3-Isopropylmalate dehydrogenase was purified to homogeneity from the acidophilic autotroph ... The crystal structure of T. ferrooxidans 3-isopropylma-.
JOURNAL
OF BIOSCIENCE
AND BIOENGINEERING
Vol. 90, No. 4, 459-461. 2000
Purification and Characterization of 3-Isopropylmalate Dehydrogenase from Thiobacillus thiooxidans HIROSHI
KAWAGUCHI,*,s
KENJI INAGAKI, HIDEYUKI MATSUNAMI, TATSUO TANO, 5 and HIDEHIKO TANAKA
YUMI NAKAYAMA,
Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University, Okayama 700-8530, Japan Received17 May 2OOOIAccepted 10 July 2000 3-Isopropylmalate dehydrogenase was purified to homogeneity from the acidophilic autotroph Thiobacillus thiooxidans. The native enzyme was a dimer of molecular weight 40,000. The apparent K,,, values for 3isopropylmalate and NAD+ were estimated to be 0.13 mM and 8.7 mM, respectively. The optimum pH for activity was 9.0 and the optimum temperature was 6S’C. The properties of the enzyme were similar to those of the Thiobacillus ferrooxidans enzyme, expect for substrate specificity. T. thiooxidans 3-isopropylmalate dehydrogenase could not utilize malate as a substrate. [Key words: Thiobacillus thiooxiduns, 3-isopropylmalate dehydrogenase, purification]
fermentor (MSJ-U2W 5OL, B.E.Marubishi, Tokyo) (17). The pH of the broth and the Na&03 concentration were kept at 5.0 using K&O3 and at 0.25% (w/v) by feeding, respectively. The standard enzyme assay mixture consisted of 1OOmM Tris-HCl buffer (pH9.0), 0.5 mM Mg&, 50mM KCl, 6.7 mM NAD+, 0.67 mM 3-isopropylmalate, and appropriately diluted enzyme in a final volume of 1.5 ml (18). Enzyme activity was measured by monitoring the production of NADH at 340 nm using a Beckman DU-65 spectrophotometer. All the purification procedures described below were carried out at 4°C. Frozen cells (76g) were suspended in 50 mM potassium phosphate buffer (pH 7.5) containing 0.01% 2-mercaptoethanol and 10% glycerol. The suspension was disrupted by ultrasonic oscillation (Kubota Insonator Model 201 M) in an ice bath for 10 min. Cell debris and unbroken cells were removed by centrifugation at 105,OOOxg for 60min. The extract was dialyzed at 4°C against 1OmM potassium phosphate buffer (pH 7.5) containing 0.01% 2-mercaptoethanol and 10% glycerol (Buffer A). The dialyzed solution (185 ml) was applied onto a column of DEAE-Toyopearl 650M ($5.0 x 20 cm) (Tosoh) equilibrated with Buffer A containing 50mM KCl. After washing with the same buffer, the enzyme was eluted with Buffer A containing 1OOmM KC1 (Buffer B). The enzyme solution was applied to a QSepharose Fast Flow column ($3.0 X 10 cm) (Pharmacia, Uppsala, Sweden) equilibrated with Buffer B. After washing with Buffer B, the active fractions were eluted with Buffer A containing 200mM KC1 (Buffer C). The fractions were combined and concentrated (45 ml). The enzyme solution was brought to 25% saturation with ammonium sulfate and then applied to a Butyl-Toyopearl 650M column (42.0 x 10 cm) (Tosoh, Tokyo) equilibrated
In bacteria, leucine is biosynthesized by the actions of four enzymes, 2-isopropylmalate synthetase encoded by IeuA, isopropylmalate isomerase encoded by IeuC and ZeuD, 3-isopropylmalate dehydrogenase encoded by leuB, and aminotransferase. 3-Isopropylmalate dehydrogenase (EC 1.1.1.85), a key enzyme in leucine biosynthesis, catalyzes the oxidative decarboxylation of 3-isopropylmalate to 2-oxoisocaproate simultaneously with dehydrogenation. This enzyme has been found in a wide variety of bacteria and the amino acid sequences of the enzymes from various bacteria show high homology (l10). Thiobacillus thiooxidans is a chemolithotrophic, acidophilic bacterium that obtains energy from the oxidation of reduced inorganic sulfur compounds. This bacterium is one of the most important microorganisms for the bacterial leaching of sulfide ores and for the cycling of inorganic sulfur compounds in the natural environment. We previously cloned the 1euB gene encoding the 3-isopropylmalate dehydrogenase