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374 a.a.
_CA
Key reference
DOI no: 10.1074/jbc.M804119200 J Biol Chem 283:23819-23828 (2008) PubMed id: 18574239
Structural and Mutational Characterization of the Catalytic A-module of the Mannuronan C-5-epimerase AlgE4 from Azotobacter vinelandii. H.J.Rozeboom, T.M.Bjerkan, K.H.Kalk, H.Ertesvåg, S.Holtan, F.L.Aachmann, S.Valla, B.W.Dijkstra. 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.
Selected figure(s)
Figure 5.
FIGURE 5. Superimposition of the active site residues of ALY-1 in green (His^119, Gln^117, Arg^72, and Tyr^195), A1-III (His^192, Asn^191, Arg^239, Tyr^246, and trisaccharide) in lilac, and AlgE4A (His^154, Asp^152, Lys^117, and Tyr^149 and trisaccharide) in CPK colors. The image was constructed in PyMOL (52).Figure 7.
FIGURE 7. Proposed catalytic mechanism of AlgE4. A and B, the alginate polymer enters the catalytic site. B and C, the carboxylate moiety of the mannuronic acid in subsite +1 is protonated, enabling it to form a hydrogen bond with Asp^152 (and/or 178), which stabilizes the substrate-enzyme complex. C, upon deprotonation of Tyr^149 (via Arg^195) the alkoxide ion group extracts H-5 from the re-face of the mannuronic acid in subsite +1. C and D, a double bond is formed, which makes the conformation of the +1 mannuronic acid partially planar. D, the protonated His^154 performs a nucleophilic attack on the C-5 atom of the +1 sugar from the si-face with the concomitant flip of the +1 sugar ring into the ^1C[4] chair conformation of guluronic acid. D and E, the carboxylic acid moiety on sugar +1 is deprotonated. E and F, the epimerized sugar leaves the active site and His^154 is protonated again. F, the epimerase is ready to perform a new reaction.The above figures are reprinted by permission from the ASBMB: J Biol Chem (2008, 283, 23819-23828) copyright 2008. Figures were selected by an automated process.
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