) Google Scholar, 3Linker A. Jones R.S. Nature. 1964; 204: 187-188Crossref PubMed Scopus (102) Google Scholar, 4Gorin P.A.J Spencer J.F.T Can. J. Chem. 1966; 44: 993-998Crossref Google Scholar). The M- and G-moieties are distributed irregularly as blocks of homopolymeric regions (M- or G-blocks) interspersed with blocks of alternating structure (MG-blocks). The relative amounts and sequence distributions of M and G vary considerably between alginates from different sources, and this variation in sequence and composition imparts different properties to alginates. G-blocks are rather stiff, and form strong, brittle gels with divalent cations such as Ca2+, whereas M-blocks are less rigid and do not form cationic gels. MG-blocks are the most flexible and have been shown to bind Ca2+ and form junction zones (5Donati I. Holtan S. Mørch Y.A. Borgogna M. Dentini M. Skjåk-Bræk G. Biomacromolecules. 2005; 6: 1031-1040Crossref PubMed Scopus (273) Google Scholar).In Azotobacter vinelandii, alginate constitutes a major part of the coat surrounding cells differentiated into resting stage cysts (6Lin L.P. Sadoff H.L. J. Bacteriol. 1969; 100: 480-486Crossref PubMed Google Scholar, 7Sadoff H.L. Bacteriol. Rev. 1975; 39: 516-539Crossref PubMed Google Scholar), and alginate negative strains do not encyst (8Campos M.E. Martínez-Salazar J.M. Lloret L. Moreno S. Núñez C. Espín G. Soberón-Chávez G. J. Bacteriol. 1996; 178: 1793-1799Crossref PubMed Google Scholar). Nevertheless, the polymer is also produced during vegetative growth, and under such conditions it may play a role in protecting the bacterium from oxidative stress (9Sabra W. Zeng A.P. Lünsdorf H. Deckwer W.D. Appl. Env. Microbiol. 2000; 66: 4037-4044Crossref PubMed Scopus (137) Google Scholar).The alginate polymer is produced as polymannuronic acid, and the guluronic acid residues are introduced at the polymer level by the action of mannuronan C-5-epimerases (10Valla S. Li J.P. Ertesvåg H. Barbeyron T. Lindahl U. Biochimie (Paris). 2001; 83: 819-830Crossref PubMed Scopus (41) Google Scholar). All known alginate-producing bacteria possess a periplasmic C-5-epimerase, AlgG (11Rehm B.H. Ertesvåg H. Valla S. J. Bacteriol. 1996; 178: 5884-5889Crossref PubMed Google Scholar), but A. vinelandii also secretes a family of seven extracellular, Ca2+-dependent mannuronan C-5-epimerases, algE1–7 (12Ertesvåg H. Høidal H.K. Hals I.K. Rian A. Doseth B. Valla S. Mol. Microbiol. 1995; 16: 719-731Crossref PubMed Scopus (114) Google Scholar, 13Svanem B.I.G Skjåk-Bræk G. Ertesvåg H. Valla S. J. Bacteriol. 1999; 181: 68-77Crossref PubMed Google Scholar). These enzymes are highly homologous and consist of one or two catalytic A-modules and one to seven regulatory R-modules, of which at least one is needed for full activity (14Ertesvåg H. Valla S. J. Bacteriol. 1999; 181: 3033-3038Crossref PubMed Google Scholar). Each of the extracellular A. vinelandii enzymes produces a distinctive M/G-pattern (14Ertesvåg H. Valla S. J. Bacteriol. 1999; 181: 3033-3038Crossref PubMed Google Scholar); AlgE2 and AlgE5 make relatively short G-blocks, whereas AlgE6 makes longer G-blocks (13Svanem B.I.G Skjåk-Bræk G. Ertesvåg H. Valla S. J. Bacteriol. 1999; 181: 68-77Crossref PubMed Google Scholar, 15Ramstad M.V. Ellingsen T.E. Josefsen K.D. Høidal H.K. Valla S. Skjåk-Bræk G. Levine D.W. Enzyme Microb. Technol. 1999; 24: 636-646Crossref Scopus (18) Google Scholar). AlgE4 almost exclusively produces alternating MG sequences (16Høidal H. Ertesvåg H. Skjåk-Bræk G. Stokke B.T. Valla S. J. Biol. Chem. 1999; 274: 12316-13322Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar).AlgE4 is the smallest of the extracellular epimerases with a molecular mass of 57.7 kDa (553 amino acids). It contains one A-module, which is active on its own, comprising the 385 N-terminal amino acids, and one R-module of 142 residues, connected by a 7-residue linker. At the C terminus it has a small "S motif" of 19 residues of unknown function (12Ertesvåg H. Høidal H.K. Hals I.K. Rian A. Doseth B. Valla S. Mol. Microbiol. 1995; 16: 719-731Crossref PubMed Scopus (114) Google Scholar). Atomic force microscopy studies have revealed that the AlgE4 A-module binds more strongly to the alginate chain than the complete enzyme, indicating that the R-module is involved in the regulation of the enzyme-substrate binding strength (17Sletmoen M. Skjåk-Bræk G. Stokke B.T. Biomacromolecules. 2004; 5: 1288-1295Crossref PubMed Scopus (40) Google Scholar). Nevertheless, the R-module on its own is also able to bind to the alginate chain (18Aachmann F.L. Svanem B.I.G Güntert P. Petersen S.B. Valla S. Wimmer R. J. Biol. Chem. 2006; 281: 7350-7356Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar).AlgE4 has its optimum activity around pH 6.5–7.0 in the presence of 1–3 mm Ca2+ and at temperatures close to 37 °C (16Høidal H. Ertesvåg H. Skjåk-Bræk G. Stokke B.T. Valla S. J. Biol. Chem. 1999; 274: 12316-13322Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). It is highly sensitive to alkaline pH, being inactive at pH > 8. It binds a minimum of 6 sugar residues in the catalytic site, and for short chains the third residue from the non-reducing end is the first to be epimerized (19Campa C. Holtan S. Nilsen N. Bjerkan T.M. Stokke B.T. Skjåk-Bræk G. Biochem. J. 2004; 381: 155-164Crossref PubMed Scopus (70) Google Scholar). For longer chains the reaction proceeds toward the reducing end in a non-random, processive manner (20Hartmann M. Holm O.B. Johansen G.A.B Skjåk-Bræk G. Stokke B.T. Biopolymers. 2002; 63: 77-88Crossref PubMed Scopus (42) Google Scholar). On average, the enzyme epimerizes 10 units ((MG)10) in each reaction before leaving the chain (19Campa C. Holtan S. Nilsen N. Bjerkan T.M. Stokke B.T. Skjåk-Bræk G. Biochem. J. 2004; 381: 155-164Crossref PubMed Scopus (70) Google Scholar).An alignment of all known mannuronan C-5-epimerase sequences from bacteria and algae showed that they share an YG(F/I)DPH(D/E) motif (residues 149–155 in AlgE4) (21Svanem B.I.G Strand W.I. Ertesvåg H. Skjåk-Bræk G. Hartmann M. Barbeyron T. Valla S. J. Biol. Chem. 2001; 276: 31542-31550Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 22Nyvall P. Corre E. Boisset C. Barbeyron T. Rousvoal S. Scornet D. Kloareg B. Boyen C. Plant Physiol. 2003; 133: 726-735Crossref PubMed Scopus (64) Google Scholar, 23Douthit S.A. Dlakic M. Ohman D.E. Franklin M.J. Bacteriol. J. 2005; 187: 4573-4583Crossref PubMed Scopus (24) Google Scholar). Asp152 of this motif, which is also conserved in some pectate lyases, has been shown to be important for activity in AlgE7 (21Svanem B.I.G Strand W.I. Ertesvåg H. Skjåk-Bræk G. Hartmann M. Barbeyron T. Valla S. J. Biol. Chem. 2001; 276: 31542-31550Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The reaction mechanism of C-5-epimerases has been proposed to be similar to the lyase reaction (24Gacesa P. FEBS Lett. 1987; 212: 199-202Crossref Scopus (161) Google Scholar), involving three steps. First, the target uronic acid charge is neutralized, after which the proton at C-5 is abstracted. An enolate anion intermediate is formed, which is stabilized by resonance. In the lyase case, the enolate anion leads to β-elimination of the 4-O-glycosidic bond, whereas in the epimerase case β-elimination is prevented by donation of a proton to the opposite face of the sugar ring. Indeed in the bifunctional epimerase/lyase AlgE7 mutation