Identification of the covalently bound flavins of d-gluconate dehydrogenases from Pseudomonas aeruginosa and Pseudomonas fluorescens and of 2-keto-d-gluconate dehydrogenase from Gluconobacter melanogenus

Abstract

An improved method is presented for the purification of 8 α-(N1-histidyl)riboflavin, 8 α-(N3-histidyl)riboflavin and their 2′,5′-anhydro forms, which permits the isolation of sizeable quantities of each of these compounds from a synthetic mixture in pure form. Flavin peptides were isolated from the D-gluconate dehydrogenases of Pseudomonas aeruginosa and Pseudomonas fluorescens and from the 2-keto-D-gluconate dehydrogenase of Gluconobacter melanogenus. After conversion into the aminoacyl-riboflavin, the flavin in all three enzymes was identified as 8 α-(N3-histidyl)riboflavin. By sequential treatment with nucleotide pyrophosphatase and alkaline phosphatase, the flavin in each enzyme was shown to be in the dinucleotide form.