A novel phenylserine dehydratase from Pseudomonas pickettii PS22: purification, characterization, and sequence of its phosphopyridoxyl peptide.
A novel phenylserine dehydratase [EC 4.2.1.-], which catalyzes the deamination of L-threo-3-phenylserine to yield phenylpyruvate and ammonia, was purified to homogeneity from a crude extract of Pseudomonas pickettii PS22 isolated from soil. The enzyme was a monomer having a molecular mass of about 38 kDa and contained 1 mol of pyridoxal 5'-phosphate per mol of enzyme. The enzyme exhibited absorption maxima at 279 and 416 nm. No appreciable spectral change was observed over the pH range of 6.0 to 8.0. The maximal reactivity was obtained at about pH 7.5. The enzyme was highly specific for L-threo-3-phenylserine (Km, 0.21 mM). L-erythro-3-Phenylserine, L-threonine, L-serine, and D-serine were inert. The enzyme was inhibited by phenylhydrazine, hydroxylamine, p-chloromercuribenzoate, and HgCl2, but not by L-isoleucine, L-threonine, or L-serine. AMP, ADP, and ATP did not affect the enzyme activity. The N-terminal amino acid sequence was not similar to those of biosynthetic and biodegradative L-threonine dehydratases and L-serine dehydratases. The isolated tryptic phosphopyridoxyl peptide, however, contained a pyridoxal 5'-phosphate-binding consensus amino acid sequence of amino acid dehydratases.
Okuda
H
H
Department of Bioresources Science, Kochi University.
Nagata
S
S
Misono
H
H
D000595:Amino Acid Sequence; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006836:Hydro-Lyases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D011549:Pseudomonas / Q000201:enzymology*; D011732:Pyridoxal Phosphate / Q000032:analysis; D017421:Sequence Analysis; D012694:Serine / Q000031:analogs & derivatives / Q000378:metabolism; D013379:Substrate Specificity; D014357:Trypsin
D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D012694:Serine; D011732:Pyridoxal Phosphate; D014357:Trypsin; C101394:3-phenylserine dehydratase; D006836:Hydro-Lyases