title: 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.
authors.affiliation: Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Key Discipline of Biological Engineering of Hebei Province, College of Life Sciences, Hebei University, Baoding, 071002, China.
authors.forename: Yuxuan
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Key Discipline of Biological Engineering of Hebei Province, College of Life Sciences, Hebei University, Baoding, 071002, China.
authors.forename: Weiwei
authors.initials: W
authors.lastname: Xia
authors.affiliation: Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Key Discipline of Biological Engineering of Hebei Province, College of Life Sciences, Hebei University, Baoding, 071002, China.
authors.forename: Pucheng
authors.initials: P
authors.lastname: Yang
authors.affiliation: Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Key Discipline of Biological Engineering of Hebei Province, College of Life Sciences, Hebei University, Baoding, 071002, China.
authors.forename: Shuo
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Key Discipline of Biological Engineering of Hebei Province, College of Life Sciences, Hebei University, Baoding, 071002, China.
authors.forename: Zhihui
authors.initials: Z
authors.lastname: Shi
authors.affiliation: Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Key Discipline of Biological Engineering of Hebei Province, College of Life Sciences, Hebei University, Baoding, 071002, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Tang
authors.affiliation: Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Key Discipline of Biological Engineering of Hebei Province, College of Life Sciences, Hebei University, Baoding, 071002, China.
authors.forename: Liping
authors.initials: L
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001487:Basidiomycota / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000235:genetics; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data
chemical_list: D004247:DNA; D006867:Hydrolases; C020156:fumarylacetoacetase
keywords: Fumarylacetoacetate hydrolase; Marine yeast; Rhodosporidium diobovatum