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Pathway for Isoleucine Formation from Pyruvate by Leucine Biosynthetic Enzymes in Leucine-Accumulating Isoleucine Revertants of Serratia Marcescens

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Pathway for Isoleucine Formation from Pyruvate by Leucine Biosynthetic Enzymes in Leucine-Accumulating Isoleucine Revertants of Serratia Marcescens J. Biochem., 82, 95-103 (1977) Pathway for Isoleucine Formation from Pyruvate by Leucine Biosynthetic Enzymes in Leucine-Accumulating Isoleucine Revertants of Serratia marcescens Masahiko KISUMI, Saburo KOMATSUBARA, and Ichiro CHIBATA Research Laboratory of Applied Biochemistry, Tanabe Seiyaku Co., Ltd., Kashima, Yodogawa-ku, Osaka, Osaka 532 Received for publication, January 19, 1977 Leaky revertants isolated from isoleucine auxotrophs of Serratia marcescens mutant resistant to a-aminobutyric acid were previously reported to accumulate leucine in the medium, due to the absence of both feedback inhibition and repression of leucine biosynthesis. Growth of the revertant was accelerated by pyruvate, n(-)-citramalate, citraconate, and a-ketobutyrate, but not by threonine. Extracts of the revertant exhibited high activities of pyruvate-dependent coenzyme A liberation from acetyl-coenzyme A, hydration of citraconate, and conversion of citraconate to a-ketobutyrate, but showed no threonine-deaminating activity. In the leucine accumulating revertants the above three activities were not affected by leucine, but in the wild strain and other revertants accumulating no leucine all or one of these activities was controlled by leucine. A leucine auxotroph isolated from the leucine-accumulating revertant showed isoleucine auxotrophy as well. From these data, it is concluded that, in leucine-accumulating revertants of S. marcescens, isoleucine is synthesized from a-ketobutyrate via citramalate formed from pyruvate and acetyl-coenzyme A by leucine biosynthetic enzymes, as a result of desensitization of a-isopropylmalate synthetase to feedback inhibition. Generally isoleucine is synthesized from aspartate viously isolated leaky revertants from isoleucine via threonine in microorganisms. L-Threonine auxotrophs of S. marcescens mutants resistant to dehydratase [EC 4.2.1.16] is the rate-limiting enzyme a-aminobutyric acid. Most of the isoleucine re for isoleucine biosynthesis. Nevertheless, some vertants accumulated large amounts of leucine in microorganisms are known to form isoleucine from the medium (6). Leucine accumulation was precursors other than threonine by other pathways. ascribed to both constitutive synthesis of leucine We have been studying biosynthetic regulation biosynthetic enzymes and desensitization of a and accumulation of branched-chain amino acids isopropylmalate synthetase [EC 4.1.3.12], the first using Serratia marcescens mutants (1-6). In S. enzyme of leucine biosynthesis, to feedback inhibi marcescens as well as other enteric bacteria, iso tion. The constitutive synthesis and desensitiza leucine is synthesized from threonine by five iso tion resulted from a-aminobutyric acid resistance leucine-valine biosynthetic enzymes. We pre and reversion of isoleucine auxotrophy, respectively. Vol. 82, No. 1, 1977 95 96 M. KISUMI, S. KOMATSUBARA, and I. CHIBATA Interestingly, the partial reversion of isoleucine Enzyme Assays-The bacteria were cultured auxotrophy in leucine-accumulating revertants did in minimal medium under the conditions described not depend on the restoration of L-threonine de in the previous paper (4). The cells were harvested hydratase activity. Evidence for the absence of the from exponentially growing culture by centrifuga activity in vivo will be given in a separate paper tion and extracts were prepared by the method (in preparation). It is also suggested that a single described previously (4, 6). Protein was de mutational event influences both isoleucine auxo termined as described previously (4). Pyruvate trophy and the sensitivity of a-isopropylmalate dependent coenzyme A liberation from acetyl synthetase to feedback inhibition. It appears that coenzyme A was measured under the conditions these revertants might not synthesize isoleucine via described previously (6) using pyruvate instead of threonine; i.e., might not require the participation a-ketoisovalerate as a substrate. To assay citra of L-threonine dehydratase. conate hydration, the method for a-isopropyl This report describes a pathway for isoleucine malate isomerase [EC 4.2.1.33] (8) was modified as formation from pyruvate and acetyl-coenzyme A, follows. The reaction mixture contained citra and suppression of isoleucine auxotrophy by conate (1.5 pmol), potassium phosphate buffer (pH desensitization of the first enzyme of leucine bio 7.0, 300 pmol), and cell-free extracts (0.3-0.5 mg of synthesis. protein) in a total volume of 3.0 ml. Incubation was carried out at room temperature in a cuvette MATERIALS AND METHODS with 1 cm light path. The rate of decrease in ab sorbance was followed with a Varian Techtron Bacteria-S. marcescens strain I and its mu spectrophotometer (model 635). a-Ketobutyrate tants listed in Table I were used. formation from citraconate was assayed by a mo Isolation of Revertants from Strain 1656-Cells dification of the (ƒÀ-isopropylmalate dehydrogenase of strain 1656 were spread on agar plates