p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens. 1. Completion of the elucidation of the primary structure.
As a final step in the elucidation of the primary structure of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, the amino acid sequences of a CNBr peptide (CB1, positions 111-276), that accounts for the middle part of the sequence, and the C-terminal CNBr peptide (CB2, positions 277-394) from the enzyme were determined. Important sequence information was obtained from two subfragments that were formed by the cleavage with CNBr of the Met-Thr sequence (positions 346-347) in peptide CB2. The alignment of the two subfragments from peptide CB2 and three one-residue overlaps between peptides from one of these subfragments were confirmed by investigation of well-resolved parts of a 0.25-nm electron-density map. The sequence of residues 343-346 could not be determined with chemical methods and was assigned from the size and shape of the amino acids in the electron-density map. An important tool in the analysis of the amino acid sequence of peptide CB1 was the proteinase Lys-C from Lysobacter enzymogenes, which preferentially cleaves at lysine residues.
D015109:4-Hydroxybenzoate-3-Monooxygenase / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D055598:Chemical Phenomena; D002621:Chemistry; D002918:Chymotrypsin; D006899:Mixed Function Oxygenases / Q000302:isolation & purification*; D010446:Peptide Fragments / Q000302:isolation & purification; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D014357:Trypsin
D010446:Peptide Fragments; D006899:Mixed Function Oxygenases; D015109:4-Hydroxybenzoate-3-Monooxygenase; D002918:Chymotrypsin; D014357:Trypsin