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

被引：51

作者：

JIA, S ^{[1
]}

MCGINNIS, K ^{[1
]}

VANDUSEN, WJ ^{[1
]}

BURKE, CJ ^{[1
]}

KUO, A ^{[1
]}

GRIFFIN, PR ^{[1
]}

SARDANA, MK ^{[1
]}

ELLISTON, KO ^{[1
]}

STERN, AM ^{[1
]}

FRIEDMAN, PA ^{[1
]}

机构：

[1] MERCK SHARP & DOHME LTD,RES LABS,W POINT,PA 19486

来源：

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA | 1994年 / 91卷 / 15期

关键词：

D O I：

10.1073/pnas.91.15.7227

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

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[C-14]acetamide labeling by Fe2+/alpha-ketoglutarate whereas the H675A mutant protein tein is not, suggesting that this mutant does not bind Fe2+/alpha-ketoglutarate.

引用

页码：7227 / 7231

页数：5

共 31 条

[1] ATG VECTORS FOR REGULATED HIGH-LEVEL EXPRESSION OF CLONED GENES IN ESCHERICHIA-COLI [J].