Cloning and expression of fumarylacetoacetate hydrolase derived from marine yeast Rhodosporidium diobovatum.

Abstract

Fumarylacetoacetate hydrolase (FAH) catalyzes the hydrolytic cleavage of a carbon-carbon bond in fumarylacetoacetate to yield acetoacetate and fumarate as the final step in tyrosine degradation. In this study, the FAH genomic DNA and cDNA of Rhodosporidium. diobovatum were cloned by using the methods of degenerate polymerase chain reaction and RACE method. The results showed that the gene contained six exons, and that the encoded polypeptide held a sequence of 308 amino acid residues, containing a 75 residue N-terminal prosequence, a 216 residue proteinase domain, and a 17 residue C-terminal domain. Then, the expression vectors pET28a-FAH were constructed and expressed in Escherichia coli. The enzymatic activity of FAH protein was determined by monitoring the decrease in optical density at 330 nm of the substrate fumarylacetoacetate. The values for Km and Vmax of the purified enzyme was 1.4 µM and 2.3 µmol · min(-1) · mg(-1), respectively.