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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

Academic Article
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Overview

authors

  • Jia, S.
  • McGinnis, K.
  • Vandusen, W. J.
  • Burke, C. J.
  • Kuo, A.
  • Griffin, Patrick
  • Sardana, M. K.
  • Elliston, K. O.
  • Stern, A. M.
  • Friedman, P. A.

publication date

  • July 1994

journal

  • Proceedings of the National Academy of Sciences of the United States of America  Journal

abstract

  • The alpha-ketoglutarate-dependent dioxygenase aspartyl (asparaginyl) beta-hydroxylase (EC 1.14.11.16) 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.

subject areas

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

International Standard Serial Number (ISSN)

  • 0027-8424

Digital Object Identifier (DOI)

  • 10.1073/pnas.91.15.7227

PubMed ID

  • 8041771
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Additional Document Info

start page

  • 7227

end page

  • 7231

volume

  • 91

issue

  • 15

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