of Asp152 eliminated both activities, suggesting that they occur at the same site (21Svanem B.I.G Strand W.I. Ertesvåg H. Skjåk-Bræk G. Hartmann M. Barbeyron T. Valla S. J. Biol. Chem. 2001; 276: 31542-31550Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Here we report the three-dimensional structure of the AlgE4 A-module, which is responsible for generating MG-alginates, and discuss the location of the active site and a possible catalytic mechanism.EXPERIMENTAL PROCEDURESGrowth of Bacteria—The bacterial strains and plasmids used in this study are listed in Table 1. Unless stated otherwise, bacteria were maintained at 37 °C in L broth (10 g of tryptone, 5 g of yeast extract, and 5 g of NaCl per liter) or on L agar (L broth supplemented with 15 g of agar per liter). Protein was expressed in 3× L broth (30 g of tryptone, 15 g of yeast extract, and 5 g of NaCl per liter). Media were supplemented with 200 μg/ml ampicillin or 12.5 μg/ml tetracycline when appropriate.TABLE 1Bacterial strains and plasmidsStrains or plasmidsRelevant characteristicsaThe abbreviations used are: Aprampicillin resistanceTcrtetracycline resistance. Plasmids pTB61-194 and pHE186-187 are identical to pBL5 with the exception of the introduced mutation in algE4.Mutagenic oligonucleotidebThese are the forward primers; the reverse primers were complementary to them. Changed bases are shown in lower case. In pTB87, pTB89, pTB158, and pTB192, pBL5 was not used as the template.Refs.E. coli DH5α25Sambrook J. Russell D. Molecular Cloning: A Laboratory Manual. Third Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001Google Scholar ER2566Encodes T7 RNA polymeraseNew England BiolabsPlasmids pTYB4IMPACT-CN fusion vector containing a C-terminal chitin binding TagNew England BioLabs pUC7TcpUC7 containing the tetAR genes from RK2, Apr, Tcr53Blatny J.M. Brautaset T. Winther-Larsen H.C. Karunakaran P. Valla S. Plasmid. 1997; 38: 35-51Crossref PubMed Scopus (136) Google Scholar pBL5Derivative of pTrc99A encoding algE4 from A. vinelandii, Apr51Bjerkan T.M. Lillehov B.E. Strand W.I. Skjåk-Bræk G. Valla S. Ertesvåg H. Biochem. J. 2004; 381: 813-821Crossref PubMed Scopus (19) Google Scholar pTB22Derivative of pTYB4 in which a NcoI-XmaI fragment of pBL5 containing algE4 was inserted into the corresponding sites of the vectorThis study pTB26Derivative of pTB22 in which the tetAR genes from a pUC7Tc BamHI-fragment was blunt-end ligated into the unique Eco47III site of the vector TcrThis study pTB61algE4 H154F (removing BssSI)CGGCTTCGACCCCttCGAGCAGACCThis study pTB62algE4 Y149F (inserting BspEI)CGAGATGTCCGGaTtCGGCTTCThis study pTB63algE4 Y149H (inserting BspEI)CGAGATGTCCGGacACGGCTTCThis study pTB64algE4 K117E (removing SalI)ACCAGCGGCgAaGTtGACGGCTGGTThis study pTB65algE4 K117R (inserting NotI)AACACCAGCGGCcgcGTCGACGGCTGGTTCThis study pTB67algE4 F122Y (inserting RsaI)CGACGGCTGGTaCAACGGCTATATCThis study pTB69algE4 D173V (removing BglI)CAACGGCCTCGtgGGaTTCGTCGCCGACThis study pTB70algE4 R195L (inserting BspMI)GCCAACGACCtgCACGGCTTCAACThis study pTB71algE4 D152E (inserting BstBI)ACGGCTTCGAaCCCCACGAGThis study pTB72algE4 D152N (removing BssSI)CTACGGCTTCaACCCCCAtGAGCAGACCThis study pTB73algE4 H154Y (removing BssSI)CTTCGACCCCtACGAGCAGACCThis study pTB74algE4 R195K (removing BtgI)CGCCAACGACaagCACGGCTTCThis study pTB75algE4 D173N (inserting BsaI)GACAACGGtCTCaACGGCTTCGTCThis study pTB76algE4 D173E (inserting XhoI)CAACGGCCTCGAgGGCTTCThis study pTB78algE4 P153A (inserting BspHI)CGGCTTCGACgCtCAtGAGCAGACCThis study pTB79algE4 R249A (inserting Eco57I)GACAACGCCgctGAAGGCGTGCTGThis study pTB80algE4 F122V (inserting HpaI)CGACGGCTGGgTtAACGGCTATATCThis study pTB82algE4 D178E (removing SexAI)CGGCTTCGTCGCCGAaTAtCTGGTCThis