Does A2 encode a dioxygenase?
--Adriane Menssen, Heinz Saedler and Alfons Gierl
The A2 gene encodes an enzyme acting late in anthocyanin biosynthesis in the aleurone tissue of kernels or other parts of the maize plant. The biochemical role of the A2 enzyme in anthocyanin biosynthesis is not well understood. Inter-tissue complementation assays (Reddy and Coe, Science 138:149-150, 1962) suggest that the A2 enzyme acts after the A1 gene product (dihydroflavonol 4-reductase), and converts cis-leucoanthocyanidin to anthocyanidin (cis-leucocyanidin to cyanidin or cis-leucopelargonidin to pelargonidin depending on Pr), the first colored component of the anthocyanin enzyme cascade. This step requires two biochemical reactions: trans-elimination of H2O and oxidation of carbon No. 2.
The A2 gene was cloned by transposon-tagging and its transcription unit was determined (Menssen et al. EMBO J. 9:3051-3057, 1990). The A2 gene is intronless and the open reading frame encodes a putative protein of 43.5kD. An amino acid sequence homology search revealed a possible function. A 26% identity and 48% similarity was found with the pTOM13 mRNA encoded protein. pTOM13 probably encodes a polypeptide involved in the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene by the ethylene forming enzyme (ACC-oxidase, Hamilton et al., Nature 346:384-387). A similar amount of homology (29% identity and 53% similarity) was found to the flavanone 3-hydroxylase cDNAs of Antirrhinum majus (C. Martin and A. Prescott, personal communication) and Petunia (G. Forkmann, personal communication). The flavanone 3-hydroxylase is a dioxygenase that is 2-oxoglutarate and Fe2+ dependent (Forkmann et al., Z. Naturforsch. 35c:691-695, 1980). In this case, flavanone is converted to dihydroflavonol by introduction of a hydroxyl group in the 3-position. Therefore, it is assumed that the A2 enzyme is also a 2-oxoglutarate dependent dixoygenase, as well as the ACC-oxidase. In the case of the A2 reaction the oxidation of carbon No. 2 may occur via introduction of a hydroxyl group. The resulting triol might be an unstable intermediate, which is spontaneously dehydrated, probably because the activation energy for the elimination of water is reduced by the generation of the aromatic system. Another possibility is that in addition to the A2 enzyme, a dehydratase is involved in the synthesis of anthocyanidin. However, no corresponding mutant allele has been found.
The inc3 (candica) gene of A. majus probably represents the homologue of the A2 gene, since both gene products share 60% amino acid identity and 70% similarity (C. Martin and A. Prescott, personal communication), as would be expected for monocots vs. dicots. Scattered patches of amino acid motifs are identical in all five proteins mentioned above. These motifs may represent conserved binding-sites of the common co-substrate 2-oxoglutarate or the common cofactor Fe2+. Experiments are now on the way to unravel this last unknown step in anthocyanidin biosynthesis.
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