Cloning and characterization of a 4-hydroxyphenylacetate 3-hydroxylase from the thermophile Geobacillus sp. PA-9.
A 4-hydroxyphenylacetic acid (4-HPA) hydroxylase-encoding gene, on a 2.7-kb genomic DNA fragment, was cloned from the thermophile Geobacillus sp. PA-9. The Geobacillus sp. PA-9 4-HPA hydroxylase gene, designated hpaH, encodes a protein of 494 amino acids with a predicted molecular mass of 56.269 Da. The deduced amino-acid sequence of the hpaH gene product displayed <30% amino-acid sequence identity with the larger monooxygenase components of the previously characterized two-component 4-HPA 3-hydroxylases from Escherichia coli W and Klebsiella pneumoniae M5a1. A second oxidoreductase component was not present on the 2.7-kb genomic DNA fragment. The deduced amino-acid sequence of a second C-terminal truncated open reading frame, designated hpaI, exhibited homology to extradiol oxygenases and displayed the highest amino-acid sequence identity (43%) with the 3,4-dihydroxyphenylacetate 2,3-dioxygenase of Arthrobacter globiformis, encoded by mndD. These results, along with catalytic activity observed in crude intracellular extracts prepared from Escherichia coli cells expressing hpaH, is in support of a role for hpaH in the 4-HPA degradative pathway of Geobacillus sp. PA-9.
Hawumba
J F
JF
Department of Microbiology and Plant Pathology, University of Pretoria, 0002 Pretoria, South Africa.
Brözel
V S
VS
Theron
J
J
D000595:Amino Acid Sequence; D001406:Bacillaceae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010648:Phenylacetates / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA