A novel type of covalently bound coenzyme in trimethylamine dehydrogenase

Bacterial trimethylamine dehydrogenase contains a covalently bound yellow coenzyme, the properties of which distinguish it from all known riboflavin, pyridoxine, and pteridine derivatives. A pure dodecapeptide containing the covalently linked coenzyme has been isolated from tryptic-chymotryptic digests. Treatment with aminopeptidase M converts it to a ninhydrin-positive aminoacyl coenzyme, which has also been isolated in chromatographically pure form. The coenzyme as isolated is virtually nonfluorescent but, being extremely photolabile, it is rapidly converted on illumination to products, the predominant one being highly fluorescent. Analysis for phosphate and the influence of phosphatase treatment on electrophoretic mobility show that the coenzyme is isolated as a monophosphate, while periodate titrations prove the presence of a pentityl side chain, to which the phosphate is attached. Oxidation of the aminoacyl coenzyme with performic acid, followed by acid hydrolysis, yields 2',5'-anhydroriboflavin and some free riboflavin, products also obtained on similar treatment of FMN. These observations and the very tight binding to apoflavodoxin show that the coenzyme is a flavin derivative. The absorption and NMR spectra, however, clearly set it apart from 8alpha-substituted flavins and suggest that the coenzyme is the first representative of a new class of covalently bound flavins.