study pTB83algE4 D178N (inserting NruI)GACGGCTTCGTCGCgaAtTACCTGThis study pTB84algE4 Q225E (removing BsgI, inserting BsrBI)CCTGGTGGTGgAGCGGGGTCTGThis study pTB87algE4 D152N, D173E; derivative of pTB72 (inserting XhoI)CAACGGCCTCGAgGGCTTCThis study pTB89algE4 D173E, P153A; derivative of pTB76 (inserting BspHI)CGGCTTCGACgCtCAtGAGCAGACCThis study pTB92algE4 Q225N (removing BsgI)CCTGGTGGTGaAtCGGGGTCTGGThis study pTB93algE4 K255R (inserting BspEI)GTGCTGCTCcgGATGACCAGCGACATCACThis study pTB147algE4 E155A (removing BssSI)GACCCCCACGcGCAGACCATCAACCThis study pTB153algE4 Y149A (inserting NgoMIV)CGAGATGTCCGGCgcCGGCTTCGACCCThis study pTB154algE4 D152A (inserting BseRI)GGCTACGGCTTCGctCCtCACGAGCAGACCThis study pTB155algE4 H154A (removing BssSI, inserting PvuII)GGCTACGGCTTCGACCCagctGAGCAGACCATCThis study pTB156algE4 D173A (inserting NaeI)CAACGGCCTCGcCGGCTTCGTCGCThis study pTB157algE4 D178A (removing SexAI)CTTCGTCGCCGcaTAtCTGGTCGACAGThis study pTB158algE4 D173V, R195L; derivative of pTB69 (inserting BspMI)GCCAACGACCtgCACGGCTTCAACThis study pHE186algE4 H154R (removing BssSI)GCTTCGACCCaCgCGAGCAGACCATCAACThis study pHE187algE4 H154K (removing BssSI)CGGCTTCGACCCtaAgGAGCAGACCATCThis study pTB185algE4 H196A (inserting NaeI)CTACGCCAACGACCGCgcCGGCTTCAACThis study pTB186algE4 H196F (inserting PvuI)CTACGCCAACGAtCGCttCGGCTTCAACThis study pTB187algE4 H196Y (inserting PvrI)CTACGCCAACGAtCGCtACGGCTTCAACThis study pTB188algE4 K117A (removing SalI)CAACACCAGCGGCgcGGTtGACGGCTGGTTCThis study pTB190algE4 Q156A (removing BssSI)CTTCGACCCCCACGAagcGACCATCAACCTGThis study pTB191algE4 E155A, Q156A (removing BssSI)CTTCGACCCCCACGcGgcGACCATCAACCTGThis study pTB192algE4 Q225A, K255A; derivative of pTB193 (inserting NruI)GAAGGCGTGCTGCTCgcGATGACCAGCGThis study pTB193algE4 Q225A (inserting SmaI)GCCTGGTGGTGgccCGGGGTCTGGAGGThis study pTB194algE4 K255A (removing NruI)GAAGGCGTGCTGCTCgcGATGACCAGCGThis studya The abbreviations used are:Aprampicillin resistanceTcrtetracycline resistance. Plasmids pTB61-194 and pHE186-187 are identical to pBL5 with the exception of the introduced mutation in algE4.b These are the forward primers; the reverse primers were complementary to them. Changed bases are shown in lower case. In pTB87, pTB89, pTB158, and pTB192, pBL5 was not used as the template. Open table in a new tab Recombinant DNA Technology—Standard recombinant DNA procedures were done according to Sambrook and Russell (25Sambrook J. Russell D. Molecular Cloning: A Laboratory Manual. Third Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001Google Scholar), whereas transformations were carried out according to the RbCl transformation protocol (New England BioLabs). Plasmids were isolated using the Wizard® Plus SV minipreps DNA Purification System (Promega). All gene manipulations were done in Escherichia coli DH5α, and the pTB26 plasmid was later transferred to E. coli ER2566 (New England BioLabs). DNA sequencing was performed using the BigDye® Terminator version 1.1 Cycle Sequencing Kit (Applied Biosystems).Production and Purification of Mannuronan Oligomers—Partial acid hydrolysis of mannuronan was performed essentially as described by Campa et al. (19Campa C. Holtan S. Nilsen N. Bjerkan T.M. Stokke B.T. Skjåk-Bræk G. Biochem. J. 2004; 381: 155-164Crossref PubMed Scopus (70) Google Scholar). Briefly, after pre-hydrolysis at pH 5.6, the sample was incubated for 6 h at 95°C and pH 3.5, and then neutralized with NaOH. The resulting oligomannuronic acid hydrolysis mixture was fractionated on three serially connected columns of Superdex 30 preparative grade (HiLoad 2.6 × 60 cm, GE Healthcare) at a flow rate of 0.8 ml/min with 0.1 m NH4Ac (pH 6.9) at room temperature. Fractions of 5 ml were collected from