HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chen, H. Articles by McCormick, D. B. Search for Related Content PubMed PubMed Citation Articles by Chen, H. Articles by McCormick, D. B. Social Bookmarking                      What's this?

Volume 272, Number 32, Issue of August 8, 1997 pp. 20077-20081
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Riboflavin 5-Hydroxymethyl Oxidation
MOLECULAR CLONING, EXPRESSION, AND GLYCOPROTEIN NATURE OF THE 5-ALDEHYDE-FORMING ENZYME FROM SCHIZOPHYLLUM COMMUNE

(Received for publication, April 21, 1997)

From the Department of Biochemistry, Rollins Research Center, Emory University, Atlanta, Georgia 30322-3050

Vitamin B₂-aldehyde-forming enzyme catalyzes oxidation of the 5-hydroxymethyl of riboflavin to the formyl group. We have purified the enzyme from the culture media of Schizophyllum commune (ATCC 38719) by modifying the procedure of Tachibana and Oka (Tachibana, S., and Oka, M. (1981) J. Biol. Chem. 256, 6682-6685) for cell-free extract. By SDS-polyacrylamine gel electrophoresis, the enzyme appears to be 78 kDa. The enzyme has a blocked amino terminus, so fragments were obtained by cleaving the purified enzyme with lysyl endopeptidase. Selected peptides were sequenced from their amino termini. We have isolated a full-length 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 the cDNA clone, it is evident that the enzyme has a Ser/Thr-rich fragment near the COOH-terminal Asp. The enzyme was determined to be a glycoprotein; however, O-deglucosylation only slightly affects activity. Computer searches showed that the B₂-aldehyde-forming enzyme has little homology with other proteins, but domain motifs may reflect N-myristoylation of a dehydrogenase with a signature similar to 4Fe-4S ferredoxins. The enzyme cDNA was subcloned into a Pichia expression vector pPIC9K to produce a recombinant protein which exhibited B₂-aldehyde-forming enzyme activity.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				D. B. McCormick A Trail of Research on Cofactors: An Odyssey with Friends J. Nutr., February 1, 2000; 130(2): 323 - 323. [Abstract] [Full Text]

				J. R. Miller and D. E. Edmondson Influence of Flavin Analogue Structure on the Catalytic Activities and Flavinylation Reactions of Recombinant Human Liver Monoamine Oxidases A and B J. Biol. Chem., August 13, 1999; 274(33): 23515 - 23525. [Abstract] [Full Text] [PDF]