Operon structure and functional analysis of the genes encoding thermophilic desulfurizing enzymes of Paenibacillus sp. A11-2.
Paenibacillus A11-2 can efficiently cleave two carbon&bond;sulfur bonds in dibenzothiophene (DBT) and alkyl DBTs, which are refractory by conventional petroleum hydrodesulfurization, to remove sulfur atom at high temperatures. An 8.7-kb DNA fragment containing the genes for the DBT desulfurizing enzymes of A11-2 was cloned in Escherichia coli and characterized. Heterologous expression analysis of the deletion mutants identified three open reading frames that were required for the desulfurization of DBT to 2-hydroxybiphenyl (2-HBP). The three genes were designated tdsA, tdsB, and tdsC (for thermophilic desulfurization). Both the nucleotide sequences and the deduced amino acid sequences show significant homology to dszABC genes of Rhodococcus sp. IGTS8, but there are several local differences between them. Subclone analysis revealed that the product of tdsC oxidizes DBT to DBT-5,5'-dioxide via DBT-5-oxide, the product of tdsA converts DBT-5,5'-dioxide to 2-(2-hydroxyphenyl) benzene sulfinate, and the product of tdsB converts 2-(2-hydroxyphenyl)benzene sulfinate to 2-HBP. Cell-free extracts of a recombinant E. coli harboring all the three desulfurization genes converted DBT to 2-HBP at both 37 and 50 degrees C. In vivo and in vitro exhibition of desulfurization activity of the recombinant genes derived from a Paenibacillus indicates that an E. coli oxidoreductase can be functionally coupled with the monooxygenases of a gram-positive thermophile.
Ishii
Y
Y
Bio-Refining Process Laboratory, Advanced Technology and Research Institute, Petroleum Energy Center, 1, 900 Sodeshi-Cho, Shimizu-Shi, Shizuoka, 424-0037, Japan. yishii@brpl.pecj.or.jp
Konishi
J
J
Okada
H
H
Hirasawa
K
K
Onaka
T
T
Suzuki
M
M
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology / Q000235:genetics*; D001426:Bacterial Proteins; D001483:Base Sequence; D001673:Biodegradation, Environmental; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009876:Operon; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D050862:Oxidoreductases Acting on Sulfur Group Donors; D011994:Recombinant Proteins / Q000378:metabolism; D012240:Rhodococcus / Q000201:enzymology / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013876:Thiophenes / Q000378:metabolism*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D013876:Thiophenes; D010088:Oxidoreductases; D050862:Oxidoreductases Acting on Sulfur Group Donors; C407142:TdsA protein, Paenibacillus; C407143:TdsB protein, Paenibacillus; C407144:TdsC protein, Paenibacillus; C016366:dibenzothiophene