Molecular cloning and analysis of the genes encoding the 4-hydroxyphenylacetate hydroxylase from Klebsiella pneumoniae.
The Klebsiella pneumoniae genes encoding the hydroxylase involved in the meta-cleavage pathway of 4-hydroxyphenylacetic acid (4-HPA) were cloned, and the DNA fragment from the region essential for hydroxylase activity was sequenced. K. pneumoniae 4-HPA hydroxylase was composed of two proteins (HpaA and HpaH) with different molecular masses. HpaA seems to be a flavin-containing hydroxylase with a molecular mass of 58,781 Da. HpaH, with a molecular mass of 18,680 Da, seems to be a "helper" protein required for productive hydroxylation of the substrate. The hpa genes were expressed and the hydroxylase was active in Escherichia coli. Comparison of the enzyme with other monooxygenases indicates that K. pneumoniae 4-HPA hydroxylase is a member of a new family of hydroxylases.
Gibello
A
A
Departamento de Microbiología (Patología Animal I), Facultad de Veterinaria, Universidad Complutense, Avenida Puerta de Hierro s/n, 28040-Madrid, Spain. gibelloa@eucmax.sim.ucm.es
Suárez
M
M
Allende
J L
JL
Martín
M
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D006900:Hydroxylation; D007711:Klebsiella pneumoniae / Q000235:genetics; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010648:Phenylacetates / Q000378:metabolism; D010957:Plasmids; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D001426:Bacterial Proteins; D010648:Phenylacetates; C008070:4-hydroxyphenylacetic acid; D006899:Mixed Function Oxygenases; C026252:4-hydroxyphenylacetate 3-monooxygenase