Structural and Mutational Characterization of the Catalytic A-module of the Mannuronan C-5-epimerase AlgE4 from Azotobacter Vinelandii

Overview

Journal J Biol Chem

Specialty Biochemistry

Date 2008 Jun 25

PMID 18574239

Citations 19

Authors

Henriette J Rozeboom

Tonje M Bjerkan

Kor H Kalk

Helga Ertesvag

Synnove Holtan

Finn L Aachmann

Svein Valla

Bauke W Dijkstra

Affiliations

Soon will be listed here.

Abstract

Alginate is a family of linear copolymers of (1-->4)-linked beta-d-mannuronic acid and its C-5 epimer alpha-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-A resolution. AlgE4A folds into a right-handed parallel beta-helix structure originally found in pectate lyase C and subsequently in several polysaccharide lyases and hydrolases. The beta-helix is composed of four parallel beta-sheets, comprising 12 complete turns, and has an amphipathic alpha-helix near the N terminus. The catalytic site is positioned in a positively charged cleft formed by loops extending from the surface encompassing Asp(152), an amino acid previously shown to be important for the reaction. Site-directed mutagenesis further implicates Tyr(149), His(154), and Asp(178) as being essential for activity. Tyr(149) probably acts as the proton acceptor, whereas His(154) is the proton donor in the epimerization reaction.

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References

Blatny J, Brautaset T, Winther-Larsen H, Karunakaran P, Valla S . Improved broad-host-range RK2 vectors useful for high and low regulated gene expression levels in gram-negative bacteria. Plasmid. 1997; 38(1):35-51. DOI: 10.1006/plas.1997.1294. View

Ertesvag H, Valla S . The A modules of the Azotobacter vinelandii mannuronan-C-5-epimerase AlgE1 are sufficient for both epimerization and binding of Ca2+. J Bacteriol. 1999; 181(10):3033-8. PMC: 93757. DOI: 10.1128/JB.181.10.3033-3038.1999. View

Morris R, Perrakis A, Lamzin V . ARP/wARP and automatic interpretation of protein electron density maps. Methods Enzymol. 2003; 374:229-44. DOI: 10.1016/S0076-6879(03)74011-7. View

Brunger A, Adams P, Clore G, DeLano W, Gros P, Grosse-Kunstleve R . Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr. 1998; 54(Pt 5):905-21. DOI: 10.1107/s0907444998003254. View

Aachmann F, Svanem B, Guntert P, Petersen S, Valla S, Wimmer R . NMR structure of the R-module: a parallel beta-roll subunit from an Azotobacter vinelandii mannuronan C-5 epimerase. J Biol Chem. 2006; 281(11):7350-6. DOI: 10.1074/jbc.M510069200. View

Otwinowski Z, Minor W . Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 1997; 276:307-26. DOI: 10.1016/S0076-6879(97)76066-X. View

Yoon H, Hashimoto W, Miyake O, Murata K, Mikami B . Crystal structure of alginate lyase A1-III complexed with trisaccharide product at 2.0 A resolution. J Mol Biol. 2001; 307(1):9-16. DOI: 10.1006/jmbi.2000.4509. View

Nyvall P, Corre E, Boisset C, Barbeyron T, Rousvoal S, Scornet D . Characterization of mannuronan C-5-epimerase genes from the brown alga Laminaria digitata. Plant Physiol. 2003; 133(2):726-35. PMC: 219047. DOI: 10.1104/pp.103.025981. View

Sabra W, Zeng A, Lunsdorf H, Deckwer W . Effect of oxygen on formation and structure of Azotobacter vinelandii alginate and its role in protecting nitrogenase. Appl Environ Microbiol. 2000; 66(9):4037-44. PMC: 92256. DOI: 10.1128/AEM.66.9.4037-4044.2000. View

10.

Lin L, SADOFF H . Chemical composition of Azotobacter vinelandii cysts. J Bacteriol. 1969; 100(1):480-6. PMC: 315417. DOI: 10.1128/jb.100.1.480-486.1969. View

11.

Huang W, Boju L, Tkalec L, Su H, Yang H, Gunay N . Active site of chondroitin AC lyase revealed by the structure of enzyme-oligosaccharide complexes and mutagenesis. Biochemistry. 2001; 40(8):2359-72. DOI: 10.1021/bi0024254. View

12.

Campos M, Lloret L, Moreno S, Nunez C, Espin G, Soberon-Chavez G . Characterization of the gene coding for GDP-mannose dehydrogenase (algD) from Azotobacter vinelandii. J Bacteriol. 1996; 178(7):1793-9. PMC: 177871. DOI: 10.1128/jb.178.7.1793-1799.1996. View

13.

Sletmoen M, Skjak-Braek G, Stokke B . Single-molecular pair unbinding studies of Mannuronan C-5 epimerase AlgE4 and its polymer substrate. Biomacromolecules. 2004; 5(4):1288-95. DOI: 10.1021/bm0345211. View

14.

Yamasaki M, Moriwaki S, Miyake O, Hashimoto W, Murata K, Mikami B . Structure and function of a hypothetical Pseudomonas aeruginosa protein PA1167 classified into family PL-7: a novel alginate lyase with a beta-sandwich fold. J Biol Chem. 2004; 279(30):31863-72. DOI: 10.1074/jbc.M402466200. View

15.

Jabs A, Weiss M, Hilgenfeld R . Non-proline cis peptide bonds in proteins. J Mol Biol. 1999; 286(1):291-304. DOI: 10.1006/jmbi.1998.2459. View

16.

Donati I, Holtan S, Morch Y, Borgogna M, Dentini M, Skjak-Braek G . New hypothesis on the role of alternating sequences in calcium-alginate gels. Biomacromolecules. 2005; 6(2):1031-40. DOI: 10.1021/bm049306e. View

17.

Douthit S, Dlakic M, Ohman D, Franklin M . Epimerase active domain of Pseudomonas aeruginosa AlgG, a protein that contains a right-handed beta-helix. J Bacteriol. 2005; 187(13):4573-83. PMC: 1151786. DOI: 10.1128/JB.187.13.4573-4583.2005. View

18.

Bradford M . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72:248-54. DOI: 10.1016/0003-2697(76)90527-3. View

19.

Jenkins J, Shevchik V, Hugouvieux-Cotte-Pattat N, Pickersgill R . The crystal structure of pectate lyase Pel9A from Erwinia chrysanthemi. J Biol Chem. 2003; 279(10):9139-45. DOI: 10.1074/jbc.M311390200. View

20.

Svanem B, Skjak-Braek G, Ertesvag H, Valla S . Cloning and expression of three new Aazotobacter vinelandii genes closely related to a previously described gene family encoding mannuronan C-5-epimerases. J Bacteriol. 1998; 181(1):68-77. PMC: 103533. DOI: 10.1128/JB.181.1.68-77.1999. View