HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Onishi, A. Articles by Benkovic, S. J. Search for Related Content PubMed PubMed Citation Articles by Onishi, A. Articles by Benkovic, S. J. Social Bookmarking                      What's this?

J. Biol. Chem., Vol. 266, Issue 28, 18454-18459, Oct, 1991

Cloning and expression of Chromobacterium violaceum phenylalanine hydroxylase in Escherichia coli and comparison of amino acid sequence with mammalian aromatic amino acid hydroxylases

A Onishi, LJ Liotta and SJ Benkovic
Department of Chemistry, Pennsylvania State University, University Park 16802.

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.
CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				J. Song, T. Xia, and R. A. Jensen PhhB, a Pseudomonas aeruginosa Homolog of Mammalian Pterin 4a-Carbinolamine Dehydratase/DCoH, Does Not Regulate Expression of Phenylalanine Hydroxylase at the Transcriptional Level J. Bacteriol., May 1, 1999; 181(9): 2789 - 2796. [Abstract] [Full Text]

				D. Chen and P. A. Frey Phenylalanine Hydroxylase from Chromobacterium violaceum. UNCOUPLED OXIDATION OF TETRAHYDROPTERIN AND THE ROLE OF IRON IN HYROXYLATION J. Biol. Chem., October 2, 1998; 273(40): 25594 - 25601. [Abstract] [Full Text] [PDF]