Cloning, nucleotide sequence, and expression of a p-hydroxybenzoate hydroxylase isozyme gene from Pseudomonas fluorescens.
A gene encoding for a putative isozyme of p-hydroxy-benzoate hydroxylase (PHBH) has been isolated from Pseudomonas fluorescens (ATCC 13525). A comparison of the translated amino acid sequence with that of the known PHBH from P. fluorescens revealed that the new enzyme contains 3 additional amino acids and has 73% absolute homology to the previously known enzyme; conservation of secondary and active-site structures implied that the isozyme and known enzyme share the same general tertiary structure. Subsequent expression of the isozyme in Escherichia coli produced an enzyme with a specific activity about half that of the previously characterized PHBHs from P. fluorescens and Pseudomonas aeruginosa; in addition, somewhat weaker binding affinities for both NADPH and p-hydroxybenzoate were observed. Speculations are made on the reason for the existence of the isozyme, which does not appear to be expressed routinely in P. fluorescens.
Shuman
B
B
Department of Chemistry, University of California, Irvine 92717.
Dix
T A
TA
D000595:Amino Acid Sequence; D000693:Anaerobiosis; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011551:Pseudomonas fluorescens / Q000201:enzymology / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
D004269:DNA, Bacterial; D007527:Isoenzymes; D006899:Mixed Function Oxygenases; C021385:3-hydroxybenzoate-4-monooxygenase