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CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector Volume 216, number 1, 133-139 FEB 04718 May 1987 The prephenate dehydrogenase component of the bifunctional T-protein in enteric bacteria can utilize L-arogenate Suhail Ahmad and Roy A. Jensen Department of Microbiology and Cell Science, McCarty Hall, IFAS, University of Florida, Gainesville, FL 32611, USA Received 3 February 1987; revised version received 19 March 1987 The prephenate dehydrogenase component of the bifunctional T-protein (chorismate mutase:prephenate de- hydrogenase) has been shown to utilize L-arogenate, a common precursor of phenylalanine and tyrosine in nature, as a substrate. Partially purified T-protein from Klebsiellapneumoniae and from Escherichia coli strains K12, B, C and W was used to demonstrate the utilization of L-arogenate as an alternative substrate for prephenate in the presence of nlcotinamide adenine dinucleotide as cofactor. The formation of L-tyro- sine from L-arogenate by the T-protein dehydrogenase was confirmed by high-performance liquid chroma- tography. As expected of a common catalytic site, dehydrogenase activity with either prephenate or L-aroge- nate was highly sensitive to inhibition by L-tyrosine. Tyrosine synthesis; T-protein; Enteric bacteria; Regulatory enzyme 1. INTRODUCTION specifying chorismate mutase [2]. Cyclohexadienyl dehydrogenase and cyclohexadienyl dehydratase Escherichia coli and Klebsiella pneumoniae, (often referred to as arogenate dehydrogenase and closely related enteric bacteria, possess a pair of arogenate dehydratase in earlier papers) are able to bifunctional proteins that compete for chorismate accept either prephenate or L-arogenate as as initial substrate molecules in reactions leading to substrates. Within the Superfamily-B cluster, a biosynthesis of aromatic amino acids. Fig.1 il- phylogenetic assemblage comprising one of three lustrates that the P-protein (chorismate mutase- major groups of Gram-negative bacteria (includes P : prephenate dehydratase) channels chorismate to enteric genera, Oceanospirillum, Xanthomonas, L-phenylalanine, while the T-protein (chorismate fluorescent pseudomands and Acinetobacter) all mutase-T : prephenate dehydrogenase) channels lineages possess the P-protein and most retain a chorismate to L-tyrosine at a metabolic branch cyclohexadienyl dehydratase as well [6]. In con- point [ 1,2]. Each bifunctional protein has been trast, the T-protein evolved very recently within purified to homogeneity from E. coli and K. Superfamily B, being present only within the pneumoniae [3-51. These proteins were postulated enteric lineage [2]. Superfamily-B organisms to have evolved by fusion of genes specifying possess either cyclohexadienyl dehydrogenase (e.g. cyclohexadienyl dehydrogenase and cyclohexa- Acinetobacter, fluorescent pseudomonads and dienyl dehydratase with one of two gene duplicates Xanthomonas) or the T-protein, but not both [6]. L-Arogenate has become recognized as a widely utilized precursor of tyrosine and/or phenyl- Correspondence address: R.A. Jensen, Department of alanine in nature [7]. However, the exclusive for- Microbiology and Cell Science, 1059 McCarty Hall, mation of prephenate within the two channeling IFAS, University of Florida, Gainesville, FL 32611, systems of E. coli and K. pneumoniae essentially USA. excludes prephenate as a substrate for one or more Published by Elsevier Science Publishers B. V. (Biomedical Division) 00145793/87/$3.50 0 1987 Federation of European Biochemical Societies 133 Volume 216, number 1 FEBS LETTERS May 1987 NH; NH; I OH OH OH Fig.1. Operation of two bifunctional proteins in biosynthesis of phenylalanine (PHE) and tyrosine (TYR) in enteric bacteria. Chorismate (CHA) is competed for as substrate by both the P-protein (enzymes [2a] and [2b]) and the T- protein (enzymes [la] and [lb]). Phenylpyruvate (PPY) and 4-hydroxyphenylpyruvate (HPP) are transaminated to PHE and TYR, respectively, in the presence of glutamate (GLU). The catalytic activities of the P-protein are chorismate mutase-P [2a] and prephenate dehydratase [2b]; the catalytic activities of the T-protein are chorismate mutase-T [la] and prephenate dehydrogenase [lb]. The reaction of arogenate (AGN) dehydrogenase which is catalyzed by the [lb] component of the T-protein is shown at the bottom. The section of arogenate dehydratase which is not catalyzed by component [2b] of the P-protein is shown (dashed lines) at the top. Enzymes able to utilize both PPA and AGN as cyclohexadienyl substrates have been referred to as cyclohexadienyl dehydratases or as cyclohexadienyl dehydrogenases. Thus, the T-protein is a cyclohexadienyl dehydrogenase, but the P-protein is not a cyclohexadienyl dehydratase. aminotransferases able to form L-arogenate from We have indeed found that the P-protein de- prephenate in vivo. Hence, the competing channel- hydratase is specific for prephenate, whereas the ing systems essentially preclude the endogenous T-protein dehydrogenase still functions as a cyclo- formation