Trp17 and Glu20 residues in conserved WMN(D/E)PN motif are essential for Aspergillus ficuum endoinulinase (EC 3.2.1.7) activity.
The importance of the WMN(D/E)PN motif, which is well conserved among beta-fructofuranosidases grouped in the glycosylhydrolase family 32, in Aspergillus ficuum endoinulinase was accessed. Each mutant enzyme generated by site-directed mutagenesis of Trp17 in the conserved motif to Gln, Leu, Ser, Pro, Thr, or Met had an activity of less than 1% of the wild type. Another mutant enzyme obtained by mutation of Glu20 in the motif to Ser, Leu, Thr, Gln, Ala, or Val had an enzyme activity of less than 1% of the wild type. Furthermore, the E20D mutant enzyme, in which Glu20 in the conserved motif was replaced with Asp, had 1.1% of the wild type activity. These results clearly indicated that Trp17 and Glu20 are essential for the enzyme activity.
Park
Seockyu
S
Division of Biological Sciences and Research Center for Industrial Development of Biofood Materials, Chonbuk National University, Chonju, Chonbuk 561-756, Republic of Korea.
Han
Yeonsoo
Y
Kim
Heeun
H
Song
Songyi
S
Uhm
Tai-Boong
TB
Chae
Keon-Sang
KS
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001230:Aspergillus / Q000201:enzymology*; D017124:Conserved Sequence; D017931:DNA Primers / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics*; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D014364:Tryptophan / Q000235:genetics*
D017931:DNA Primers; D011994:Recombinant Proteins; D018698:Glutamic Acid; D014364:Tryptophan; D006026:Glycoside Hydrolases; C023086:inulinase