of an acidophilic chemolithotrophic bacterium, Thiobacillus ferrooxidans, in E. coli (11) and purified the enzyme to homogeneity from E. coli cells harboring a recombinant plasmid containing the leuB gene (12). The T. ferrooxidans enzyme utilizes various alkyl-malate compounds as substrate, in addition to 3-isopropylmalate (13), which is similar to an extreme thermophile, Thermus thermophilus (14) and thermoacidophilic archaeon, Sulfolobus sp. strain 7 (15). The crystal structure of T. ferrooxidans 3-isopropylmalate dchydrogenase complexed with 3-isopropylmalate at 2.OA resolution was also determined (16). The structure exhibits a fully closed conformation. The r-isopropyl group of 3-isopropylmalate is recognized by a unique hydrophobic pocket which includes Glu88, Leu91, Leu92, and Va193’. In this paper, we describe the purification and characterization of the 3-isopropylmalate dehydrogenase from T. thiooxidans. For enzyme preparation, T. thiooxidans ON107 was
with Buffer A containing 25% ammonium sulfate (Buffer D). After a thorough wash with the same buffer, the enzyme was eluted using a linear ammonium sulfate gradient of 25 to 0% in Buffer A. The active fractions
grown on an iron-based medium with 0.25% NazSz03 as the sole energy source at 30°C for 60 h using a 50-I jar
were pooled, concentrated and dialyzed against Buffer C and the dialyzate was applied to a Sephacryl S-200 column ($1.6 x 95 cm) (Pharmacia) equilibrated with Buffer
* Correspondingauthor. * Present address: Kurashiki Sakuyo University, Kurashiki, Okayama710-0292, Japan.
C. The enzyme was eluted with the same buffer. The concentrated enzyme solution was brought to 25% satura459
460
KAWAGUCHI TABLE
ET AL.
J. BIOSCI. BIOENG.,
1. Purification of 3Gsopropylmalate dehydrogenase from T. thiooxidans
Crude extract DEAE-Toyopearl650M Q-Sepharose Butyl-Toyopearl650M Sephacryl S-200 Phenyl-Toyopearl650M Mono Q
Total protein (mg) 1810
Total Specific activity activity= (units) (units/mg)
58.7 38.3 5.07 1.59 0.13 0.09
11.3 21.2 21.9 14.4 4.28 0.88 0.67
0.0428 0.361 0.572 2.84 2.69 6.69 7.44
Yield (%, 100 27.4 28.3 18.6 5.5
w
1.1 0.9
a Specific activity is defined as pmol of NADH formed per mg of protein per min.
tion with ammonium sulfate and applied to a PhenylToyopearl 650M column ($1 .Ox 3.8 cm) (Tosoh) equilibrated with Buffer D. After washing with the same buffer, the enzyme was eluted with Buffer A containing 17.5% ammonium sulfate. The concentrated active fraction was applied to a Mono Q column ($3.0 x 7.5 cm) (Pharmacia) equilibrated with 20 mM Tris-HCl buffer (pH 7.5). The enzyme solution was eluted with a linear KC1 gradient of 120 to 140mM in Tris-HCl buffer (pH 7.5). The purification procedure is summarized in Table 1. The purified enzyme was homogeneous as judged from the results of disc gel electrophoresis using a 7.5% polyacrylamide gel according to the method of Davis (19) (Fig. 1). The molecular weight of the native enzyme was determined to be 70,000 by gel filtration with a Pharmacia fast-protein liquid chromatography system in a Hiload 16/60 Superdex 200 column ($1.6 x 60 cm). The subunit structure was determined by SDS-polyacrylamide gel electrophoresis. The molecular weight of the denatured enzyme using 0.1% SDS was estimated to be about 40,000. The difference in apparent molecular weight obtained using gel filtration and SDS-polyacrylamide electrophoresis is thought to be due to the interaction of subunits. Thus, the enzyme appeared to be a dimer composed of two identical subunits, a structure similar to that of the T. ferrooxidans 3-isopropylmalate dehydrogenase. Except for the archaeon Sulfolobus sp. enzyme (15), prokaryotic 3-isopropylmalate dehydrogenase is homodimeric. The enzyme exhibited maximum activity at pH 9.0.
,A 20
FIG. 1. Purity of 3-isopropylmalate dehydrogenase from T. thiooxidans. The purified protein was subjected to 7.5% polyacrylamide gel electrophoresis. Staining was with Coomassie Brilliant Blue R-250.
