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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publication date

  • July 1994

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.