Structural characterization of glucooligosaccharide oxidase from Acremonium strictum.
Glucooligosaccharide oxidase from Acremonium strictum was screened for potential applications in oligosaccharide acid production and carbohydrate detection. This protein is a unique covalent flavoenzyme which catalyzes the oxidation of a variety of carbohydrates with high selectivity for cello- and maltooligosaccharides. Kinetic measurements suggested that this enzyme possesses an open carbohydrate-binding groove, which is mainly composed of two glucosyl-binding subsites. The encoding gene was subsequently cloned, and one intron was detected in the genomic DNA. Large amounts of active enzymes were expressed in Pichia pastoris, with a yield of 300 mg per liter medium. The protein was predicted to share structural homology with plant cytokinin dehydrogenase and related flavoproteins that share a conserved flavin adenine dinucleotide (FAD)-binding domain. The closest sequence matches are those of plant berberine bridge enzyme-like proteins, particularly the characteristic flavinylation site. Unexpectedly, mutation of the putative FAD-attaching residue, H70, to alanine, serine, cysteine, and tyrosine did not abolish the covalent FAD linkage and had little effect on the Km. Instead, the variants displayed kcat values that were 50- to 600-fold lower, indicating that H70 is crucial for efficient redox catalysis, perhaps through modulation of the oxidative power of the flavin.
Lee
Meng-Hwan
MH
Institute of Biochemistry, National Yang-Ming University, 155, Sec. 2, Li-Nong St., Pei-Tou, Taipei 11221, Taiwan.
Lai
Wen-Lin
WL
Lin
Shuen-Fuh
SF
Hsu
Cheng-Sheng
CS
Liaw
Shwu-Huey
SH
Tsai
Ying-Chieh
YC
D000164:Acremonium / Q000201:enzymology*; D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D017931:DNA Primers; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D016385:TATA Box
D017931:DNA Primers; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; C072250:glucooligosaccharide oxidase