[Cloning and expression of a 2-naphthoate monooxygenase gene (nmo) in Burkholderia sp. JT1500].
A 4.8kb DNA fragment from one blue colony of the pLARF1 gene library of Burkholderia sp. JT1500 was subcloned to pUC18, designated as pEK123. The sequence of the inserted 4.8kb DNA' of pEK123 was analyzed and submitted to EMBL nucleotide database, the accession # is AJ566333. The transformants of pEK123 could also become blue in LB agar and sequence analysis showed that three open reading frames and a putative promoter sequence were located in this inserted fragment. Then the 4.4kb insert fragment of pEK123 was double digested with Xba I / Kpn I and EcoR I / Xba I respectively to construct plamsids pXK3 and pEX12. The pXK3 contained only one 1158bp open reading frame (ORF) and pEX12 with other two ORFs. Unlike pEK123, the colonies of pEX12 did not show any blue color even incubated for 72h in LB agar, but the transformants of pXK3 did oxidize indole into indigo. The deduced 43kD protein of 1158bp ORF showed 64% homology of amino acid composition to Ralstonia eutropha HF39 hydroxylase (bec). Results of substrate transformation analysis showed that the transformants of pEK123 was able to catalyze the oxidation of 2-naphthoate but not other key intermediates in 2-naphthoate metabolic pathway. These results confirmed that the product of 1158bp ORF is 2-naphthoate monooxygenase. Though the oxygenase activity of pEK123 is much higher than that of pXK3, SDS-PAGE analysis found no difference between the amount of the band of monooxygenase produced by pXK3 or pEK123, but one more band was found produced by pEK123. According to the difference of substrate analysis between pXK3 and pEK123, it is supposed that the products of two open reading frames up stream of nmo gene had strong influence on the activity of the monooxygenase. Benzoate was oxidized by free-cell extracts of the transformants of pEK123 in the transformation experiment with different aromatic substrates. As the DNA sequence and amino acid sequence of 2-naphthoate monoxygenase (nmo) did no show any homology with the DNA sequence and amino acid sequence of benzoate oxygenases reported, the pathway of benzoate oxidation conducted by nmo is on the investigation.
Fang
Xiangping
X
Guangdong Institute of Microbiology, Guangzhou 510070, China.
Qiu
Xiaoying
X
Cen
Yinghua
Y
Sun
Guoping
G
D019117:Burkholderia / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D015870:Gene Expression; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D013379:Substrate Specificity
D006899:Mixed Function Oxygenases