The C-terminal region controls correct folding of genus Trametes pyranose 2-oxidases.
The pyranose 2-oxidases from Trametes ochracea and Trametes pubescens share markedly similar amino acid sequences with identity of 93.4%. When expressed from the recombinant plasmids based on the same vector in the Escherichia coli host strain BL21(DE3) at higher growth temperatures, they differ strikingly in the formation of the inclusion bodies. Upon overexpression in the cultures performed at 28 degrees C, the specific activity of pyranose 2-oxidase from T. pubescens was eight times higher than that from T. ochracea: 93% of pyranose 2-oxidase from T. ochracea and only 15% of that from T. pubescens was present in the form of inclusion bodies. To ascertain the cause of this difference, both cloned genes were shuffled. Site-directed recombination of p2o cDNAs revealed that DNA constructs ending with 3' end of p2o cDNA from T. pubescens code for proteins that are folded into an active form to the greater extent, regardless of the gene expression level. "In silicio" analysis of physico-chemical properties of the protein sequences of pyranose 2-oxidases revealed that the sequence of amino acid residues 368-430, constituting the small, head domain of pyranose 2-oxidase from T. pubescens, affects positively the enzyme folding at higher cultivation temperatures. The domain differs in six amino acid residues from that of T. ochracea.
Maresová
Helena
H
Laboratory of Enzyme Technology, Institute of Microbiology v.v.i., Academy of Sciences of the Czech Republic, Vídenská 1083, 142 20 Prague, Czech Republic.
Palyzová
Andrea
A
Kyslík
Pavel
P
D000595:Amino Acid Sequence; D001487:Basidiomycota / Q000201:enzymology*; D002237:Carbohydrate Dehydrogenases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D019295:Computational Biology; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D004926:Escherichia coli; D002479:Inclusion Bodies / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D011995:Recombination, Genetic / Q000235:genetics; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions / Q000378:metabolism
D002237:Carbohydrate Dehydrogenases; C022489:pyranose oxidase