Structure of two fungal beta-1,4-galactanases: searching for the basis for temperature and pH optimum

Abstract

beta-1,4-Galactanases hydrolyze the galactan side chains that are part of the complex carbohydrate structure of the pectin. They are assigned to family 53 of the glycoside hydrolases and display significant variations in their pH and temperature optimum and stability. Two fungal beta-1,4-galactanases from Myceliophthora thermophila and Humicola insolens have been cloned and heterologously expressed, and the crystal structures of the gene products were determined. The structures are compared to the previously only known family 53 structure of the galactanase from Aspergillus aculeatus (AAGAL) showing approximately 56% identity. The M. thermophila and H. insolens galactanases are thermophilic enzymes and are most active at neutral to basic pH, whereas AAGAL is mesophilic and most active at acidic pH. The structure of the M. thermophila galactanase (MTGAL) was determined from crystals obtained with HEPES and TRIS buffers to 1.88 A and 2.14 A resolution, respectively. The structure of the H. insolens galactanase (HIGAL) was determined to 2.55 A resolution. The thermostability of MTGAL and HIGAL correlates with increase in the protein rigidity and electrostatic interactions, stabilization of the alpha-helices, and a tighter packing. An inspection of the active sites in the three enzymes identifies several amino acid substitutions that could explain the variation in pH optimum. Examination of the activity as a function of pH for the D182N mutant of AAGAL and the A90S/ H91D mutant of MTGAL showed that the difference in pH optimum between AAGAL and MTGAL is at least partially associated with differences in the nature of residues at positions 182, 90, and/or 91.

Figure 1.

Illustration of the overall fold of HIGAL. Made with Swiss PDB-Viewer (Guex and Peitsch 1997).

Figure 2.

Close-up of a TRIS molecule in the MTGAL active site (molecule A). Produced with Swiss PDB-Viewer (Guex and Peitsch 1997).

Figure 3.

Structure-based sequence alignment of MTGAL, HIGAL, and AAGAL. Created using Indonesia (D. Madsen and G. Kleywegt, in prep.). Shading indicates the degree of conservation.

Figure 4.

Comparison of α-helix stabilization in MTGAL, HIGAL, and AAGAL. Stabilized/destabilized helices were classified from the observation of favorable or unfavorable interactions between the α-helix dipole, the three first N-terminal residues, and the three last C-terminal residues as in Teixeira et al. (2001). Positively charged residues (Lys, Arg, His) were considered favorable at the C-terminus and unfavorable at the N-terminus. For negatively charged residues (Asp, Glu), the opposite was true. Helices where favorable interactions outnumbered unfavorable interactions were considered stabilized. Where unfavorable interactions outnumbered the favorable ones, the helix was considered destabilized. Only barrel helices were considered.

Figure 5.

Superposition of the MTGAL (in red) and AAGAL (in green) active sites. The possible position of Asp 181 at pH 4.0 is represented by dashed lines. Produced with Swiss PDB-Viewer (Guex and Peitsch 1997).

Figure 6.

(A) pH activity profile of native and D182N-substituted AAGAL. (B) pH activity profile of native and A90S/H91D-substituted MTGAL.