Cyclohexadienyl dehydrogenase from Pseudomonas stutzeri exemplifies a widespread type of tyrosine-pathway dehydrogenase in the TyrA protein family.
The uni-domain cyclohexadienyl dehydrogenases are able to use the alternative intermediates of tyrosine biosynthesis, prephenate or L-arogenate, as substrates. Members of this TyrA protein family have been generally considered to fall into two classes: sensitive or insensitive to feedback inhibition by L-tyrosine. A gene (tyrA(c)) encoding a cyclohexadienyl dehydrogenase from Pseudomonas stutzeri JM300 was cloned, sequenced, and expressed at a high level in Escherichia coli. This is the first molecular-genetic and biochemical characterization of a purified protein representing the feedback-sensitive type of cyclohexadienyl dehydrogenase. The catalytic-efficiency constant k(cat)/K(m) for prephenate (7.0x10(7) M/s) was much better than that of L-arogenate (5.7x10(6) M/s). TyrA(c) was sensitive to feedback inhibition by either L-tyrosine or 4-hydroxyphenylpyruvate, competitively with respect to either prephenate or L-arogenate and non-competitively with respect to NAD(+). A variety of related compounds were tested as inhibitors, and the minimal inhibitor structure was found to require only the aromatic ring and a hydroxyl substituent. Analysis by multiple alignment was used to compare 17 protein sequences representing TyrA family members having catalytic domains that are independent or fused to other catalytic domains, that exhibit broad substrate specificity or narrow substrate specificity, and that possess or lack sensitivity to endproduct inhibitors. We propose that the entire TyrA protein family lacks a discrete allosteric domain and that inhibitors act competitively at the catalytic site of different family members which exhibit individuality in the range and extent of molecules recognized as substrate or inhibitor.
Xie
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G
Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611, USA.
Bonner
C A
CA
Jensen
R A
RA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002384:Catalysis; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003470:Culture Media; D004269:DNA, Bacterial / Q000235:genetics; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes; D010957:Plasmids / Q000235:genetics; D011303:Prephenate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D003470:Culture Media; D004269:DNA, Bacterial; D009097:Multienzyme Complexes; C076398:TyrA protein, Bacteria; D017886:Durapatite; D011303:Prephenate Dehydrogenase; C027017:cyclohexadienyl dehydrogenase