Cloning, sequence analysis, and purification of choline oxidase from Arthrobacter globiformis: a bacterial enzyme involved in osmotic stress tolerance.
Choline oxidase catalyzes the four-electron oxidation of choline to glycine betaine, one of a limited number of compounds that accumulate to high levels in the cytoplasm of cells to prevent dehydration and plasmolysis in adverse hyperosmotic environments. In the present study, the highly GC rich codA gene encoding for choline oxidase was cloned from genomic DNA of Arthrobacter globiformis strain ATCC 8010 and expressed to high yields in Escherichia coli strain Rosetta(DE3)pLysS. The resulting enzyme was purified to high levels in a single chromatographic step using DEAE-Sepharose, as shown by SDS-PAGE analysis. Denaturation and mass spectroscopic analyses showed that the covalent linkage between the FAD cofactor and the protein is preserved in recombinant choline oxidase, consistent with protein flavinylation being a self-catalytic process. The enzyme was shown to be a homodimer of 120,000 Da by size-exclusion chromatography and to be active with both choline and betaine aldehyde as substrate. Sequencing analysis indicated that the nucleotide sequence of codA originally reported in GenBank contains seven flaws, resulting in a translated protein with a significantly altered amino acid sequence between position 298 and 410.
Fan
Fan
F
Department of Biology, Georgia State University, Atlanta, GA 30302-4098, USA.
Ghanem
Mahmoud
M
Gadda
Giovanni
G
D000222:Adaptation, Physiological; D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001173:Arthrobacter / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D008969:Molecular Sequence Data; D009997:Osmotic Pressure; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D001426:Bacterial Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; C019888:choline oxidase