The active site topology of Aspergillus niger endopolygalacturonase II as studied by site-directed mutagenesis.
Strictly conserved charged residues among polygalacturonases (Asp-180, Asp-201, Asp-202, His-223, Arg-256, and Lys-258) were subjected to site-directed mutagenesis in Aspergillus niger endopolygalacturonase II. Specific activity, product progression, and kinetic parameters (K(m) and V(max)) were determined on polygalacturonic acid for the purified mutated enzymes, and bond cleavage frequencies on oligogalacturonates were calculated. Depending on their specific activity, the mutated endopolygalacturonases II were grouped into three classes. The mutant enzymes displayed bond cleavage frequencies on penta- and/or hexagalacturonate different from the wild type endopolygalacturonase II. Based on the biochemical characterization of endopolygalacturonase II mutants together with the three-dimensional structure of the wild type enzyme, we suggest that the mutated residues are involved in either primarily substrate binding (Arg-256 and Lys-258) or maintaining the proper ionization state of a catalytic residue (His-223). The individual roles of Asp-180, Asp-201, and Asp-202 in catalysis are discussed. The active site topology is different from the one commonly found in inverting glycosyl hydrolases.
Armand
S
S
Section Molecular Genetics of Industrial Microorganisms, Wageningen Agricultural University, Dreijenlaan 2, 6703 HA, Wageningen, The Netherlands.
Wagemaker
M J
MJ
Sánchez-Torres
P
P
Kester
H C
HC
van Santen
Y
Y
Dijkstra
B W
BW
Visser
J
J
Benen
J A
JA
D001234:Aspergillus niger / Q000201:enzymology*; D020134:Catalytic Domain / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000378:metabolism; D011096:Polygalacturonase / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D013499:Surface Properties
D009844:Oligosaccharides; D011994:Recombinant Proteins; C400187:endopolygalacturonase II; D011096:Polygalacturonase