Biosci. Biotechnol. Biochem., 70 (5), 1102–1109, 2006 Purification and Characterization of Fumarase from Corynebacterium glutamicum y Tomoko GENDA,1 Shoji WATABE,2 and Hachiro OZAKI1; 1Biological Institute, Faculty of Education, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8513, Japan 2Basic Laboratory Science, Faculty of Health Sciences, Yamaguchi University School of Medicine, 1-1-1 Minami-Kogushi, Ube 755-8505, Japan Received August 2, 2005; Accepted December 10, 2005 Fumarase (EC 4.2.1.2) from Corynebacterium gluta- fumarases.9–12) micum (Brevibacterium flavum) ATCC 14067 was puri- Corynebacterium glutamicum is an industrially im- fied to homogeneity. Its amino-terminal sequence (res- portant microorganism widely used in the production idues 1 to 30) corresponded to the sequence (residues 6 of various amino acids. TCA-cycle members of the to 35) of the deduced product of the fumarase gene of enzymes of this bacterium, citrate synthase (EC C. glutamicum (GenBank accession no. BAB98403). The 4.1.3.7),13) isocitrate dehydrogenase (EC 1.1.1.42),14,15) molecular mass of the native enzyme was 200 kDa. The 2-oxoglutarate dehydrogenase (EC 1.2.4.2),16) malate protein was a homotetramer, with a 50-kDa subunit dehydrogenase (EC 1.1.1.37),17–19) and a membrane- molecular mass. The homotetrameric and stable prop- associated malate:quinone oxidoreductase (MQO) (EC erties indicated that the enzyme belongs to a family of 1.1.99.16)18,19) have been studied with respect to Class II fumarase. Equilibrium constants (Keq) for the metabolic regulation. As for fumarase of the bacterium, enzyme reaction were determined at pH 6.0, 7.0, and its amino-acid sequence deduced from the gene has been 8.0, resulting in Keq ¼ 6:4, 6.1, and 4.6 respectively in reported (GenBank accession no. BAB98403). Hence, phosphate buffer and in 16, 19, and 17 in non-phosphate we investigated the biochemical properties of fumarase buffers. Among the amino acids and nucleotides tested, of this bacterium. ATP inhibited the enzyme competitively, or in mixed- Recent studies on fumarase have been concentrated type, depending on the buffer. Substrate analogs, meso- upon unraveling the reaction mechanisms in the three- tartrate, D-tartrate, and pyromellitate, inhibited the dimensional structures of the active center using com- enzyme competitively, and D-malate in mixed-type. petitive inhibitors for fumarases of pig heart,20) E. coli,21) and yeast.22) This paper deals with purification Key words: fumarase; Corynebacterium glutamicum; and characterization of fumarase from C. glutamicum, Brevibacterium flavum; equilibrium con- including inhibition by substrate-analogs. stant; homotetramer Materials and Methods Fumarase or fumarate hydratase (EC 4.2.1.2), which catalyzes the reversible hydration of fumarate to L- Materials. Sodium fumarate, sodium L-malate, so- malate, is an integral part of the tricarboxylic acid dium L-tartrate, D-tartaric acid, meso-tartaric acid, urea, (TCA) cycle. Most studies have been concerned with DTT, and DTNB were products of Wako Pure Chemical mammalian fumarases.1–8) Fumarases from microorgan- Industries (Osaka, Japan). D-Malic acid was purchased isms have been studied less extensively. In Escherichia from Sigma (St. Louis, MO). L-Amino acids were coli, three kinds of fumarases, fumarase A (FumA), B products of Ajinomoto (Tokyo). Toyopearl-Phenyl- (FumB), and C (FumC), have been found.9) FumA and 650M was a product of Tosoh (Tokyo). Pyromellitic FumB are fumarases of a class I form, which are acid was a product of Tokyo Kasei Chemical (Tokyo). homodimeric, labile, and iron-dependent enzymes hav- All other chemicals used were the same as reported in ing a molecular mass of 120 kDa. FumC is of a class II the previous paper.17) form, which is a homotetrameric, stable, and iron- independent enzyme having a molecular mass of Microorganism and culture. A wild-type strain of 200 kDa. The biochemical properties of FumC have C. glutamicum ATCC14067 (Brevibacterium flavum been reported to be homologous to the mammalian no. 2247) was used. The nutrient agar medium (CM-2) y To whom correspondence should be addressed. Fax: +81-83-933-5357; E-mail: [email protected] or [email protected] Abbreviations: DTT, 1,4-dithiothreitol; DTNB, 5,50-dithiobis(2-dinitrobenzoic acid); MES, 2-(N-morpholino)ethanesulfonic acid; TES, N- tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid Fumarase from C. glutamicum 1103 was composed, in g/l, of Polypepton, 10; yeast extract, of fumarase preparation properly diluted with 10 mM of 10; NaCl, 5; and agar, 20 (pH 7.0), and was sterilized at the buffer in the total volume of 1 ml, was used. The 120 C for 20 min. The medium for seed and main reaction was started by adding the enzyme. The initial culture (G30) was composed, in weight/l, of glucose, absorbance change at 250 nm was measured on a 36 g; urea, 10 g; KH2PO4, 1 g; MgSO4.4H2O, 0.4 g; spectrophotometer (Ultrospec 3000, Amersham Phar- FeSO4.7H2O, 10 mg; MnSO4.4H2O, 8.1 mg; thiamine. macia Biotech, Cambridge UK) at room temperature HCl, 100 mg; d-biotin, 30 mg; and 6 N HCl, 7 ml. For the (20–23 C). The activities were expressed in units (mmol À1 seed and main cultures, 50 ml and 3 liters of medium G30 fumarate formed min ) using an extinction coefficient were autoclaved in a 500-ml flask and a 5-liter jar of 1.45 mMÀ1 cmÀ1 at 250 nm.1) The reverse reaction respectively at 115 C for 10 min (final pH, 7.0). A was assayed in the same way except that 10 mM sodium loopful of cells of the bacterium grown on CM-2 at fumarate was used as substrate, and the absorbance 30 C for 24 h was inoculated into the flask and cultured change at 290 nm was measured. The activities were with shaking at 120 rpm at 30 C for 24 h as seed culture. calculated using an extinction coefficient of 0.109 À À The broth of one flask of the seed culture was inoculated mM 1 cm 1 at 290 nm.1) Protein was measured by the into the main-culture jar with 200 rpm of agitation and method of Lowry et al.,23) with bovine serum albumin as 1 liter/liter/min of aeration at 30 C for 20–24 h on a jar the standard. fermentor (model MD-300, Marubishi, Tokyo). The cells were collected by centrifugation, washed with Gel filtration. One ml of 50 mM Tris–HCl buffer, 0.2% of KCl, and stored at À20 C. pH 7.5, containing 3 mg of thyroglobulin (bovine, 670 kDa), 1 mg of urease (soybean, 480–490 kDa), 2 mg of Purification of fumarase. The washed cells (20 g wet catalase (bovine, 248 kDa), 0.5 mg of alcohol dehydro- weight) were suspended in 30 ml of 50 mM sodium genase (yeast, 150 kDa), 0.5 mg of peroxidase (horse phosphate buffer, pH 7.0 (buffer A) and disrupted in radish, 40 kDa), and 20 ml of purified fumarase was gel- a sonic oscillator (Kubota 201, 9 kHz, Kubota Shoji, filtered through a Toyopearl HW-60 column (1:9 Â Tokyo) under cooling (below 6 C) for 20 min. The 40 cm) using the same buffer. sonic extracts from 160 g of the cells were pooled and centrifuged at 100;000 Â g for 40 min. The supernatant Native polyacrylamide gel electrophoresis (PAGE). was treated with 2.0% (w/v) streptomycin sulfate for Native PAGE was done in 7.5% gel using the ATTO- 60 min, and the precipitate was removed by centrifuga- Compact PAGE system (Atto, Tokyo). The gel was tion (30;000 Â g for 20 min). The resulting supernatant stained with Coomassie Brilliant Blue R-250. was fractionated by ammonium sulfate precipitation and centrifugation. The pellet obtained between 40% and SDS–PAGE. SDS–PAGE was done in 11% gel using 60% was dissolved in buffer A and dialyzed against the ATTO-Compact PAGE system and a kit for mo- 10 mM Tris–acetate buffer, pH 7.3 (buffer B). The lecular weight purchased from Sigma (St. Louis, MO). dialyzed sample was chromatographed using a DEAE- Toyopearl 650M column (2:64 Â 15 cm) and 500 ml of Amino-terminal sequence analysis. The amino-termi- buffer B containing 0.1 M NaCl and 500 ml of buffer B nal amino-acid sequence of the purified enzyme was containing 0.4 M NaCl. The fractions showing fumarase determined by use of a protein sequencer (ABI, Model activity were pooled and dialyzed against buffer B. The 476A). dialyzed sample was rechromatographed in the same way except for the use of a smaller column (1:9 Â Results and Discussion 11 cm) and 100 ml each of the buffer. The fumarase fractions obtained on the second chromatography were Purification of fumarase pooled, added to solid ammonium sulfate to give a final Fumarase was purified from strain no. 2247 by concentration of 2.0 M, and applied to a Phenyl-Toyo- ammonium sulfate precipitation, DEAE-Toyopearl, and pearl 650M column (1:5 Â 10 cm) equilibrated with Phenyl-Toyopearl column chromatographies. A summa- 10 mM potassium phosphate buffer, pH 7.3 (buffer C), ry of the purification of the enzyme is shown in Table 1. containing 2 M ammonium sulfate. After the column was Fumarase was purified 252-fold, with a 12% yield. washed with the buffer, chromatography was performed PAGE of the enzyme preparation showed a single with a linear concentration gradient of ammonium protein band, as shown in Fig. 1B, indicating that the sulfate, 2 to 0 M in buffer C (100 ml each).