title: Chaperonin GroESL mediates the protein folding of human liver mitochondrial aldehyde dehydrogenase in Escherichia coli.
abstract: An efficient bacterial expression system for the human mitochondrial aldehyde dehydrogenase (ALDH2) was developed using co-overexpression of heat shock chaperone gene GroESL. On the basis of the ALDH2 amino acid sequence and cDNA sequences a full-length cDNA encoding wild-type ALDH2 was cloned from a human liver library. A mutant-type ALDH2 (ALDH2(2)) was developed using site-directed mutagenesis of the ALDH2 cDNA and also cloned. Both types of ALDH2 cDNA were subcloned for expression in Escherichia coli (E. coli), recombinant ALDH2 and ALDH2(2) were successfully expressed as soluble active enzymes following co-expression with a second plasmid construct producing GroES and GroEL, E. coli chaperonin proteins. Purified wild-type ALDH2 and mutant ALDH2(2) had a K(m) for acetaldehyde of 0.65 and 25.73 microM, respectively. Co-expression of ALDH2 with ALDH2(2) in the presence of E. coli chaperonins produced a soluble enzyme with a K(m) for acetaldehyde of 8.79 microM, suggesting that the product was a heteromer. Mitochondrial matrix hsp60 and hsp10 chaperonins are then thought to act on imported ALDH2 and are essential for accurate protein folding and multisubunit formation. Protein-protein interactions between ALDH2s and various chaperones were investigated using the yeast two-hybrid system. The wild-type and mutant-type enzymes strongly interacted with each other and GroEL and ALDH2s also interacted but only weakly. Chaperone hsp10 also interacted with hsp60 and ALDH2(1) and ALDH2(2), but again the interactions were weak ones.
authors.affiliation: Department of Biochemistry, College of Medicine, Hanyang University, 17 Haengdang-dong, Seongdong-Gu, Seoul 133-791, Republic of Korea. leekihwan@gaiagene.com
authors.forename: Ki-Hwan
authors.initials: KH
authors.lastname: Lee
authors.forename: Ho-Seung
authors.initials: HS
authors.lastname: Kim
authors.forename: Han-Seung
authors.initials: HS
authors.lastname: Jeong
authors.forename: Yong-Sung
authors.initials: YS
authors.lastname: Lee
mesh_terms: D000444:Aldehyde Dehydrogenase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000071396:Aldehyde Dehydrogenase, Mitochondrial; D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology*; D018835:Chaperonin 10 / Q000235:genetics / Q000378:metabolism; D018833:Chaperonins / Q000235:genetics / Q000502:physiology*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D008930:Mitochondria, Liver / Q000201:enzymology*; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014169:Transformation, Bacterial; D020798:Two-Hybrid System Techniques
chemical_list: D001426:Bacterial Proteins; D018835:Chaperonin 10; C097970:GroESL protein, Bacteria; D024101:Mitochondrial Proteins; D011994:Recombinant Proteins; C096127:ALDH2 protein, human; D000444:Aldehyde Dehydrogenase; D000071396:Aldehyde Dehydrogenase, Mitochondrial; D018833:Chaperonins