RIBOFLAVIN 5'-HYDROXYMETHYL OXIDATION - MOLECULAR-CLONING, EXPRESSION, AND GLYCOPROTEIN NATURE OF THE 5'-ALDEHYDE-FORMING ENZYME FROM SCHIZOPHYLLUM-COMMUNE

Vitamin B-2-aldehyde-forming enzyme catalyzes oxidation of the 5'-hydr oxymethyl of riboflavin to the formyl group. We have purified the enzy me from the culture media of Schizophyllum commune (ATCC 38719) by mod ifying the procedure of Tachibana and Oka (Tachibana, S., and Oka, M. (1981) J. Biol. Chem. 256, 6682-6685) for cell-free extract. By SDS-po lyacrylamine gel electrophoresis, the enzyme appears to be 78 kDa, The enzyme has a blocked amino terminus, so fragments were obtained by cl eaving the purified enzyme with lysyl endopeptidase. Selected peptides were sequenced from their amino termini, We have isolated a full-leng th cDNA clone using a DNA hybridization probe amplified by polymerase chain reaction with two degenerate oligonucleotide primers, the design of which was based on one of the partial amino acid sequences. From t he cDNA clone, it is evident that the enzyme has a Ser/Thr-rich fragme nt near the COOH-terminal Asp, The enzyme was determined to be a glyco protein; however, O-deglucosylation only slightly affects activity, Co mputer searches showed that the B-2-aldehyde-forming enzyme has little homology with other proteins, but domain motifs may reflect N-myristo ylation of a dehydrogenase with a signature similar to 4Fe-4S ferredox ins. The enzyme cDNA was subcloned into a Pichia expression vector pPI C9K to produce a recombinant protein which exhibited B-2-aldehyde-form ing enzyme activity.