When the enzyme was assayed at various temperatures, maximum activity was found at 65°C (Fig. 2). The enzyme retained about 90% of the original activity after heating at 60°C for 30min (Fig. 2). These properties of the T. thiooxidans enzyme were similar to those of the acidophilic autotroph T. ferrooxidans enzyme (12). The presence of a divalent cation (e.g., Mg2+ or Co2+, but not Mn2+) was required for enzymatic activity. These divalent cations may be part of the substrate, forming a salt with 3-isopropylmalate. Double reciprocal plots of initial velocity against NAD+ concentration in the presence of various fixed concentrations of 3-isopropylmalate gave intersecting straight lines. From the secondary plots of the intercepts against the reciprocal of the fixed NAD+ concentrations, the apparent Km value for 3isopropylmalate was estimated to be 0.13 mM and that for NAD+ 8.7 mM. These values were about lo-fold higher than the values for the T. ferrooxidans enzyme. The ability of the enzyme to catalyze the dehydrogenation of various derivatives of 3-isopropylmalate was investigated, but none of them could be utilized as the substrate except for 3-isopropylmalate (Table 2). In addition to NAD+, various derivatives of NAD+ were investigated as coenzyme candidates, but none of them
40
Temperature (‘C) FIG. 2. Optimum temperature (left) and heat stability (right) of 3-isopropylmalate dehydrogenase from T. thiooxidans. (Left) The enzyme reaction was carried out in 100 mM Tris-HCl buffer @H 9.0). The reaction mixture without enzyme was preincubated for 10 min at each temperature. (Right) The enzyme was subjected to heat treatment for 30 min in 0.1 M Tris-HCl buffer (PH 9.0). The enzyme solution was then cooled and the activity remaining was assayed.
VOL.
NOTES
90, 2000 TABLE 2. Substrate and coenzyme specificities of 3-isopropylmalate dehydrogenase of T. thiooxidans Relative activity (%)
Substrates 3-Isopropylmalate D-Malate L-Malate 2-Isopropylmalate Dimethylcitraconate Citrate DL-Isocitrate Coenzymes NAD + NADPDeamino-NAD+ Thio-NAD’ 3-Acetylpyridine-NAD+ 3-Pyridinealdehyde-NAD+ 3-Pyridinealdehyde-deamino-NAD+
100 0 0 0 0 0 0 100 0 0 0 0 0 0
could be utilized (Table 2). The enzyme from SuZfolobus sp. is distinct from the enzymes from other microorganisms in respect to subunit composition and utilization of NADP+. Salmonella typhimurium (18), T. thermophilus (20), Sulfolobus sp. (15), and T. ferrooxidans (12) 3-isopropylmalate dehydrogenases have also been purified and their properties studied. The T. ferrooxidans (15) and T. thermophilus (14) enzymes are able to utilize alkyl-malate as a substrate in addition to 3-isopropyImaIate. However, the enzyme from T. thiooxidans is only able to utilize 3-isopropylmalate as the substrate. This indicates that the structure of the substrate binding site is different from that of the T. ferrooxidans and T. thermophilus enzymes. Following SDS-polyacrylamide gel electrophoresis, the separated protein was transferred to a PVDF membrane using Sartoblot II-S (Sartorius). The transferred protein was detected with 0.1% Coomassie Brilliant Blue and sequenced by Edman degradation using an Applied Biosysterns Model 477A gas liquid phase protein sequencer, The amino acid sequences of 3-isopropylmalate dehydrogenases from various microorganisms have been reported. The N-terminal amino acid sequence of the T. thiooxidans enzyme (MKKIAIFPGDGIGPEIVDAA) exhibits approximately 60% homology with the enzymes from other microorganisms. In particular, it exhibits 90% homology with the enzyme of T. ferrooxidans (MKKIAIFAGDGIGPEIVAAA), a species from the same genus. However, as mentioned previously, the T. thiooxidans enzyme was not able to utilize alkyl-malate as a substrate. Gene analysis of T. thiooxidans IeuB should yield more information about the structure of T. thiooxidans 3-isopropylmalate dehydrogenase. REFERENCES Kirino, H., Aoki, M., Aoshima, M., Hayashi, Y., Ohba, M., Yamagishi, A., Wakagi, T., and Oshima, T.: Hydrophobic interaction at the subunit interface contributes to the thermostability of 3-isopropyhnalate dehydrogenase from an extreme thermophile. Eur. J. Biochem., 220, 275-281 (1994). Kryger, G., WaIlon, G., Lovett, S., Ringe, D., and Petsko, G. A.: Revision of the amino-acid sequence of 3-isopropylmalate dehydrogenase from Salmonella typhimurium by means of X-ray crystallography. Gene, 164, 85-87 (1995).
