Identification of active site carboxylic residues in Bacillus licheniformis 1,3-1,4-beta-D-glucan 4-glucanohydrolase by site-directed mutagenesis.
Active site residues of 1,3-1,4-beta-D-glucan 4-glucanohydrolase (EC 3.2.1.73) from Bacillus licheniformis have been identified by site-directed mutagenesis. Previous work revealed that Glu-134 was essential for enzymatic activity, and it was proposed as the catalytic nucleophile by affinity labeling of the highly homologous Bacillus amyloliquefaciens enzyme. To search for the general acid catalyst, the Asp and Glu residues conserved among the Bacillus isozymes have been mutated to Asn and Gln, respectively. Out of the 14 positions studied, only the E138Q mutation yielded an inactive enzyme, whereas the E134Q and D136N mutants retained less than 0.5% of the wild type activity. Based on the three-dimensional structure of a hybrid B. amyloliquefaciens-Bacillus macerans 1,3-1,4-beta-D-glucan 4-glucanohydrolase, Glu-134, Asp-136, and Glu-138 are the only carboxylic acid residues that are properly located into the active site cleft to participate in catalysis. Glu-138 appears as the most likely candidate to function as the general acid catalyst, while Asp-136 may affect the pK alpha of the catalytic residues.
Juncosa
M
M
Institut de Biologia Fonamental V. Villar Palasi, Universitat Autonoma de Barcelona, Spain.
Pons
J
J
Dot
T
T
Querol
E
E
Planas
A
A
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017931:DNA Primers; D004795:Enzyme Stability; D004926:Escherichia coli; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D017931:DNA Primers; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C028020:licheninase