Cloning and Characterization of a 4-Hydroxyphenylacetate 3-Hydroxylase From the Thermophile <Emphasis Type="Italic">Geobacillus</Emphasis> sp. PA-9 |
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Authors: | J F Hawumba V S Brözel J Theron |
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Institution: | (1) Department of Microbiology and Plant Pathology, University of Pretoria, 0002 Pretoria, South Africa;(2) Biochemistry Department, Makerere University, P. O. Box 7062, Kampala, Uganda;(3) Department of Biology and Microbiology, South Dakota State University, Brookings, SD 57007, USA |
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Abstract: | 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. |
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