Covalent cofactor binding to flavoenzymes requires specific effectors.
Modification by covalent FAD attachment to a histidine residue via an 8 alpha-(N3-histidyl)-riboflavin linkage occurs in several flavoenzymes. Among them is 6-hydroxy-D-nicotine oxidase (6-HDNO) of Arthrobacter oxidans and the flavoprotein subunits of the fumarate reductase and succinate dehydrogenase complex of Escherichia coli and other bacterial and eukaryotic cells. We found that 6-HDNO holoenzyme formation from apo-6-HDNO, monitored by [14C]FAD incorporation and increase in enzyme activity, can be mediated not only by phosphoenolpyruvate [Nagursky, H., Bichler, V. and Brandsch, R. (1988) Eur. J. Biochem. 177, 319-325], but also by one of the glycolytic intermediates glyceraldehyde-3-P, glycerate-3-P, or the intermediate in glycerol utilization by bacteria, glycerol-3-P. Apoflavoprotein of fumarate reductase and succinate dehydrogenase was obtained in an E. coli riboflavin-requiring strain (E. coli RR28rf) overexpressing the frdABCD or the sdhCDAB operon from the recombinant plasmids pGS39 and pGS141, respectively. In extracts obtained from these cells, flavoprotein flavinylation, analyzed as covalent [14C]FAD incorporation into the apoflavoprotein polypeptide by polyacrylamide gel electrophoresis and fluorography, was stimulated severalfold by the citric acid cycle intermediates citrate, isocitrate, succinate and fumarate. Our results suggest that covalent modification and thus activation of these enzymes is dependent on specific metabolic intermediates which may act as allosteric effectors in the reaction.
Brandsch
R
R
Biochemisches Institut, Universität Freiburg, Federal Republic of Germany.
Bichler
V
V
D002951:Citrates / Q000494:pharmacology; D002952:Citric Acid Cycle; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D005420:Flavoproteins / Q000378:metabolism*; D005650:Fumarates / Q000494:pharmacology; D007523:Isocitrates / Q000494:pharmacology; D010957:Plasmids; D011773:Pyruvates / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence; D013385:Succinate Dehydrogenase / Q000378:metabolism*; D013386:Succinates / Q000494:pharmacology
D002951:Citrates; D005420:Flavoproteins; D005650:Fumarates; D007523:Isocitrates; D011773:Pyruvates; D013386:Succinates; D005182:Flavin-Adenine Dinucleotide; C032005:fumaric acid; D013385:Succinate Dehydrogenase