Jia S, McGinnis K, VanDusen W J, Burke C J, Kuo A, Griffin P R, Sardana M K, Elliston K O, Stern A M, Friedman P A
Merck Research Laboratories, West Point, PA 19486.
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 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.
α-酮戊二酸依赖性双加氧酶天冬氨酰（天冬酰胺酰）β-羟化酶（EC 1.14.11.16）特异性地羟基化多种蛋白质某些表皮生长因子样结构域中的一个天冬氨酸或天冬酰胺残基。本文描述了该酶在大肠杆菌中的表达、纯化、全活性催化结构域的表征以及活性位点区域鉴定的证据。对脊椎动物α-酮戊二酸依赖性双加氧酶进行了序列比对分析，并开展了化学修饰研究，旨在定位参与底物结合和/或催化的52 kDa重组天冬氨酰（天冬酰胺酰）β-羟化酶的特定区域。基于这些研究，提出了脯氨酰羟化酶、赖氨酰羟化酶和天冬氨酰（天冬酰胺酰）β-羟化酶C端区域的比对。当位于该比对同源区域的不变残基组氨酸-675在天冬氨酰（天冬酰胺酰）β-羟化酶中突变为丙氨酸残基（H675A）时，未检测到酶活性。化学修饰研究表明，野生型蛋白可被Fe2+/α-酮戊二酸保护免受碘[14C]乙酰胺标记，而H675A突变蛋白则不能，这表明该突变体不结合Fe2+/α-酮戊二酸。
