Biochem. J. (2004) 382
(279–291) (Printed in Great Britain)
A core catalytic domain of the TyrA protein family: arogenate dehydrogenase from Synechocystis
Carol A. BONNER*, Roy A. JENSEN*†‡, John E. GANDER* and Nemat O. KEYHANI*1
*Department of Microbiology and Cell Science, Bldg 981, PO Box 110700, University of Florida, Gainesville, FL 32611, U.S.A., †Biosciences Division, Los Alamos National Laboratory, Los Alamos, NM 87544, U.S.A., and ‡Department of Chemistry, City College of New York, New York, NY 10031, U.S.A.
The TyrA protein family includes prephenate dehydrogenases, cyclohexadienyl dehydrogenases and TyrAas (arogenate dehydrogenases). tyrAa from Synechocystis sp. PCC 6803, encoding a 30 kDa TyrAa protein, was cloned into an overexpression vector in Escherichia coli. TyrAa 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 (Km=331 µM) and NADP+ (Km=38 µM), being unable to substitute prephenate or NAD+ respectively. L-Tyrosine was a potent inhibitor of the enzyme (Ki=70 µM). 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 TyrAa, were present in many diverse cyanobacteria and in a variety of eukaryotic red and green algae.
Key words: arogenate dehydrogenase, enzyme specificity, prephenate, Synechocystis, TyrA, tyrosine.
Abbreviations used: AGN, L-arogenate; DTT, dithiothreitol; LB, Luria–Bertani; PHE, L-phenylalanine; TYR, L-tyrosine; TyrAa, arogenate dehydrogenase; for brevity, single-letter code has been used for amino acids; for example, H197 stands for His-197.
1To whom correspondence should be addressed (email keyhani@ufl.edu).
The nucleotide and the deduced amino acid sequences corresponding to the Synechocystis sp. PCC 6803 cloned tyrAa gene reported in this paper have been submitted to the DDBJ, EMBL, GenBank® and GSDB Nucleotide Sequence Databases under the accession number AF482689.
Received 25 November 2003/11 May 2004; accepted 1 June 2004
Published as BJ Immediate Publication 1 June 2004, DOI 10.1042/BJ20031809
The Biochemical Society, London ©2004
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