N-Acetyl-D-glucosamine 2-epimerase from Anabaena sp. CH1 contains a novel ATP-binding site required for catalytic activity

Hui Fen Liao, Chao Hung Kao, Wei De Lin, Nai Wan Hsiao, Wen Hwei Hsu, Yen Chung Lee

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

ATP is required as a structural activator for the reversible epimerization of N-acetyl-d-glucosamine to N-acetyl-d-mannosamine by N-acetyl-d-glucosamine 2-epimerase (AGE); however, the ATP-binding site on AGE has not been clearly identified. This study aimed to investigate the specific region of Anabaena sp. CH1 AGE (bAGE) that is required for ATP binding. In the absence of ATP, tryptic digest of bAGE resulted in the production of 2 segments of 17 and 26 kDa, while in the presence of 1 mM ATP, the enzyme was resistant to trypsin. ADP also displayed protective effects against trypsin digestion. A trypsin-mediated ATP-footprinting assay identified a deviant ATP-protected region, 156-GKYTK-160, which is located within the flexible loop of bAGE. Site-directed mutagenesis of residues in the loop region was performed, and both K151A and K160A variants greatly decreased the enzymatic activity as well as the ATP-binding ability of bAGE, indicating that residues K151 and K160 may be critical for ATP binding. This study demonstrated that the ATP-binding site (151-KDNPKGKYTK-160) of bAGE was a novel rather than a classical Walker motif A. This is the first ATP-binding site reported for AGEs.

Original languageEnglish
Pages (from-to)948-952
Number of pages5
JournalProcess Biochemistry
Volume47
Issue number6
DOIs
Publication statusPublished - 2012 Jun 1

Fingerprint

Glucosamine
Anabaena
Adenosinetriphosphate
Binding sites
Catalyst activity
Adenosine Triphosphate
Binding Sites
Racemases and Epimerases
Trypsin
N-acyl-D-glucosamine 2-epimerase
Administrative data processing
Mutagenesis
Site-Directed Mutagenesis
Adenosine Diphosphate
Digestion
Assays
Enzymes

All Science Journal Classification (ASJC) codes

  • Bioengineering
  • Biochemistry
  • Applied Microbiology and Biotechnology

Cite this

Liao, Hui Fen ; Kao, Chao Hung ; Lin, Wei De ; Hsiao, Nai Wan ; Hsu, Wen Hwei ; Lee, Yen Chung. / N-Acetyl-D-glucosamine 2-epimerase from Anabaena sp. CH1 contains a novel ATP-binding site required for catalytic activity. In: Process Biochemistry. 2012 ; Vol. 47, No. 6. pp. 948-952.
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N-Acetyl-D-glucosamine 2-epimerase from Anabaena sp. CH1 contains a novel ATP-binding site required for catalytic activity. / Liao, Hui Fen; Kao, Chao Hung; Lin, Wei De; Hsiao, Nai Wan; Hsu, Wen Hwei; Lee, Yen Chung.

In: Process Biochemistry, Vol. 47, No. 6, 01.06.2012, p. 948-952.

Research output: Contribution to journalArticle

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