RGD Reference Report - Molecular cloning and nucleotide sequence of cDNA encoding rat kidney long-chain L-2-hydroxy acid oxidase. Expression of the catalytically active recombinant protein as a chimaera. - Rat Genome Database

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Molecular cloning and nucleotide sequence of cDNA encoding rat kidney long-chain L-2-hydroxy acid oxidase. Expression of the catalytically active recombinant protein as a chimaera.

Authors: Belmouden, A  Le, KH  Lederer, F  Garchon, HJ 
Citation: Belmouden A, etal., Eur J Biochem 1993 May 15;214(1):17-25.
RGD ID: 70759
Pubmed: (View Article at PubMed) PMID:8508789

Long-chain L-alpha-hydroxy acid oxidase from rat kidney is a member of the family of FMN-dependent alpha-hydroxy-acid-oxidizing enzymes. With the knowledge of the recently determined amino acid sequence, the cDNA encoding the enzyme has now been cloned using the polymerase chain reaction. The 1648-bp cDNA contains an open reading frame coding for the 352 residues of the previously determined sequence, preceded by a methionine codon. In addition, several clones were found to present a nine-base insertion, predicting the existence of an isoform with a tripeptide VRK inserted between residues 188 and 189 of the mature protein. The presence of about 10% of this isoform in the oxidase purified from rat kidney was indeed identified by amino acid sequencing. A recombinant active enzyme was obtained as a protein fused to glutathione S-transferase using the bacterial expression plasmid pGEX-3X. Physico-chemical characterization indicated, for the fused enzyme, properties similar to those of the rat kidney protein. When the chimaera was submitted to factor Xa, proteolysis at the engineered cleavage point was poor. Separation of hydroxy acid oxidase from glutathione S-transferase could not be achieved with trypsin either. With both proteases, the initial cleavage point appeared to be in a peptide loop internal to the hydroxy acid oxidase sequence, close to or in the tripeptide insertion locus and not at the engineered factor-Xa-cleavage point. Comparative tryptic proteolysis of the rat kidney enzyme yielded a form cleaved in the same loop.

Annotation

Gene Ontology Annotations    

Biological Process

Molecular Function

Objects Annotated

Genes (Rattus norvegicus)
Hao2  (hydroxyacid oxidase 2)


Additional Information