title: The mycobacterial binuclear iron monooxygenases require a specific chaperonin-like protein for functional expression in a heterologous host.
abstract: The mimABCD gene clusters in Mycobacterium smegmatis strain mc(2) 155 and Mycobacterium goodii strain 12523 encode binuclear iron monooxygenases that oxidize propane and phenol. In this study, we attempted to express each mimABCD gene cluster in a heterologous host. The actinomycetous strain Rhodococcus opacus B-4, which is phylogenetically close to Mycobacterium, was selected as the host. Each mimABCD gene cluster was cloned into the Rhodococcus-Escherichia coli shuttle vector, pTip-QC2, and then introduced into R. opacus cells. Although whole-cell assays were performed with phenol as a substrate, the transformed R. opacus cells did not oxidize this substrate. SDS/PAGE analysis revealed that the oxygenase large subunit MimA was expressed in the insoluble fraction of R. opacus cells. We found that a gene designated mimG, which lies downstream of mimABCD, exhibits similarity in the amino acid sequence of its product with the products of genes encoding the chaperonin GroEL. When the mimG gene was cloned and coexpressed with each mimABCD gene cluster in R. opacus 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.
authors.affiliation: Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University, Tokyo, Japan.
authors.forename: Toshiki
authors.initials: T
authors.lastname: Furuya
authors.forename: Mika
authors.initials: M
authors.lastname: Hayashi
authors.forename: Hisashi
authors.initials: H
authors.lastname: Semba
authors.forename: Kuniki
authors.initials: K
authors.lastname: Kino
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D018834:Chaperonin 60 / Q000235:genetics / Q000378:metabolism; D018833:Chaperonins / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D007501:Iron / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D005810:Multigene Family; D009161:Mycobacterium / Q000201:enzymology* / Q000235:genetics; D020102:Mycobacterium smegmatis / Q000201:enzymology / Q000235:genetics; D010084:Oxidation-Reduction; D019800:Phenol / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012240:Rhodococcus / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D018834:Chaperonin 60; D011994:Recombinant Proteins; D019800:Phenol; D007501:Iron; D006899:Mixed Function Oxygenases; D018833:Chaperonins