Purification and Mechanism of Human Aldehyde Oxidase Expressed in Escherichia coli

被引：39

作者：

Alfaro, Joshua F. ^{[1
]}

Joswig-Jones, Carolyn A. ^{[1
]}

Ouyang, Wenyun ^{[1
]}

Nichols, Joseph ^{[1
]}

Crouch, Gregory J. ^{[1
]}

Jones, Jeffrey P. ^{[1
]}

机构：

[1] Washington State Univ, Dept Chem, Pullman, WA 99163 USA

来源：

DRUG METABOLISM AND DISPOSITION | 2009年 / 37卷 / 12期

基金：

美国国家卫生研究院;

关键词：

AMINO-ACID-RESIDUES; RATE-LIMITING STEP; XANTHINE-OXIDASE; MOLYBDENUM HYDROXYLASES; ACTIVE-SITE; GUINEA-PIG; SUBSTRATE-SPECIFICITY; ENZYMIC REACTIONS; IN-VITRO; LIVER;

D O I：

10.1124/dmd.109.029520

中图分类号：

R9 [药学];

学科分类号：

1007 ;

摘要：

Human aldehyde oxidase 1 (AOX1) has been subcloned into a vector suitable for expression in Escherichia coli, and the protein has been expressed. The resulting protein is active, with sulfur being incorporated in the molybdopterin cofactor. Expression levels are modest, but 1 liter of cells supplies enough protein for both biochemical and kinetic characterization. Partial purification is achieved by nickel affinity chromatography through the addition of six histidines to the amino-terminal end of the protein. Kinetic analysis, including kinetic isotope effects and comparison with xanthine oxidase, reveal similar mechanisms, with some subtle differences. This expression system will allow for the interrogation of human aldehyde oxidase structure/function relationships by site-directed mutagenesis and provide protein for characterizing the role of AOX1 in drug metabolism.

引用

页码：2393 / 2398

页数：6

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