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

The alpha-ketoglutarate-dependent dioxygenase aspartyl (asparaginyl) b eta-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, purifica tion, characterization of a fully active catalytic domain, and evidenc e for the identification of an active-site region of this enzyme are d escribed. Sequence alignment analyses among the vertebrate alpha-ketog lutarate-dependent dioxygenases and chemical modification studies were undertaken aimed at locating specific regions of 52-kDa recombinant a spartyl (asparaginyl) beta-hydroxylase involved in substrate binding a nd/or catalysis. Based upon these studies, an alignment of the C-termi nal regions of prolyl and lysyl hydroxylase and of aspartyl (asparagin yl) beta-hydroxylase is proposed. When histidine-675, an invariant res idue located in a region of homology within this alignment, was mutate d to an alanine residue in aspartyl (asparaginyl) beta-hydroxylase (H6 75A), no enzymatic activity was detected. Chemical modification studie s show that the wild-type protein is protected from iodo[C-14]acetamid e labeling by Fe2+/alpha-ketoglutarate whereas the H675A mutant protei n tein is not, suggesting that this mutant does not bind Fe2+/alpha-ke toglutarate.