three successive column runs and pooled. The pooled samples were stored at 4 °C prior to three cycles of freeze-drying to remove all traces of NH4Ac and to provide solid, purified oligomers of mannuronic acid.Protein Expression and Purification—The A-module of AlgE4 was recombinantly expressed from pTB26, a derivative of the IMPACT-CN expression vector pTYB4 (New England BioLabs) with the sequence encoding the first 378 amino acids of AlgE4 inserted (Table 1). AlgE4A was expressed in E. coli ER2566 at 15 °C and purified in a single affinity chromatography step, using the IMPACT-CN Protein Fusion and Purification System (New England BioLabs), as described in the manual.Secondary Structure Determination—Circular dichroism studies of AlgE4A were carried out with a protein concentration of 5 μm in 100 μm HEPES (pH 6.9), at 293 K, and 5 mm CaCl2 on a JASCO J-175 spectropolarimeter. The resulting spectrum was the average of 12 scans recorded at a rate of 50 nm/min; it was analyzed by the program K2d (26Andrade M.A. Chacón P. Merelo J.J. Morán F. Protein Eng. 1993; 6: 383-390Crossref PubMed Scopus (946) Google Scholar).Crystallization—Crystallization conditions were difficult to find; over 500 different conditions were tested before the first protein crystals appeared. AlgE4A crystals were obtained after two months from hanging drop experiments with 1.6–1.7 m sodium malonate (pH 7–8) as precipitant at room temperature, using drops of 3 μl of protein (11 mg/ml) and 3 μl of reservoir solution (27McPherson A. Protein Sci. 2001; 10: 418-422Crossref PubMed Scopus (100) Google Scholar). The lozenge-shaped crystals had a typical size of 0.3 × 0.2 × 0.1 mm3.X-ray Data Collection—Before data collection, crystals were soaked for 1 min in a cryoprotectant solution containing 1.7 m malonate and 20% glycerol, directly followed by flash cooling. Data were collected in house at 100 K on a DIP2030H image plate detector (Bruker-AXS, Delft, The Netherlands) using Cu-Kα radiation from a Bruker-AXS FR591 rotating-anode generator equipped with Franks mirrors. Intensity data were processed using DENZO and SCALEPACK (28Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38349) Google Scholar). A highly redundant native data set to 2.1-Å resolution was obtained. The crystals belong to space group P21212 (Table 2). With two monomers of 39.8 kDa in the asymmetric unit, the VM is 3.0 Å3/Da (29Matthews B.W. J. Mol. Biol. 1968; 33: 491-497Crossref PubMed Scopus (7899) Google Scholar), and the calculated solvent content is 59%. Preparation of heavy atom derivatives was done by conventional soaking experiments (30Bluhm M.M. Bodo G. Dintzis H.M. Kendrew J.C. Proc. R. Soc. A. 1958; 246: 369-389Google Scholar) with 1–5 mm of the heavy atom compound. For substrate binding studies crystals were soaked for 18 h in a saturated solution of M4 (an oligomer of four (1→4)-linked β-d -mannuronic acid residues) in 50% (w/v) polyethylene glycol 3350, and 10 mm CaCl2 at pH 6.5. The M4 dataset was collected at Bruker-AXS, Delft, using Cu-Kα radiation from a Bruker-AXS Microstar generator with Montel200 multilayer graded optics, and a Proteum-R diffractometer system with a 3-axis goniostat, at a temperature of 100 K.TABLE 2Data collection and phasing statisticsCrystal/derivativeNativeEMP (C2H5HgPO4)PtCl4M4Cell (Å)174.0, 121.3, 44.7174.0, 121.2, 44.6174.0, 121.3, 44.7169.6, 121.8, 45.1Resolution range (Å)50.0-2.1 (2.18-2.1)50.0-2.8 (2.9-2.8)50.0-3.2 (3.31-3.2)43.6-2.7No. of unique reflections51,691 (4,598)19,544 (1,886)14,451 (1,368)26,719Completeness (%)92.3 (83.6)81.2 (80.7)88.5 (87.4)99.2 (93.8)Overall I/σ (I)12.4 (1.8)11.6 (3.5)11.8 (5.3)10.7 (1.9)Rsym (%)9.8 (63.8)9.6 (30.6)13.0 (29.7)10.0 (56.1)No. of sites21- Open table in a new tab Structure Determination—The structure of AlgE4A was determined by multiple isomorphous replacement with two heavy atom derivatives (Table 2). Heavy atom positions were located with the program SOLVE (31Terwilliger T.C. Methods Enzymol. 