Isoforms of human O-GlcNAcase show distinct catalytic efficiencies

O-GlcNAcase (OGA) is a family 84 glycoside hydrolase catalyzing the hydrolytic cleavage of O-linked β-N-acetylglucosamine (O-GlcNAc) from serine and threonine residues of proteins. Thus far, three forms of OGA have been identified in humans. Here we optimized the expression of these isoforms in E. c...

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Bibliographic Details
Published in:Biochemistry (Moscow) 2010-07, Vol.75 (7), p.938-943
Main Authors: Li, Jing, Huang, Cai-luan, Zhang, Lian-wen, Lin, Lin, Li, Zhong-hua, Zhang, Fu-wu, Wang, Peng
Format: Article
Language:English
Subjects:
Amino Acid Motifs
Amino Acid Sequence
Amino acids
Antigens, Neoplasm - chemistry
Antigens, Neoplasm - genetics
Antigens, Neoplasm - metabolism
Biocatalysis
Biochemistry
Biomedical and Life Sciences
Biomedicine
Bioorganic Chemistry
Catalysis
Catalytic Domain
E coli
Enzyme Stability
Enzymes
Escherichia coli
Gene expression
Histone Acetyltransferases - chemistry
Histone Acetyltransferases - genetics
Histone Acetyltransferases - metabolism
Humans
Hyaluronoglucosaminidase - chemistry
Hyaluronoglucosaminidase - genetics
Hyaluronoglucosaminidase - metabolism
Hydrolases
Isoenzymes - chemistry
Isoenzymes - genetics
Isoenzymes - metabolism
Kinetics
Life Sciences
Microbiology
Molecular Sequence Data
Proteins
Substrate Specificity
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Summary:O-GlcNAcase (OGA) is a family 84 glycoside hydrolase catalyzing the hydrolytic cleavage of O-linked β-N-acetylglucosamine (O-GlcNAc) from serine and threonine residues of proteins. Thus far, three forms of OGA have been identified in humans. Here we optimized the expression of these isoforms in E. coli and characterized their kinetic properties. Using Geno 3D, we predicted that N-terminal amino acids 63-342 form the catalytic site for O-GlcNAc removal and characterized it. Large differences are observed in the K m value and catalytic efficiency (k cat/K m) for the three OGA variants, though all of them displayed O-GlcNAc hydrolase activity. The full-length OGA had the lowest K m value of 0.26 mM and the highest catalytic efficiency of 3.51·10³. These results reveal that the N-terminal region (a.a. 1-350) of OGA contains the catalytic site for glycoside hydrolase and the C-terminal region of the coding sequence has the ability to stabilize the native three-dimensional structure and further affect substrate affinity.
ISSN:0006-2979
1608-3040
DOI:10.1134/s0006297910070175