Sequencing and expression of the L-phenylalanine oxidase gene from Pseudomonas sp. P-501. Proteolytic activation of the proenzyme.
The nucleotide sequence encoding L-phenylalanine oxidase (deaminating and decarboxylating) (PAO) from Pseudomonas sp. P-501 was determined. The open reading frame is arranged in the order of prosequence, alpha subunit, dipeptide and beta subunit from the 5'- to 3'-end. Expression of the gene in Escherichia coli showed that without the prosequence, PAO is produced in small quantity as a soluble form with no visible absorption, but with the prosequence (proPAO), PAO is highly expressed and yellow. The purified proPAO contained one mol of FAD per mol of proPAO polypeptide, but had no catalytic activity. Treatment of proPAO with a mixture of Pronase and trypsin converted the noncatalytic proPAO to the catalytic form, and the Pronase-trypsin-treated proPAO showed kinetic and spectral properties comparable to the native enzyme. These results suggest that in Pseudomonas, PAO is expressed as a proenzyme that is processed by proteolysis to the active form.
Suzuki
Haruo
H
Division of Biosciences, Graduate School of Fundamental Life Science, School of Science, Kitasato University, Kitasato 1-15-1, Sagamihara, Kanagawa 228-8555, Japan. suzuki@sci.kitasato-u.ac.jp
Higashi
Yuhki
Y
Asano
Mai
M
Suguro
Masaya
M
Kigawa
Masayuki
M
Maeda
Masahiro
M
Katayama
Satoshi
S
Mukouyama
Etsuko B
EB
Uchiyama
Koji
K
D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
D004792:Enzyme Precursors; D000594:Amino Acid Oxidoreductases; C022342:phenylalanine oxidase