Release Time:2019-03-13  Hits:

Indexed by: Journal Article

Date of Publication: 2016-02-01

Journal: BIOSCIENCE TRENDS

Included Journals: Scopus、SCIE、PubMed

Volume: 10

Issue: 1

Page Number: 79-84

ISSN: 1881-7815

Key Words: Aspartate aminotransferase (AspAT); plasma membrane fatty acid binding protein (FABPpm); kynurenine aminotransferase-IV; crystallization; moonlighting protein

Abstract: Mitochondrial aspartate aminotransferase (mAspAT) was recognized as a moonlighting enzyme because it has not only aminotransferase activity but also a high-affinity long-chain fatty acids (LCFA) binding site. This enzyme plays a key role in amino acid metabolism, biosynthesis of kynurenic acid and transport of the LCFA. Therefore, it is important to study the structure-function relationships of human mAspAT protein. In this work, the mature form of human mAspAT was expressed to a high level in Escherichia coli periplasmic space using pET-22b vector, purified by a combination of immobilized metal-affinity chromatography and cation exchange chromatography. Optimal activity of the enzyme occurred at a temperature of 47.5 degrees C and a pH of 8.5. Crystals of human mAspAT were grown using the hanging-drop vapour diffusion method at 277K with 0.1 M HEPES pH 6.8 and 25%(v/v) Jeffamine (R) ED-2001 pH 6.8. The crystals diffracted to 2.99 angstrom and belonged to the space group P1 with the unit-cell parameters a = 56.7, b = 76.1, c = 94.2 angstrom, alpha = 78.0, beta = 85.6, gamma = 78.4 degrees. Elucidation of mAspAT structure can provide a molecular basis towards understanding catalysis mechanism and substrate binding site of enzyme.