The mycobacterial binuclear iron monooxygenases require a specific chaperonin‐like protein for functional expression in a heterologous host. (2nd January 2013)

• Record Type: Journal Article
• Authors: Furuya, Toshiki; Hayashi, Mika; Semba, Hisashi; Kino, Kuniki
• Is Part Of: FEBS journal. Volume 280:Number 3(2013)
• Page Start: 817
• Page End: 826
• Publication Date: 2013-01-02
• Subjects: Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pathology, Molecular -- Periodicals; 572
• DOI: 10.1111/febs.12070
• Languages: English
• ISSNs: 1742-464X

Abstract

<abstract abstract-type="main" xml:lang="en" id="febs12070-abs-0001"> <title> <x xml:space="preserve">Abstract</x> </title> <p>The <italic>mimABCD</italic> gene clusters in <italic>Mycobacterium smegmatis</italic> strain mc<sup>2</sup>155 and <italic>Mycobacterium goodii</italic> strain 12523 encode binuclear iron monooxygenases that oxidize propane and phenol. In this study, we attempted to express each <italic>mimABCD</italic> gene cluster in a heterologous host. The actinomycetous strain <italic>Rhodococcus opacus</italic> B‐4, which is phylogenetically close to <italic>Mycobacterium</italic>, was selected as the host. Each <italic>mimABCD</italic> gene cluster was cloned into the <italic>Rhodococcus</italic>–<italic>Escherichia coli</italic> shuttle vector, pTip‐QC2, and then introduced into <italic>R. opacus</italic> cells. Although whole‐cell assays were performed with phenol as a substrate, the transformed <italic>R. opacus</italic> cells did not oxidize this substrate. SDS/PAGE analysis revealed that the oxygenase large subunit MimA was expressed in the insoluble fraction of <italic>R. opacus</italic> cells. We found that a gene designated <italic>mimG</italic>, which lies downstream of <italic>mimABCD</italic>, exhibits similarity in the amino acid sequence of its product with the products of genes encoding the chaperonin GroEL. When the <italic>mimG</italic> gene was cloned and coexpressed with each <italic>mimABCD</italic> gene cluster in <italic>R. opacus</italic> strain B‐4, this host successfully acquired oxidation activity towards phenol. SDS/PAGE and western blotting analyses demonstrated that MimA was clearly soluble when in the presence of MimG. These results indicated that MimG played essential roles in the productive folding of MimA, and that the resulting soluble MimA protein led to the active expression of MimABCD.</p> </abstract>