title: Cloning and expression of Chromobacterium violaceum phenylalanine hydroxylase in Escherichia coli and comparison of amino acid sequence with mammalian aromatic amino acid hydroxylases.
abstract: The complete amino acid sequence (296 amino acids) of Chromobacterium violaceum phenylalanine hydroxylase (PAH) was determined by nucleotide analysis of a DNA clone isolated using both a synthetic oligonucleotide probe based on the NH2-terminal amino acid sequence and an antibody against this enzyme. The ApaL I fragment (approximately 1.9 kilobase pairs) containing the entire PAH gene was subcloned in pBluescript II and induced by isopropyl-beta-D-thiogalactopyranoside. In order to eliminate fusion proteins the XbaI/ClaI fragment which contained the PAH gene from the Bluescript construct was subcloned into pMAC 5-8 containing the TAC promoter. The recombinant protein reacts with antibody raised to authentic C. violaceum PAH and its NH2-terminal 20-amino acid sequence and COOH-terminal amino acid residue were identical with the wild-type protein. Key physical and chemical characteristics of the recombinant protein, i.e. its copper content and Michaelis-Menten parameters, were the same as wild-type. Comparison of amino acid sequences revealed a highly conserved region between C. violaceum PAH and three different mammalian aromatic amino acid hydroxylases. This conserved area may well be a catalytically important domain of these pterin- and metal-requiring aromatic amino acid hydroxylases. The over-expression of C. violaceum PAH in Escherichia coli will facilitate the analysis of the enzyme mechanism by various spectroscopic methods.
authors.affiliation: Department of Chemistry, Pennsylvania State University, University Park 16802.
authors.forename: A
authors.initials: A
authors.lastname: Onishi
authors.forename: L J
authors.initials: LJ
authors.lastname: Liotta
authors.forename: S J
authors.initials: SJ
authors.lastname: Benkovic
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002861:Chromobacterium / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010651:Phenylalanine Hydroxylase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D015183:Restriction Mapping; D016415:Sequence Alignment
chemical_list: D004269:DNA, Bacterial; D010651:Phenylalanine Hydroxylase