461
3. Imai, R., Sekiguchi, T., Nosoh, Y., and Tsuda, K.: The nucleotide sequence of 3-isopropylmalate dehydrogenase from Bacillus subtilis. Nucl. Acids Res., 15, 4988 (1987). 4. Sekiguchi, T., Ortega-Cessna, J., Nosoh, Y., Ohashi, S., Tsuda, K., and Shigenori, K.: DNA and amino-acid sequence of 3isopropyhnalate dehydrogenase of Bacillus coagulans. Comparison with the enzymes of Saccharomyces cerevisiae and Thermus thermophilus. Biochim. Biophys. Acta, 867, 36-44 (1986). 5. Sekiguchi. T.. Suda, M., IshIi, T., Nosoh, Y., and Tsuda, K.: Thenucleotide sequence of 3-isopropylmalate dehydrogenase from Bacillw cardotenax. Nucl. Acids Res., 15, 853 (1987). 6. Kagawa, Y., Nojima, H., Nukiwa, N., Ishizuka, M., Nakajima, T., Yasuhara, T., Tanaka, T., and Oshima, T.: High guanine plus cytosine content in the third letter of codons of an extreme thermophile. DNA sequence of the isopropylmalate dehydrogenase of Thermus thermophilus. J. Biol. Chem., 259, 2956-2960 (1984). 7. Kirino. H. and Oshima, T.: Molecular cloning and nucleotide sequence of 3-isopropylmalate dehydrogenase gene (leuB) from an __.. extreme ___ _ thermophile, __ Thermus aauaticus YT-1. J. Biothem., 109, 85%857.(1991). 8. Godon, J. J., Chopin, M. C., and Ehrlich, S. D.: Branchedchain amino acid biosynthesis genes in Lactococcus lactis subsp. lactis. J. Bacterial., 174, 6580-6589 (1992). 9. Hamasawa, K., Kobayashi, Y., Harada, S., Yoda, K., Yamasaki, M., and Tamura, G.: Molecular cloning and nucleotide sequence of the 3-isopropylmalate dehydrogenase gene of Candida utilis. J. Gen. Microbial., 133, 1089-1097 (1987). 10. Andreadis, A., Hsu, Y. P., Hermodson, M., Koblhaw, G., and Schimmel, P.: Yeast LEU2 repression of mRNA levels by leucine and primary structure of the gene product. J. Biol. Chem., 259, 8059-8062 (1984). 11. Inagaki, K., Kawaguchi, H., Kuwata, Y., Sugio, T., Tanaka, H., and Tano, T.: Cloning and expression of the Thiobacillus ferrooxidans 3-isopropylmalate dehydrogenase gene in Escherichia coli. J. Ferment. Bioeng., 70, 71-74 (1990). 12. Kawaguchi, H., Inagaki, K., Kuwata, Y., Tanaka, H., and Tano, T.: 3-Isopropyhnalate dehydrogenase from chemolithoautotroph Thiobacillus ferrooxidans: DNA sequence, enzyme purification, and characterization. J. Biochem., 114, 370-377 (1993). 13. Matsunami, H., Kawaguchi, H., Inagaki, K., Egucbi, T., Kakinuma. K.. and Tanaka, H.: Overproduction and substrate specificity ‘of i-isopropylmalate dehydrogenase from Thiobacillus ferrooxid~ns. Biosci. Biotech. Biochem., 62. 372-373 (1998). 14. Miyazaki, K., KakInuma, K., Terasawa, H., &d Oshima, T:: Kinetic analysis on the substrate specificity of 3-isopropylmaLate dehydrogenase. FEBS Lett., 332, 35-36 (1993). 15. Suzuki, T., Inoki, Y., Yamagishi, A., Iwasaki, T., Wakagi, T., and Oshima, T.: Molecular and phylogenetic characterization of isopropylmalate dehydrogenase of a thermoacidophilic archaeon, Sulfolobus sp. strain 7. J. Bacterial., 179, 1174-l 179 (1997). ” 16. Imada, K., Inagaki, K., Matsunami, H., Kawaguchi, H., Tanaka. H.. Tanaka, N., and Namba, K.: Structure of 3isoprop$malate dehydrogenase in complex with 3-isopropylmalate at 2.OA resolution: the role of Glu88 in the unique substrate-recognition mechanism. Structure, 6, 971-982 (1998). 17. Silverman, M. P. and Lundgren, D. G.: Studies on the Thiobacillus ferrooxidans, I. An improved medium for harvesting procedure for securing high cell yields. J. Bacterial., 77, 642647 (1959). 18. Parsons, S. J. and Barns, R. 0.: Purification and properties of B-isopropylmalate dehydrogenase. J. Biol. Chem., 244, 9961003 (1969). 19. Davis, B. J.: Disc-electrophoresis, II. Method and application to human serum proteins. Ann. N. Y. Acad. Sci., 121, 404-427 (1964). 20. Yamada, T., Akutsu, N., Miyazaki, K., Kakinuma, K., Yoshida, M., and Oshima, T.: Purification, catalytic properties, and thermal stability of ihreo-Ds-3-isopropylmalate dehydrogenase coded by leuB gene from an extreme thermophile, Thermus thermophilus strain HB8. J. Biochem., 108, 449-456 (1990). ---
r
-
---.--..
