CHARACTERIZATION OF ENZYMES INVOLVED IN FLAVIN COFACTOR METABOLISM

Joseph Lynch

The cofactors FMN and FAD are required in all living organisms. Both cofactors are derived from riboflavin, which is synthesized from GTP and ribulose-5-phosphate. The pathway for synthesis of riboflavin in plants has been identified, and most of the enzymes involved have been cloned and biochemically characterized. However, many of the enzymes which interconvert between riboflavin, FMN, and FAD in plants have yet to be identified. We identified a putative FAD synthetase, AtFADS1 encoded by the genome of Arabidopsis thaliana which has two distinct domains. The first domain has sequence homology the previously characterized FAD synthetase from Saccharomyces cerevisiae, FAD1. The second has sequence homology to a second protein from S. cerevisiae. To determine the function of the second protein, it was overexpressed in yeast, purified, and biochemically characterized. It is demonstrated here to be a FAD pyrophosphatase with a strict requirement for potassium, and is designated FPY1. Humans also possess a protein with individual domains homologous to FAD1 and FPY1, though with the orientation of the two domains reversed relative to plants, which is encoded by the FLAD1 gene. It has previously been shown to be an active FAD synthetase. Following recombinant expression in S. cerevisiae and purification, the human protein is shown to also possess FAD pyrophosphatase activity. This demonstrates the first ever known fusion between an FAD synthetase and FAD pyrophosphates. The AtFADS1 gene from A. thaliana was cloned and expressed in S. cerevisiae. It was found to be an active FAD synthetase, but no FAD pyrophatase activity was detected. Analysis suggests mutation of catalytic residues in the lineage leading to vascular plants. In total, these results indicate that S. cerevisiae proteins FAD1 and FPY1 represent an ancestoral FAD synthetase and an ancestoral FAD pyrophosphatase, respectively, that the lineage that gave rise to animals resulted in a genetic fusion between these two proteins, and that the lineage that gave rise to vascular plants resulted in first a genetic fusion between the two proteins, then a loss of the FAD pyrophosphatase activity. These results illuminate the mechanisms by which different organisms maintain flavin cofactor homeostasis.

CHARACTERIZATION OF ENZYMES INVOLVED IN FLAVIN COFACTOR METABOLISM

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