The crystal structure of Aquifex aeolicus prephenate dehydrogenase reveals the mode of tyrosine inhibition.
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.
Sun
Warren
W
Department of Cell and Systems Biology, University of Toronto, Ontario, Canada.
Shahinas
Dea
D
Bonvin
Julie
J
Hou
Wenjuan
W
Kimber
Matthew S
MS
Turnbull
Joanne
J
Christendat
Dinesh
D
D001419:Bacteria / Q000145:classification / Q000201:enzymology*; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D009243:NAD / Q000378:metabolism; D011303:Prephenate Dehydrogenase / Q000737:chemistry* / Q000502:physiology*; D011487:Protein Conformation; D014443:Tyrosine / Q000378:metabolism*
D009243:NAD; D014443:Tyrosine; D006639:Histidine; D011303:Prephenate Dehydrogenase