A core catalytic domain of the TyrA protein family: arogenate dehydrogenase from Synechocystis.
The TyrA protein family includes prephenate dehydrogenases, cyclohexadienyl dehydrogenases and TyrA(a)s (arogenate dehydrogenases). tyrA(a) from Synechocystis sp. PCC 6803, encoding a 30 kDa TyrA(a) protein, was cloned into an overexpression vector in Escherichia coli. TyrA(a) was then purified to apparent homogeneity and characterized. This protein is a model structure for a catalytic core domain in the TyrA superfamily, uncomplicated by allosteric or fused domains. Competitive inhibitors acting at the catalytic core of TyrA proteins are analogues of any accepted cyclohexadienyl substrate. The homodimeric enzyme was specific for L-arogenate (K(m)=331 microM) and NADP+ (K(m)=38 microM), being unable to substitute prephenate or NAD+ respectively. L-Tyrosine was a potent inhibitor of the enzyme (K(i)=70 microM). NADPH had no detectable ability to inhibit the reaction. Although the mechanism is probably steady-state random order, properties of 2',5'-ADP as an inhibitor suggest a high preference for L-arogenate binding first. Comparative enzymology established that both of the arogenate-pathway enzymes, prephenate aminotransferase and TyrA(a), were present in many diverse cyanobacteria and in a variety of eukaryotic red and green algae.
Bonner
Carol A
CA
Department of Microbiology and Cell Science, Bldg 981, PO Box 110700, University of Florida, Gainesville, FL 32611, USA.
Jensen
Roy A
RA
Gander
John E
JE
Keyhani
Nemat O
NO
D000595:Amino Acid Sequence / Q000235:genetics; D000600:Amino Acids, Dicarboxylic / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics*; D000460:Chlorophyta / Q000201:enzymology; D003001:Cloning, Molecular / Q000379:methods; D000458:Cyanobacteria / Q000201:enzymology; D053138:Cyclohexenes; D030562:Databases, Protein; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry*; D011303:Prephenate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics; D000461:Rhodophyta / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013379:Substrate Specificity; D046939:Synechocystis / Q000201:enzymology*; D000637:Transaminases / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D000600:Amino Acids, Dicarboxylic; D001426:Bacterial Proteins; D053138:Cyclohexenes; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C076398:TyrA protein, Bacteria; D014443:Tyrosine; C015478:pretyrosine; D011303:Prephenate Dehydrogenase; C027017:cyclohexadienyl dehydrogenase; D000637:Transaminases; C044954:prephenate aminotransferase