Monofunctional chorismate mutase from Bacillus subtilis: purification of the protein, molecular cloning of the gene, and overexpression of the gene product in Escherichia coli.
The monofunctional chorismate mutase from Bacillus subtilis has been purified 2200-fold to homogeneity. The enzyme is a homodimer of subunit Mr = 14,500 and is the smallest natural chorismate mutase that has been characterized. The purified enzyme follows Michaelis-Menten kinetics with a Km of 100 microM and a kcat of 50 s-1, carries no other associated enzymic activities, and is unaffected by any of the aromatic amino acids. The N-terminal amino acid sequence of the protein has been determined, and this information has been used to construct a precise oligonucleotide probe for the gene by means of in vitro DNA amplification from total chromosomal DNA by the polymerase chain reaction. The cloned aroH gene encodes a protein of 127 amino acid residues and is expressed in Escherichia coli. The cloned gene product is indistinguishable from that purified from Bacillus. The aroH coding region was directly subcloned into a phagemid expression vector by means of the polymerase chain reaction. The resulting construct, with the aroH gene positioned behind efficient transcription and translation initiation sequences of E. coli, results in the production of the monofunctional mutase at levels of 30-35% of the soluble cell protein in E. coli transformants. Chorismate mutases comprise a set of functionally related proteins that show little sequence similarity to each other. This diversity stands in contrast to other chorismate-utilizing enzymes.
Gray
J V
JV
Department of Chemistry, Harvard University, Cambridge, Massachusetts 02138.
Golinelli-Pimpaneau
B
B
Knowles
J R
JR
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D001483:Base Sequence; D002826:Chorismate Mutase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D007535:Isomerases / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012689:Sequence Homology, Nucleic Acid; D014169:Transformation, Bacterial
D015345:Oligonucleotide Probes; D007535:Isomerases; D002826:Chorismate Mutase