Crystal Structure and Mutational Analysis of Isomalto-dextranase, a Member of Glycoside Hydrolase Family 27

Abstract

Arthrobacter globiformis T6 isomalto-dextranase (AgIMD) is an enzyme that liberates isomaltose from the non-reducing end of a polymer of glucose, dextran. AgIMD is classified as a member of the glycoside hydrolase family (GH) 27, which comprises mainly α-galactosidases and α-N-acetylgalactosaminidases, whereas AgIMD does not show α-galactosidase or α-N-acetylgalactosaminidase activities. Here, we determined the crystal structure of AgIMD. AgIMD consists of the following three domains: A, C, and D. Domains A and C are identified as a (β/α)8-barrel catalytic domain and an antiparallel β-structure, respectively, both of which are commonly found in GH27 enzymes. However, domain A of AgIMD has subdomain B, loop-1, and loop-2, all of which are not found in GH27 human α-galactosidase. AgIMD in a complex with trisaccharide panose shows that Asp-207, a residue in loop-1, is involved in subsite +1. Kinetic parameters of the wild-type and mutant enzymes for the small synthetic saccharide p-nitrophenyl α-isomaltoside and the polysaccharide dextran were compared, showing that Asp-207 is important for the catalysis of dextran. Domain D is classified as carbohydrate-binding module (CBM) 35, and an isomaltose molecule is seen in this domain in the AgIMD-isomaltose complex. Domain D is highly homologous to CBM35 domains found in GH31 and GH66 enzymes. The results here indicate that some features found in GH13, -31, and -66 enzymes, such as subdomain B, residues at the subsite +1, and the CBM35 domain, are also observed in the GH27 enzyme AgIMD and thus provide insights into the evolutionary relationships among GH13, -27, -31, -36, and -66 enzymes.

Keywords: carbohydrate metabolism; enzyme kinetics; glycoside hydrolase; phylogenetics; x-ray crystallography.

FIGURE 1.

Overall structure of AgIMD. A, ribbon model of AgIMD-isomaltose. The N-terminal extra loop (magenta), domain A (yellow), subdomain B (cyan), domain C (blue), domain D (green), and two isomaltose molecules (red) are indicated. B–H, F_o − F_c omit maps contoured at 3 σ level for isomaltose in domain A of AgIMD-isomaltose (B), isomaltose in domain D of AgIMD-isomaltose (C), panose in domain A of AgIMD-panose (D), a molecule modeled as isomaltose in domain D of AgIMD-panose (E), Sme243 in unliganded AgIMD (F), Sme243 in AgIMD-isomaltose (G), and Sme243 in AgIMD-panose (H) are shown.

FIGURE 2.

Stereo views of the active site of AgIMD-isomaltose. A, active sites of unliganded AgIMD (green) and AgIMD-isomaltose (red and magenta). In AgIMD-isomaltose, hydrogen bonds linking atom O2 of Glc −1 to oxygen atom of Val-242 via a water molecule (red ball) are indicated as red dashed lines. B, comparison of the residues comprising tunnel-1 of unliganded AgIMD (green) and AgIMD-isomaltose (red and magenta). Red dashed line, hydrogen bond; red ball, water molecule. Arrows indicate conformational changes from the unliganded form to the ligand-bound form. The subsite numbers of isomaltose are labeled.

FIGURE 3.

Active site of AgIMD-panose. A, stereo view of the active sites of unliganded AgIMD (green), AgIMD-isomaltose (magenta), and AgIMD-panose (blue). B, two tunnels (tunnel-1 and tunnel-2) are found near the active site of AgIMD-panose. The routes of the tunnels calculated with the program Caver are shown in blue. Red balls inside the tunnels represent water molecules. The panose molecule (Glc +1, Glc −1, and Glc −2) as well as residues Sme243, His-245, and Phe-247 are indicated. C, schematic drawing of the amino acid residues interacting with panose in domain A of AgIMD-panose. White circle, oxygen atom; black circle, carbon atom; gray circle, nitrogen atom; circle with S, sulfur atom; dashed line, hydrogen bond. The subsite numbers are labeled. Phe-156 and Phe-198 are involved in hydrophobic interactions with panose.

FIGURE 4.

Comparison of domain A and subdomain B of AgIMD and related enzymes. A, stereo view of the Cα backbones of AgIMD-panose (pink and red) and hGAL (PDB code 3HG3; cyan and blue). In AgIMD, subdomain B, loop-1 and loop-2 are shown in red. In hGAL, subdomain B is shown in blue. The pyranose rings at subsite −1 alone are indicated by an arrow. B, stereo view of the superimposition of the Cα backbones of AgIMD-panose (magenta), TVAI-glucopentasaccharide (PDB code 2D0F; green), EcYicI-fluoroxylopyranosyl intermediate (PDB code 1XSK; blue), and LaMel36A-galactose (PDB code 2XN2; orange). The pyranose rings at subsite −1 alone are indicated by an arrow. C–E, comparison of the folds of β3-subdomain B-α3 of AgIMD (C), EcYicI (D), and TVAI (E). The Cα backbones are rainbow-colored from blue to red from the N to the C termini.

FIGURE 5.

Some key residues in the active sites of AgIMD and comparison with those of related enzymes. Stereo views of AgIMD-panose (A), hGAL-melibiose (PDB code 3HG3) (B), EcYicI-fluoroxylopyranosyl intermediate (PDB code 1XSK) (C), and LaMel36A-galactose (PDB code 2XN2) (D). The two catalytic Asp residues (Asp-197 and Asp-265 in AgIMD), residues at subsite −1 (Tyr-40 and Asp-77 in AgIMD), and other key residues are illustrated. Colors: magenta, catalytic Asp residues; cyan, residues at subsite −1; blue, residues at plus subsites; green, other key residues. The subsite numbers of bound saccharides are labeled.

FIGURE 6.

Stereo views of CBM35(AgIMD) and comparison with other CBM35 domains. A, comparison of the Cα backbones of CBM35(AgIMD)-isomaltose (magenta), CBM35(Lmo2446)-glucose (PDB code 4WKU; blue), and CBM35(BcCIT)-IG8 (PDB code 3WNN; orange). Arrows are as follows: black arrow, Glc-(a); red arrow, long loop comprising residues 502–509 in CBM35(AgIMD); blue arrow, calcium-binding site in CBM35(Lmo2446) and CBM35(BcCIT). B, sugar-binding site of CBM35(AgIMD). Colors and symbols are as follows: green, unliganded CBM35(AgIMD); magenta, CBM35(AgIMD)-isomaltose; red dashed line, hydrogen bond; red ball, water molecule. Arrows are as in A. C, sugar-binding site of CBM35(BcCIT). Colors are as follows: gray, residues conserved in CBM35(AgIMD); orange, residues not conserved in CBM35(AgIMD). Arrows are as in A. D, comparison of the Cα backbones of CBM35 and catalytic domains of AgIMD (magenta), Lmo2446 (blue), and BcCIT (orange). CBM35 domains are superimposed. E, surface model of AgIMD-panose. Colors are as in Fig. 1A. Arrows are as follows: blue arrow, catalytic cleft; black arrow, ligand-binding site in CBM35. The potential polysaccharide-binding cleft is shown in a red box.