Purification and characterization of FAD synthetase from Brevibacterium ammoniagenes.
The bifunctional enzyme FAD synthetase from Brevibacterium ammoniagenes was purified by a method involving ATP-affinity chromatography. The final preparation was more than 95% pure. The apparent molecular weight of the enzyme was determined as 38,000 and the isoelectric point as 4.6. Although previous attempts to separate the enzymatic activities had failed, ATP:riboflavin 5'-phosphotransferase and ATP:FMN-adenylyltransferase activities in B. ammoniagenes were believed to be located on two separate proteins with similar properties, possibly joined in a complex. The following evidence, however, suggests the presence of both activities on a single polypeptide chain. The two activities copurify in the same ratio through the purification scheme as presented. Only a single band could be detected when aliquots from the final purification step were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, nondenaturing gel electrophoresis, and isoelectric focusing. Edman degradation of the protein yielded a single N-terminal sequence.
D001951:Brevibacterium / Q000201:enzymology*; D005486:Flavin Mononucleotide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D009713:Nucleotidyltransferases / Q000032:analysis / Q000302:isolation & purification*; D015032:Zinc / Q000494:pharmacology
D005486:Flavin Mononucleotide; D009713:Nucleotidyltransferases; C020143:FMN adenylyltransferase; D008274:Magnesium; D015032:Zinc