Leucine Dehydrogenase of a Thermophilic Anaerobe

Agric. BioL Chern., 51 (12), 3375-3381, 1987 3375 Leucine Dehydrogenase of a Thermophilic Anaerobe, Clostridium thermoaceticum: Gene Cloning, Purification and Characterization Hiroko Shimoi, Shinji Nagata,1" Nobuyoshi Esaki, Hidehiko Tanaka and Kenji Soda* Laboratory of Microbial Biochemistry, Institute for Chemical Research, Kyoto University, Uji, Kyoto 611, Japan Received July 30, 1987 The leucine dehydrogenase (L-leucine: NAD+ oxidoreductase, deaminating, EC 1.4. 1.9) gene of Clostridium thermoaceticum was cloned and expressed in Escherichia coli C600 with a vector plasmid, pICD242, which was constructed from pBR322 and the leucine dehydrogenase gene derived from C. thermoaceticum. The enzymeoverproduced in the clone was purified about 12 fold to homogeneity by heat treatment and another two steps with a yield of46%. The enzyme ofE. coli- pICD242was immunochemically identical with that of C. thermoaceticum. The enzyme has a molecular weight of about 350,000 and consists of six subunits identical in molecular weight (56,000). The enzyme is.not inactivated by heat treatment: at pH 7.2 and 75°C for 15min; at 55°C and various pH's between 6.0 and 10.0 for 10 min. The enzyme catalyzes the oxidative deamination of branched-chain L-amino acids and the reductive amination of their 2-oxo analogues in the presence of NAD+and NADH,respectively. The pro-S hydrogen at C-4 of the dihydronicotinamide ring of NADHis exclusively transferred to the substrate; the enzyme is B stereospecific. The enzymological properties are very similar to those of the Bacillus stearothermophilus enzyme [T. Ohshima, S. Nagata and K. Soda, Arch. Microbiol., 141, 407 (1985)]. Leucine dehydrogenase (L-leucine: NAD+ thermophilic bacteria, we have found that oxidoreductase, deaminating, EC1.4. 1.9) cat- the enzyme occurs also in several strains of alyzes the reversible deamination of L-leucine, a moderately thermophilic anaerobe, Clostri- and several other branched-chain and straight- dium thermoacticum. Wehave chosen C. chain L-amino acids.1} The enzyme occurs thermoaceticum AN28-4, which produces mainly in Bacillus species,2) and was purified leucine dehydrogenase most abundantly, as from B. cereus,3) B. subtilis^ B. sphaericus,2) an enzyme source. We here describe the and B. stearothermophilus5) to homogeneity. cloning and expression for the gene of C. The enzyme is applicable to the produc- thermoaceticum AN28-4 enzyme, in Escheri- tion3'^ and determination^ of branched chia coii, the rapid and simple purification, chain L-amino acids, and the assay of leucine and characterization of enzyme. This is the amino peptidase (EC 3.4.ll.1).8'9) The en- first report of leucine dehydrogenase of an- zyme has an antineoplastic activity against Ehrlich's ascites carcinoma.10) Ohshima et al. aerobic bacteria. have purified leucine dehydrogenase from a MATERIALS AND METHODS thermophile, B. stearothermophilus, and shown that the enzyme is very stable.5) In Materials. NAD+ and NADH were obtained from the course of screening for the enzyme in Kojin, Tokyo, Japan; 2-oxo acids (sodium salts) from * To whomall correspondence should be addressed. f Present address: Department of Agricultural Chemistry, Kochi University, Nangoku, Kochi 783, Japan. 3376 H. Shimoi et al. Sigma Chemical; amino acids from Nakarai Chemicals,phenazine methosulfate, and 0.24him nitroblue tetra- Kyoto, Japan. Other chemicals were analytical grade zolium. reagents. Purification of the enzyme. The leucine dehydrogenase of Strains and media. All the C. thermoaceticum strainsB. stearothermophilus was purified as described pre- used were isolated and identified by Dr. Akihiko viously.16) The enzyme was purified from the E. coli clone Nakayama,n) Tokai Regional Fisheries Research cells as follows. The cells were desrupted by ultrasonic Laboratory, Tokyo. C. thermoaceticum AN 28-4, whichoscillation at 0°C for lO min. The extracts were dialyzed at produces leucine dehydrogenase most abundantly, was4°C for 12hr against lOmMpotassium phosphate buffer used throughout this study. The cells were grown statically(pH 7.2) containing 0.02% 2-mercaptoethanol. This buffer at 60°C for 20h under anaerobic conditions in a mediumwas used as the standard buffer. All operations were (pH 7.2) containing 1.7% BBL-Trypticase, 0.3% BBL-performed at 0° to 5°C. Phytone, 0.6% glucose, 0.25% NaCl, 0.05% sodium thio-Step I. Preparation ofa crude extract. The washedE. glycolate, 0.025% L-cystine, 0.01% Na2SO3, and 0.07%coli clone cells (about 10 g wet weight) were suspended in agar. The cells, harvested by centrifugation, were washed20 ml of the buffer and disrupted by sonication. The intact with 0.85% NaCl and then with 10mM potassium phos-cells and debris were removedby centrifugation. phate buffer (pH 7.2) containing 0.02% 2-mercapto- Step 2. Heat treatment. The pH of the supernatant ethanol. The recombinants of E. coli C600 (F~, thi-1, solutionthr- was adjusted to 5.4 with 1 Macetate buffer (pH 1, leuB6) were grown in Luria-Bertani (L broth) medium5.0). The enzymesolution was incubated at 75°Cfor at 37°C. When necessary, ampicillin (25 jug/ml) and tetra-30 min, and then cooled in ice. The precipitate formed was cycline (15 /zg/ml) were added to the medium. removed by centrifugation. Step 3. Preparative gel electrophoresis. Preparative Enzyme andprotein assays. Leucine dehydrogenase wasslab gel electrophoresis was performed as described pre- assayed at 55°C. The standard assay mixture for oxidativeviously.