The Crystal Structure of Aquifex aeolicus Prephenate Dehydrogenase Reveals the Mode of Tyrosine Inhibition

• Published in: The Journal of biological chemistry 2009-05, Vol.284 (19), p.13223-13232
• Main Authors: Sun, Warren, Shahinas, Dea, Bonvin, Julie, Hou, Wenjuan, Kimber, Matthew S., Turnbull, Joanne, Christendat, Dinesh
• Format: Article
• Language: English
• Subjects: Aquifex aeolicus; Bacteria - classification; Bacteria - enzymology; BIOSYNTHESIS; CATALYSIS; Catalytic Domain; CRYSTAL STRUCTURE; Crystallography, X-Ray; ENGINEERING; Enzyme Catalysis and Regulation; ENZYMES; Histidine - chemistry; Histidine - metabolism; INHIBITION; LIGANDS; MATERIALS SCIENCE; Models, Molecular; MUTAGENESIS; Mutagenesis, Site-Directed; MUTANTS; Mutation - genetics; NAD; NAD - metabolism; OXIDOREDUCTASES; Prephenate Dehydrogenase - chemistry; Prephenate Dehydrogenase - physiology; Protein Conformation; PROTEINS; REGULATIONS; RESIDUES; SUBSTRATES; TARGETS; TYROSINE; Tyrosine - metabolism; USES
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M806272200

TyrA proteins belong to a family of dehydrogenases that are dedicated to l-tyrosine biosynthesis. The three TyrA subclasses are distinguished by their substrate specificities, namely the prephenate dehydrogenases, the arogenate dehydrogenases, and the cyclohexadienyl dehydrogenases, which utilize prephenate, l-arogenate, or both substrates, respectively. The molecular mechanism responsible for TyrA substrate selectivity and regulation is unknown. To further our understanding of TyrA-catalyzed reactions, we have determined the crystal structures of Aquifex aeolicus prephenate dehydrogenase bound with NAD+ plus either 4-hydroxyphenylpyuvate, 4-hydroxyphenylpropionate, or l-tyrosine and have used these structures as guides to target active site residues for site-directed mutagenesis. From a combination of mutational and structural analyses, we have demonstrated that His-147 and Arg-250 are key catalytic and binding groups, respectively, and Ser-126 participates in both catalysis and substrate binding through the ligand 4-hydroxyl group. The crystal structure revealed that tyrosine, a known inhibitor, binds directly to the active site of the enzyme and not to an allosteric site. The most interesting finding though, is that mutating His-217 relieved the inhibitory effect of tyrosine on A. aeolicus prephenate dehydrogenase. The identification of a tyrosine-insensitive mutant provides a novel avenue for designing an unregulated enzyme for application in metabolic engineering.