of Davis [EC 1.1.1] (9) as follows. The reaction mixture Mingioli minimal medium (7) modified by omitting contained citraconate (20 ƒÊmol), KCl (50 ƒÊmol), citrate and increasing glucose to 0.5 %. Incubation MgCl2 (10 ƒÊmol), nicotinamide dinucleotide (5 was carried out at 30°C for 3-5 days. Colonies ƒÊ mol), potassium phosphate buffer (pH 8.0, 100 were selected and purified by single colony isolaƒÊƒÊ mol), and cell-free extracts (0.5-1.0 mg of protein) tion. in a total volume of 1.0 ml. Incubation was carried Growth Experiments-Growth experiments out at 30•Ž and stopped by the addition of 10 were performed in minimal medium as described trichloroacetic acid. a-Ketobutyrate formed was previously (3); the medium was inoculated with 106 determined by the method of Friedemann and cells per ml. Haugen (10). Bioassay of a-ketobutyrate was also TABLE I. Strains used. a Nitrosoguanidine. b a-Aminobutyric acid-resistant. J. Biochem. PATHWAY FOR ISOLEUCINE FORMATION FROM PYRUVATE 97 performed using strain 621 (L-threonine dehydrat Moreover, a-ketobutyrate-forming activities from ase-deficient strain which exhibits growth response these amino acids were very low in the revertants to either a-ketobutyrate or isoleucine, but not to as well as in the wild strain. citraconate or other isoleucine precursors). Speci Charon et al. presented indirect evidence for fic activities were expressed as Icmol of products isoleucine biosynthesis from pyruvate in aerobic formed or substrate reacted per mg of protein per spirochetes (15). Sai et al. reported that citra min. malate accumulated in the medium is formed from Chemicals-D(-) and L(-}-)-Citramalates were pyruvate in yeast mutants (16). Sasaki et al. stated obtained from Sigma Chemical Co. (St. Louis, Mo). that citraconate is degraded to a-ketobutyrate by Unless otherwise noted, the n(-)-form was used. extracts of a Bacillus strain grown on n-methyl malate as a sole carbon source (17). We found RESULTS that various a-keto acids are converted to a-keto acids possessing one more carbon atom and to Effects of Various Compounds on the Growth of analogs of branched-chain amino acids by S. Strains 149, 149-V17, and S-11-To identify the marcescens mutants (18). Therefore, we tested isoleucine biosynthetic pathway in leucine-accumu whether isoleucine is formed from pyruvate via lating revertants, growth experiments were carried citramalate and citraconate. out. Several pathways for isoleucine formation Pyruvate accelerated the growth of strain S-11, are known in aerobic microorganisms (Fig. 1). a representative leucine-accumulating revertant, in Phillips et alL suggested a pathway from glutamate which a-isopropylmalate synthetase is insensitive via ƒÀ-methylaspartate in Escherichia coli (11, 12). to feedback inhibition by leucine (Table II). On The growth of revertants and isoleucine auxotrophs the other hand, pyruvate had no effect on the of S. marcescens was stimulated by (ƒÀ-methylas growth of strains 149 and 149-V 17, in which the partate, but not by glutamate. Glutamate mutase synthetases are sensitive. n(-)-Citramalate and [EC 5.4.99.1] activity, forming ,B-methylaspartate, citraconate had stimulating effects on the growth was not found in the cell-free extracts by the method of these three strains, though at high concentrations. of Phillips et al. (11). Therefore, it was concluded L(+)-Citramalate and mesaconate, stereoisomers that S. marcescens mutants lack the glutamate route of the two compounds, had no effect on growth. for isoleucine formation. These data strongly suggest that, in leucine-accumu Homoserine is directly converted to a-keto lating revertants, isoleucine is formed from pyruvate butyrate by extracts of Neurospora crassa (13). by the leucine biosynthetic enzymes via citramalate Homocysteine and methionine may also be de and citraconate as intermediates of a-ketobutyrate graded to a-ketobutyrate in some bacteria (14). formation. These amino acids did not stimulate the growth of Pyruvate-Dependent Coenzyme A Liberation leaky isoleucine revertants of S. marcescens. from Acetyl-Coenzyme A and Its Control by Leucine Fig. 1. Possible pathways of a-ketobutyrate and isoleucine formation in microorganisms. Vol. 82, No. 1, 1977 98 M. KISUMI, S. KOMATSUBARA, and I. CHIBATA TABLE II. Effects of various compounds on the growth of strains 149, S-11, and 149-Vl7a. a The medium was inoculated with 106 cells per ml (optical density, 0.002). b a-Ketobutyrate and the other com pounds were added to the minimal medium at concentrations of 10-1 M and 5 •~ 10-1 M, respectively. -To identify any alternative isoleucine pathway, tase, we examined whether the liberation is con enzymatic studies were performed. It was con trolled by leucine or not (Table III). The coenzyme sidered that citramalate might be formed from A-liberating activities of strains 149 and S-11, which pyruvate and acetyl-coenzyme A by a-isopropyl are derepressed for a-isopropylmalate synthetase, malate synthetase. Therefore, the pyruvate-de were high with or without the addition of leucine pendent liberation of coenzyme A was examined by to minimal medium, while that of the wild strain the method of Kohlhaw et al. (19). As expected, was low in the minimal medium and decreased on marked liberation of coenzyme A was observed addition of leucine.
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