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...

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Bibliographic Details
Published inArchives of microbiology Vol. 167; no. 2-3; p. 160
Main Authors Gibello, A, Suárez, M, Allende, J L, Martín, M
Format Journal Article
LanguageEnglish
Published Germany 01.02.1997
Subjects
Amino Acid Sequence
Bacterial Proteins - genetics
Cloning, Molecular
Electrophoresis, Polyacrylamide Gel
Escherichia coli - genetics
Gene Expression Regulation, Bacterial
Hydroxylation
Klebsiella pneumoniae - genetics
Mixed Function Oxygenases - genetics
Mixed Function Oxygenases - metabolism
Molecular Sequence Data
Phenylacetates - metabolism
Plasmids
Restriction Mapping
Sequence Analysis, DNA
Sequence Homology, Amino Acid
Substrate Specificity
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Summary: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.
ISSN:0302-8933
DOI:10.1007/s002030050429