Structural and mutational characterization of the catalytic A-module of the mannuronan C-5-epimerase AlgE4 from Azotobacter vinelandii

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.

FIGURE 1.

Structure of AlgE4A. A mannuronan trisaccharide (M₃) bound in the active cleft is shown in stick representation. A, the secondary structure elements forming the β-helix fold. The N-terminal α-helix is shown at the top. The 4 sheets forming the helix are colored blue (PB1), yellow (PB2a), green (PB2b), and red (PB3). The T1–T3 turns are also indicated. B, the same structure as in A, seen from the C-terminal end. C, stereo view of the electrostatic potential surface. Positive potential is shown in blue and negative potential in red. The figure was produced using PyMol (52).

FIGURE 2.

Circular 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.

FIGURE 3.

Stereo view of amino acids surrounding the substrate binding site. A bound mannuronan trisaccharide (M₃) molecule is shown in stick representation. Glu¹⁵⁵ and Gln¹⁵⁶ form a cis-peptide. The image was constructed in PyMOL (52).

FIGURE 4.

Characterization of AlgE4 mutants. Relative activities are shown compared with the wild type activity (100%). Enzymes showing less than 0.05% activity are considered inactive. Only enzymes with decreased activities to less than 60% are considered as being significantly different from the native enzyme.

FIGURE 5.

Superimposition of the active site residues of ALY-1 in green (His¹¹⁹, Gln¹¹⁷, Arg⁷², and Tyr¹⁹⁵), A1-III (His¹⁹², Asn¹⁹¹, Arg²³⁹, Tyr²⁴⁶, and trisaccharide) in lilac, and AlgE4A (His¹⁵⁴, Asp¹⁵², Lys¹¹⁷, and Tyr¹⁴⁹ and trisaccharide) in CPK colors. The image was constructed in PyMOL (52).

FIGURE 6.

2F_o – F_c electron density of the native active site residues generated from a simulated-annealing composite omit map contoured at 1.5 σ. The M residue in subsite –1 from the M₃ structure is superposed on the native structure, the M residue in the +1 site has been obtained by computer modeling. The figure was produced using PyMol (52).

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 Asp¹⁵² (and/or 178), which stabilizes the substrate-enzyme complex. C, upon deprotonation of Tyr¹⁴⁹ (via Arg¹⁹⁵) 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 His¹⁵⁴ 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 ¹C₄ 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 His¹⁵⁴ is protonated again. F, the epimerase is ready to perform a new reaction.