| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Centre for Crystallographic Studies, Department of Chemistry, University of Copenhagen, DK-2100 Copenhagen, Denmark
2 NOVOZYMES A/S, Bagsværd, Denmark
Reprint requests to: Sine Larsen, Centre for Crystallographic Studies, University of Copenhagen, 5 Universitetsparken, DK-2100 Copenhagen, Denmark; e-mail:
sine{at}ccs.ki.ku.dk; fax: 45-3532-0299.
ß-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 ß-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 ~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 Å and 2.14 Å resolution, respectively. The structure of the H. insolens galactanase (HIGAL) was determined to 2.55 Å resolution. The thermostability of MTGAL and HIGAL correlates with increase in the protein rigidity and electrostatic interactions, stabilization of the 
-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.
Keywords: ß-1,4-galactanase; family 53 glycoside hydrolase; thermostability; pH optimum; clan GH-A; thermophile; alkalophile
Abbreviations: RG-I, rhamnogalacturonan I • AAGAL, Aspergillus aculeatus galactanase • MTGAL, Myceliophthora thermophila galactanase • HIGAL, Humicola insolens galactanase • PEG, polyethyleneglycol • HEPES, 4-(2-hydroxyethyl)-1-1-piperazineethane sulfonic acid • TRIS, tris (hydroxymethyl) aminomethane • GlcNac, 2-N-acetyl-ß-D-glucose • SC, Simmon’s citrate • AZCL, Azurine-Cross linked
CiteULike 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  J. Ni, M. Takehara, M. Miyazawa, and H. Watanabe Random exchanges of non-conserved amino acid residues among four parental termite cellulases by family shuffling improved thermostability Protein Eng. Des. Sel., November 1, 2007; 20(11): 535 - 542. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. M. Tynan-Connolly and J. E. Nielsen Redesigning protein pKa values Protein Sci., February 1, 2007; 16(2): 239 - 249. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. W. A. Hinz, M. I. Pastink, L. A. M. van den Broek, J.-P. Vincken, and A. G. J. Voragen Bifidobacterium longum Endogalactanase Liberates Galactotriose from Type I Galactans Appl. Envir. Microbiol., September 1, 2005; 71(9): 5501 - 5510. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