^ The gel was crushed with a Teflon homogenizer, deamination contained 20 iumol of l-leucine, 2.5 /mioland ofthe enzyme was extracted with the buffer, followed by NAD+, 120 /miol of glycine-KCl-KOH buffer (pH 10.5),centrifugation. and enzyme in a final volume of 1.0ml. The assay systemStep 4. Gel filtration. The enzyme was applied to two for reductive amination consisted of 10 /imol of 2-oxoiso-tandem columns of Superose 12á" (1.0x30cm), in an caproate, 0.1/onol of NADH, 750/miol of NH4C1- Pharmacia FPLC system, with 0.15m NaCl and 0.01% 2- NH4OH buffer (pH 9.5), and enzyme in a final volumemercaptoethanol of as the mobile phase at a flow rate of 1.0ml. One unit of the enzyme is defined as the amount0.4ml/min. of The active fractions were pooled and con- enzyme that catalyzes the formation of 1 ,umol of NADHcentrated by ultrafiltration with an Amicon PM10 per min in the oxidative deamination with a molar absorption coefficient for NADH (6220M^cm"1). Spe- membrane. cific activity is expressed as units per mg of protein. Pro-Ultracentrifugal analysis and molecular weight determi- tein was assayed by the method ofLowry et al.,12) withnation. eggThe purity of the enzyme and its sedimentation albumin as a standard. Protein elution patterns were es-coefficient were determined with a Spinco model E ultra- timated by the 280-nm absorption. The concentration centrifugeof in the same manner as described previous- the purified enzyme was determined with an absorptionly.1^ The molecular weight of the enzyme was also esti- £=9.\\),coefficient which {A\° was determined by the mated with Superose 12á"under the same conditions method of Perlman and Longworth.13) described above. Pig muscle lactate dehydrogenase (Mr, 142,000), bovine liver catalase (Mr, 232,000), horse Cloning of the gene for C. thermoaceticum leucine dehy-spleen ferritih (Mr, 440,000), and bovine thyroid thyro- drogenase. C. thermoaceticum AN 28-4 cells were harvest-globulin (Mr, 669,000) were used as marker proteins. ed in the late log phase (at about 20hr). They were The subunit molecular weight was determined by the incubated with lOmg/ml of lysozyme at 37°C for 30min,method of Laemmli.18) The marker proteins used were: and then the suspension was frozen and thawed repeatedlysoybean trypsin inhibitor (Mr, 20,100), carbonic anhy- twice. The chromosomal DNA (10fig), which was isolateddrase (Mr, 30,000), ovalbumin (Mr, 43,000), and bovine essentially according to the procedure of Saito and serum albumin (Mr, 67,000). Miura,14) was digested with Hindlll, and the resulting fragments were ligated into the Hindlll site of pBR322 Immunochemical analysis. Antiserum against the C. (3 fig). The recombinant E. coli library was screened thermoaceticumfor leucine dehydrogenase was obtained from the expression of the C. thermoaceticum leucine dehy-an adult male rabbit by subcutaneous injection of the drogenase gene essentially by the method of Inagaki ethomogeneous enzyme (0.5mg in lml of 0.9% NaCl) al.15) in a reaction mixture containing 50mM L-leucine,emulsified in an equal volume of Freund's complete 50mM Tris-HCl buffer (pH 9.0), 0.625mM NAD+, 64^madjuvant (Difco). Booster injections were given twice Leucine Dehydrogenase 3377 (1 mg each), after 2 and 3 weeks. Oneweek after the final it by nick translation, and then hybridized it injection, blood was collected from the heart and serum with C. thermoaceticum and E. coli C600chro- was obtained. Immunodiffusionanalyses were performed mosomal DNAsby the method of Southern.20* according to the procedure of Ouchterlony.19) The probe could hybridize only to the 5.4kb Stereochemical analysis of hydrogen transfer from C-4 of Hindlll fragment from C. thermoaceticum. the dihydronicotinamide ring of NADH. [4S-3H] NADH, This shows that the isolated DNAfragment is with a specific activity of 2.9 x 105cpm/mmol,was pre- derived from C. thermoaceticum. The extract pared by the previous method.2) Hydrogen transfer of [4S- prepared from E. coli-plCD242 cells showed 3H] NADHto 2-oxoisocaproate was examined in a reac- about 900-fold higher leucine dehydrogenase tion mixture (2 ml) containing 2 /miol of [4S-3H] NADH, 20/miol of sodium 2-oxoisocaproate, 0.3mmol of activity than that from C. thermoaceticum cells NH4C1/NH4OH buffer (pH 9.5) and 0.01 mg of leucine (Table I). As the cell growth of E.

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