The N-terminal extension is essential for the formation of the active dimeric structure of liver peroxisomal alanine:glyoxylate aminotransferase

Autor: Christopher J. Danpure, Jackie Lewin, Carla Borri Voltattorni, Barbara Cellini, Sonia Fargue, Carlotta Zamparelli, Riccardo Montioli
Rok vydání: 2012
Předmět:
Protein Folding
Protein Conformation
Protein subunit
CHO Cells
Protein targeting
Biology
Protein aggregation
medicine.disease_cause
Biochemistry
Mice
chemistry.chemical_compound
Cricetinae
medicine
Alanine:glyoxylate aminotransferase
Pyridoxal phosphate
N-terminal extension
Intra-peroxisomal aggregates
Animals
Humans
Cloning
Molecular
Transaminases
Sequence Deletion
Alanine
chemistry.chemical_classification
Cell Biology
Peroxisome
Peptide Fragments
alanine:glyoxylate aminotransferase
intra-peroxisomal aggregates
n-terminal extension
protein aggregation
protein targeting
pyridoxal phosphate
Amino acid
Enzyme
Liver
chemistry
Hyperoxaluria
Primary
Protein Multimerization
Alanine:glyoxylate aminotransferase
Pyridoxal phosphate
N-terminal extension
Protein aggregation
Intra-peroxisomal aggregates
Protein targeting
Zdroj: The International Journal of Biochemistry & Cell Biology. 44:536-546
ISSN: 1357-2725
DOI: 10.1016/j.biocel.2011.12.007
Popis: Alanine:glyoxylate aminotransferase (AGT) is a pyridoxal-phosphate (PLP)-dependent enzyme. Its deficiency causes the hereditary kidney stone disease primary hyperoxaluria type 1. AGT is a highly stable compact dimer and the first 21 residues of each subunit form an extension which wraps over the surface of the neighboring subunit. Naturally occurring and artificial amino acid replacements in this extension create changes in the functional properties of AGT in mammalian cells, including relocation of the enzyme from peroxisomes to mitochondria. In order to elucidate the structural and functional role of this N-terminal extension, we have analyzed the consequences of its removal using a variety of biochemical and cell biological methods. When expressed in Escherichia coli, the N-terminal deleted form of AGT showed the presence of the protein but in an insoluble form resulting in only a 10% soluble yield as compared to the full-length version. The purified soluble fraction showed reduced affinity for PLP and greatly reduced catalytic activity. Although maintaining a dimer form, it was highly prone to self-aggregation. When expressed in a mammalian cell line, the truncated construct was normally targeted to peroxisomes, where it formed large stable but catalytically inactive aggregates. These results suggest that the N-terminal extension plays an essential role in allowing AGT to attain its correct conformation and functional activity. The precise mechanism of this effect is still under investigation.
Databáze: OpenAIRE
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