of L-arogenate. If abilities of the P- hexadienyl dehydrogenase. The T-protein de- protein and T-protein to use L-arogenate have lost hydrogenase is actually quite similar to the selective value, then enzymatic ability to use L- cyclohexadienyl dehydrogenases present in Super- arogenate as substrate might diminish with evolu- family-B organisms that lack the evolved T-protein tionary time. Thus, this might be expected to be [8,9]. Partially purified T-protein from E. co/i and more pronounced for the P-protein than for the T- K. pneumoniae was used to demonstrate the protein because the P-protein gene originated utilization of L-arogenate as an alternative much earlier. substrate for prephenate. Other generic members 134 Volume 216, number 1 FEBS LETTERS May 1987 belonging to the enteric lineage also possess T- Protein in the crude extract was estimated by the proteins able to utilize L-arogenate as substrate. method of Bradford [13] with bovine serum albumin as the standard. 2. MATERIALS AND METHODS 2.3. DE52 column chromatography 2.1. Bacterial strains and culture conditions Approx. 100 mg crude extract protein were ap- E. co/i K12 ATCC 10798, E. coli B ATCC plied to a DEAE-cellulose (DE52) column 23226, E. coli C ATCC 13706, E. coli W ATCC (1.5 x 20.0 cm) equilibrated in 10 mM K- 9637 and K. pneumoniae ATCC 25304 were ob- phosphate buffer (pH 7.0) containing 1 mM DTT. tained from the American Type Culture Collection The column was washed with two bed volumes of (Rockville, MD). The organisms were grown as the equilibrating buffer, and then the bound pro- described [lo]. teins were eluted with 300 ml of a linear gradient of KC1 (0.0-0.4 M) contained in the equilibration 2.2. Preparation of cell extracts and enzyme buffer. Fractions of 2.2 ml were collected. assays Crude cell-free extracts (free of small molecules) 2.4. Biochemicals and chemicals were prepared as in [lo]. Amino acids, NAD, NADP, DTT, OPA, bovine Chorismate mutase was assayed by the method serum albumin and Sephadex G-25 were obtained of Cotton and Gibson Cl]. Prephenate dehydro- from Sigma (St. Louis, MO). DE52 was purchased genase and arogenate dehydrogenase activities from Whatman (Clifton, NJ). Prephenate was were assayed by following the continuous forma- prepared as the barium salt from culture super- tion of reduced nicotinamide adenine dinucleotide natants of a tyrosine auxotroph of Salmonella (NADH). using a spectrophotofluorometer as typhimurium [14] and was converted to the described by Pate1 et al. [l 11. potassium salt before use. Chorismate was isolated To confirm the formation of L-tyrosine in the from the accumulation medium of a triple aux- arogenate dehydrogenase reaction, the enzyme was otroph of K. pneumoniae 62-l and purified as the assayed as described below followed by high- free acid [15]. L-Arogenate was prepared from a performance liquid chromatographic (HPLC) triple auxotroph of Neurospora crassa as in [16]. detection of the tyrosine formed as outlined by All other chemicals were standard reagent grade. Lindroth and Mopper [12]. The reaction mixture in a final volume of 225 ~1 contained 50 mM K 3. RESULTS AND DISCUSSION phosphate (pH 7.0) containing 1 mM dithiothrei- to1 (DTT), 0.5 mM NAD, 1.25 mM K arogenate The elution profiles of the enzyme activities par- and a suitable amount of enzyme protein. After in- ticipating in the biosynthesis of tyrosine from E. cubation at 37’C for 20 min, the contents were coli K12 (top panel), E. coli B (middle panel), and transferred to ice for HPLC analysis. A 40~1 ali- E. coli W (bottom panel) following DE52 quot was derivatized with 20081 OPA reagent chromatography of the crude extracts are shown in [54 mg o-phthalaldehyde dissolved in 1 ml fig.2. Two peaks of chorismate mutase activity methanol, followed by the addition of 9.0 ml of eluted in each case. The leading peak of chorismate 0.4 M Na-borate buffer (pH 9.5) and 200 hl of mutase activity (chorismate mutase-P) co-eluted 2-mercaptoethanol]. After 90 s, the derivatized with the prephenate dehydratase activity, thus sample was injected into a 20 ,ul loop and swept on- identifying the location of the bifunctional P- to a Cis column (Alltech Associates, Deerfield, IL) protein (not shown). The trailing activity peak of (4.6 x 250 mm) equilibrated and eluted with chorismate mutase (chorismate mutase-T) co- methanol/20 mM Na-phosphate buffer (pH 6.8) eluted with the prephenate dehydrogenase activity, (45: 55, v/v) at a flow rate of 1 ml/min. Controls thus marking the position of the bifunctional T- in which only enzyme was incubated, only protein in each case. All column fractions ex- substrates were incubated, or in which enzyme was hibiting prephenate dehydrogenase activity also added to the other components at zero time (just showed activity when arogenate was used as an before derivatization) were also included. alternative substrate. Thus, arogenate dehydro- 135 Volume 216, number 1 FEBS LETTERS May 1987 Chorlsmste mutsse 3.2 H Prephenste dehydrogenase E.

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