A fully active catalytic domain of bovine aspartyl (asparaginyl) beta-hydroxylase expressed in Escherichia coli: characterization and evidence for the identification of an active-site region in vertebrate alpha-ketoglutarate-dependent dioxygenases

Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):7227-31. doi: 10.1073/pnas.91.15.7227.


The alpha-ketoglutarate-dependent dioxygenase aspartyl (asparaginyl) beta-hydroxylase (EC specifically hydroxylates one aspartic or asparagine residue in certain epidermal growth factor-like domains of a number of proteins. The expression in Escherichia coli, purification, characterization of a fully active catalytic domain, and evidence for the identification of an active-site region of this enzyme are described. Sequence alignment analyses among the vertebrate alpha-ketoglutarate-dependent dioxygenases and chemical modification studies were undertaken aimed at locating specific regions of 52-kDa recombinant aspartyl (asparaginyl) beta-hydroxylase involved in substrate binding and/or catalysis. Based upon these studies, an alignment of the C-terminal regions of prolyl and lysyl hydroxylase and of aspartyl (asparaginyl) beta-hydroxylase is proposed. When histidine-675, an invariant residue located in a region of homology within this alignment, was mutated to an alanine residue in aspartyl (asparaginyl) beta-hydroxylase (H675A), no enzymatic activity was detected. Chemical modification studies show that the wild-type protein is protected from iodo[14C]acetamide labeling by Fe2+/alpha-ketoglutarate whereas the H675A mutant protein is not, suggesting that this mutant does not bind Fe2+/alpha-ketoglutarate.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Catalysis
  • Cattle
  • Cloning, Molecular
  • Escherichia coli
  • Humans
  • Mixed Function Oxygenases / chemistry
  • Mixed Function Oxygenases / genetics
  • Mixed Function Oxygenases / metabolism*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Sequence Homology, Amino Acid
  • Substrate Specificity
  • Vertebrates


  • Mixed Function Oxygenases
  • aspartic acid 2-oxoglutarate-dependent dioxygenase