Cloning, sequencing, and expression of the P-protein gene (pheA) of Pseudomonas stutzeri in Escherichia coli: implications for evolutionary relationships in phenylalanine biosynthesis.
The pheA gene encoding the bifunctional P-protein (chorismate mutase:prephenate dehydratase) was cloned from Pseudomonas stutzeri and sequenced. This is the first gene of phenylalanine biosynthesis to be cloned and sequenced from Pseudomonas. The pheA gene was expressed in Escherichia coli, allowing complementation of an E. coli pheA auxotroph. The enzymic and physical properties of the P-protein from a recombinant E. coli auxotroph expressing the pheA gene were identical to those of the native enzyme from P. stutzeri. The nucleotide sequence of the P. stutzeri pheA gene was 1095 base pairs in length, predicting a 365-residue protein product with an Mr of 40,844. Codon usage in the P. stutzeri pheA gene was similar to that of Pseudomonas aeruginosa but unusual in that cytosine and guanine were used at nearly equal frequencies in the third codon position. The deduced P-protein product showed sequence homology with peptide sequences of the E. coli P-protein, the N-terminal portion of the E. coli T-protein (chorismate mutase:prephenate dehydrogenase), and the monofunctional prephenate dehydratases of Bacillus subtilis and Corynebacterium glutamicum. A narrow range of values (26-35%) for amino acid matches revealed by pairwise alignments of monofunctional and bifunctional proteins possessing activity for prephenate dehydratase suggests that extensive divergence has occurred between even the nearest phylogenetic lineages.
Department of Microbiology and Cell Science, University of Florida, Gainesville 32611.
R S
RS
Fischer
G
G
Zhao
R A
RA
Jensen
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D005075:Biological Evolution; D002826:Chorismate Mutase / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism; D010649:Phenylalanine / Q000096:biosynthesis*; D011302:Prephenate Dehydratase / Q000235:genetics* / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics*; D016415:Sequence Alignment
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; C071216:P-protein, E coli; D010649:Phenylalanine; D011302:Prephenate Dehydratase; D002826:Chorismate Mutase