2003; 374: 22-37Crossref PubMed Scopus (432) Google Scholar), which was also used for refining the heavy atom parameters and for calculating phases. Density modification and phase extension to 2.1 Å was done with RESOLVE (31Terwilliger T.C. Methods Enzymol. 2003; 374: 22-37Crossref PubMed Scopus (432) Google Scholar), which was also used to automatically build fragments of the main chain. After RESOLVE, the overall figure of merit was 0.45. At this stage, a 2.1-Å electron density map was calculated and visually inspected with the program O (32Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13003) Google Scholar). The map was judged not to be interpretable; no electron density was visible for residues that had not been built by RESOLVE. Moreover, the direction of the main chain could not be deduced and no side chain density was visible. Therefore, in an iterative procedure, phases were improved with the ARP/wARP procedure (33Morris R.J. Perrakis A. Lamzin V.S. Methods Enzymol. 2003; 374: 229-244Crossref PubMed Scopus (470) Google Scholar) combined with model building by hand. When 55% of the main chain had been built in this way, the phases could not be further improved. By assuming that the two heavy atoms from the EMP derivative occupy the same positions in the protein, and by overlaying parts of the already built main chain, the non-crystallographic symmetry operators could be determined. This allowed non-crystallographic symmetry averaging during refinement with CNS (34Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16929) Google Scholar), and the gradual completion of the model building. The structure refinement converged at 2.1Åtoan Rfree value of 23.5% and a conventional Rfactor of 19.5%. The final model consists of 750 amino acids, residues 2–375 from molecule A and residues 2–377 from molecule B, 520 water molecules, 1 glycerol molecule, and 2 calcium ions. Assuming that the B-values of these calcium ions are similar to those of the neighboring protein atoms the occupancies of the calcium ions are close to 0.5 (Table 3). The stereochemistry of the final structure was evaluated using the program PROCHECK (35Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) (Table 3). For the M4 experiment the native structure was used as the starting model. Initial 2Fo – Fc and Fo – Fc maps clearly showed density for an M3 trisaccharide product as well as for calcium and chloride ions. The atomic coordinates of the final models and structure factors have been deposited in the Protein Data Bank (entries 2PYG for the native structure and 2PYH for the M4 structure).TABLE 3Refinement statisticsNativeM4Resolution (Å)50.0-2.143.6-2.7No. of reflections in working set48,99925,216No. of reflections in test set2,6241,336Rwork (%)19.423.3Rfree (%)23.527.9Root mean square deviations from ideal Bond length (Å)0.0060.007 Bond angles (deg)1.291.30Ramachandran plot Non-glycine residues in most favored regions (%)84.3 Non-glycine residues in additional allowed regions (%)14.8 Non-glycine residues in generously allowed regions (%)1.0Number of non-hydrogen atoms (average B values (A)2) total/main chain/side chain Residues in chain A2-3751-376 Protein molecule A2,782 (33.5/33.0/34.0)2,799 (49.8/49.6/50.0) Residues in chain B2-3771-377 Protein molecule B2,798 (31.0/32.5/31.7)2,806 (47.5/47.2/47.8) Water520 (42.0)61 (36.6) Calcium ions2 (33.8)7 (72.5) Glycerol1 (60.6) Mannuronic acid3 (107.0) Open table in a new tab Construction and Characterization of AlgE4 Mutants—Mutants of AlgE4 were constructed by the QuikChange™ Site-directed Mutagenesis Kit (Stratagene) according to the manufacturer's instructions using the primers shown in Table 1. AlgE4 mutant proteins (plasmids pTB61–pTB65; pTB67; pTB69–pTB76; pTB78–pTB80; pTB82–pTB84; pTB87; pTB89; pTB92–pTB93; pTB147; pTB153–pTB158; pTB185–pTB188; pTB190–pTB194; pHE186–pHE187) and the wild type AlgE4 (plasmid pBL5) were expressed in E. coli DH5α at 37 °C, partially purified by fast protein liquid chromatography on a HiTrap-Q column (GE Healthcare), and their catalytic activities measured in an isotope assay essentially as previously described (14Ertesvåg H. Valla S. J. Bacteriol. 1999; 181: 3033-3038Crossref PubMed Google Scholar), using two purification buffers and a disruption buffer containing 20 mm MOPS (pH 6.9) supplemented with 2 mm CaCl2, 2 mm CaCl2, 1 m NaCl, and 4 mm CaCl2, respectively. Protein concentrations were estimated by the Bio-Rad protein assay (36Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213288) Google Scholar).RESULTSStructure of the Mannuronan C-5-epimerase AlgE4 A-module—The crystal structure of the A-module of AlgE4 (AlgE4A) was determined at 2.1-Å resolution by the multiple isomorphous replacement method using two heavy atom derivatives (Table 2). There are two molecules, A and B, in the asymmetric unit. Residues 2–375 from molecule A and residues 2–377 from molecule B could be built into the electron density. Superimposition of the two molecules reveals that they are very similar with 373 Cα atoms overlaying with an root mean square deviation of only 0.20 Å.The AlgE4A molecule folds into a right-handed parallel β-helix structure comprising 12 complete turns with overall dimensions of 67 × 37 × 36 Å (Fig. 1). The number of amino acid residues per turn varies from 20 to 40. The β-helix turns form four parallel β-sheets, named PB1, PB2a, PB2b, and PB3, consistent with the naming of the β-sheets in polygalacturonase II (37van Santen Y. Benen J.A.E Schröter K.H. Kalk K.H. Armand S. Visser J. Dijkstra B.W. J. Biol. Chem. 1999; 274: 30474-30480Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Not being part of the β-helix, residues 307–309 and 316–318 make up a small two-stranded antiparallel β-sheet, whereas residues 19–32 form an amphipathic α-helix that caps the N-terminal end of the β-helix. The C-terminal end of the β-helix is covered by the C-terminal end of the second molecule in the asymmetric unit. This predominantly β-helical structure agrees with circular dichroism studies (Fig. 2), which suggest an α-helix content of 5%, a β-strand content of 47%, and a coil content of 48%, whereas, according to the DSSP algorithm (38Kabsch W. Sander C. Biopolymers. 1983; 22: 2577-2637Crossref PubMed Scopus (12098) Google Scholar), the crystal structure contains 3.5% α-helix, 46.5% β-strand, and 50% coiled coil.FIGURE 2Circular dichroism spectrum of 5 μm AlgE4A in 100 μm HEPES (pH 6.9), 20 °C, and 5 mm CaCl2. The spectrum of AlgE4A indicates high β-sheet structure content.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Turns connect the β-strands in adjacent β-sheets. The T1 turns between PB1 and PB2 (a or b) vary in length from 1 to 19 amino acids, whereas the T2 turns between PB2 (a or b) and PB3 are short, consisting of one amino acid residue, except in the first three turns, which comprise 8, 2, and 7 residues, respectively. Between PB3 and PB1, the T3 turns vary from 4 to 12 amino acid residues. The T1 and T3 turns are longer toward the N- and C-terminal parts of the β-helix, respectively
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