Cloning and expression of Chromobacterium violaceum phenylalanine hydroxylase in Escherichia coli and comparison of amino acid sequence with mammalian aromatic amino acid hydroxylases.
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.
Onishi
A
A
Department of Chemistry, Pennsylvania State University, University Park 16802.
Liotta
L J
LJ
Benkovic
S J
SJ
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
D004269:DNA, Bacterial; D010651:Phenylalanine Hydroxylase