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Modification of the flexible loop of tyrosine hydroxylase that determines substrate specificity

Tyrosine hydroxylase (TyrH) is one of three aromatic amino acid hydroxylases, all of which evolved from an ancient phenylalanine hydroxylase (PheH). Modern eukaryotic PheH is unable to convert tyrosine to DOPA. Tyrosine hydroxylase is able to hydroxylate phenylalanine and tyrosine. One residue in particular allows the new activity, the aspartate at position 425 in TyrH (the homologous residue in PheH is valine 379)1. Aspartate 425 lies in a flexible loop situated over the enzyme active site. Previous work showed that TyrHD425E has only 10% of the activity of wild‐type TyrH, and that hydrophobic residues substituted in position 425 abolished TyrH activity. To understand better the flexible loop's structure and role in substrate specificity, we have made shape‐altering changes to TyrHD425E, deleting the adjacent glutamine residues. These include TyrHdeltaQ424D425E (Q424 removed), TyrHD425E deltaQ426 (Q426 removed), and TyrHdeltaQ424D425EdeltaQ426 (both glutamines removed). Attempts to grow these variants in E. coli have varied in success. As assessed by SDS gel electrophoresis of crude lysates, TyrHD425EdeltaQ426 is expressed better than TyrHdeltaQ424D425E. Both enzymes are expressed comparably to wild‐type enzyme, but the variant lacking both glutamines is not expressed under routine growth conditions. The partially purified enzymes have been tested for DOPA‐synthesizing activity. These preliminary measurements suggest that both variants that lack one glutamine residue are able to convert tyrosine to DOPA at faster rates than TyrHD425E.