5AX8

Recombinant expression, purification and preliminary crystallographic studies of the mature form of human mitochondrial aspartate aminotransferase

Summary for 5AX8

• Entry DOI: 10.2210/pdb5ax8/pdb
• Related: 3PDB
• Descriptor: Aspartate aminotransferase, mitochondrial (1 entity in total)
• Functional Keywords: aspartate aminotransferase, plasma membrane fatty acid binding protein, kynurenine aminotransferase-iv, three-dimensional structure, transferase
• Biological source: Homo sapiens (Human)
• Cellular location: Mitochondrion matrix : P00505
• Total number of polymer chains: 4
• Total formula weight: 182332.34

Authors

Jiang, X.,Wang, J.,Chang, H.,Zhou, Y. (deposition date: 2015-07-20, release date: 2016-03-16, Last modification date: 2023-11-08)

Primary citation

Jiang, X.,Wang, J.,Chang, H.,Zhou, Y.
Recombinant expression, purification and crystallographic studies of the mature form of human mitochondrial aspartate aminotransferase
Biosci Trends, 10:79-84, 2016

PubMed 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 47.5ºC 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(®) ED-2001 pH 6.8. The crystals diffracted to 2.99 Å and belonged to the space group P1 with the unit-cell parameters a =56.7, b = 76.1, c = 94.2 Å, α =78.0, β =85.6, γ = 78.4º. Elucidation of mAspAT structure can provide a molecular basis towards understanding catalysis mechanism and substrate binding site of enzyme.

PubMed: 26902786
DOI: 10.5582/bst.2015.01150

Experimental method

X-RAY DIFFRACTION (2.989 Å)