Oxalate decarboxylase from Collybia velutipes. Purification, characterization, and cDNA cloning.
The oxalate-degrading enzyme oxalate decarboxylase (EC 4.1.1.2), which is inducible by oxalic acid, was purified to homogeneity from a crude extract of Collybia velutipes, a basidiomycetous fungus. Two forms of the enzyme were resolved on chromatofocusing. The two isozymes were shown to be related by amino acid composition, peptide mapping, and immunological cross-reactivity. Peak A, eluting at pH 3.3, was used for further study; the Km was found to be 4.5 mM, and the Vmax was 166 mumol/min/mg. The subunit molecular mass of the glycosylated enzyme was 64 kDa, whereas the mass of the deglycosylated protein was 55 kDa. The enzyme showed an acidic pl, was very stable over a wide pH range, and was moderately thermostable. The cDNA encoding the enzyme was obtained by immunoscreening a lambda gt11 expression library. In vitro translation of hybrid-selected mRNA gave a 55-kDa protein. Genomic Southern hybridization indicated the oxalate decarboxylase is encoded by a single gene. The cDNA probe hybridized to a single 1.5-kilobase pair species of mRNA. The mRNA was shown to be induced by oxalic acid. A temporal relationship between enzyme activity and mRNA levels was observed, thus suggesting that the expression of oxalate decarboxylase is regulated at the transcriptional level.
Mehta
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School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
Datta
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D000596:Amino Acids / Q000032:analysis; D001487:Basidiomycota / Q000201:enzymology* / Q000235:genetics; D002262:Carboxy-Lyases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D015723:Gene Library; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
D000596:Amino Acids; D004271:DNA, Fungal; D046911:Macromolecular Substances; D012333:RNA, Messenger; D002262:Carboxy-Lyases; C021915:oxalate decarboxylase