J. Mol. Biol. (1977) 116, 181-187
A Preliminary Crystallographic Study of Isocitrate Dehydrogenase from Azotobacter vinelandii Large single crystals of isocitrate dehydrogenase from Azotobacter vinelandii have been grown by vapor diffusion from ammonium sulfate and phosphate solutions. The crystals are tetragonal, space group P4,2,2 with cell dimensions a = 122.1 A, c = 163.9 a. There are two molecules of 80,000 molecular weight, per asymmetric unit. Native data to 5.5 A resolution have been collected on a diffractometer. A rotation function using data between 10 A and 6 A resolution ilrdicates three possible orientations of thr non-crystallographic S-fold axis relating the two molecules.
The NADP + -specific
isocitrate
dehydrogenase
(threo-u,-isocitrate
: NADP + oxidore-
(decarboxylating), EC 1.1.1.42) catalyzes the transfer of hydrogen atoms from isocitrate to NADP+ and decarboxylates the newly formed oxalosuccinate to produce CO, and a-ketoglutarate (Ochoa, 1948; Moyle & Dixon, 1955; Siebert et al., 1957a,b). NADP+-specific IDHasef has been isolated from various sources (Plaut, 1963). The enzyme isolated from pig heart has been the most studied among the eukaryotic sources (see Ehrlich & Colman, 1975 and the references therein). Among the prokaryotic sources the NADP+-specific IDHase from Axotobacter vinelandii is the most thoroughly characterized (Chung & Franzen, 1969; Edwards et al., 1974). We wish to report here the crystallization and preliminary X-ray crystallographic data for the enzyme from A. vinelundii. The structural study of this particular IDHase was initiated for several reasons. First, a knowledge of the three-dimensional structure of the enzyme will be vital in determining the mechanism of action at an atomic level. Second, it will be of interest, to determine the differences and the similarities between the binding of NADP+ and NAD + to proteins. There are at present four NAD +-requiring dehydrogenases from mammalian sources whose structures are known, malate dehydrogenase (Hill et aI.. 1972), lactate dehydrogenase (Adams et al., 1970)? alcohol dehydrogenase (BriindBn et al.. 1973), and glyceraldehyde 3-phosphate dehydrogenase (Buehner et al., 1974). The low-resolution structure of the NADP + -specific 6-phosphogluconate dehydrogenase has recently been reported (Adams et al., 1975). This particular dehydrogenase is of a mammalian source and functions as a dimer, whereas A. vinelundii IDHase funct)ions as a monomer. A direct’ comparison of the structures of these two NADP + requiring enzymes will also be of interest from an evolutionary standpoint. Finally, it should be noted that this is only the third crystalline nicotinamide-dependent dehydrogenasc from a prokaryotic source which has been reported to be suitable for a crystallographic structural investigation, the others being glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus (Suzuki & Harris, 1971) and Lhistidinol dehydrogenase from Salmonellu typhimurium (Yang et al., 1973). A knowledge of the structure of bacterial dehydrogenases will serve as a test for the universality of the nucleotide-binding fold observed in the mammalian NAD + -specific dehydrogenasses (Rossmann et aZ., 1974). ductase
t Al~tweviation
used:
IDHase,
ixocitfrate
dchydrogenaxo. 181
182
E. W.
C’%ERb’INSKI
I000 molecular
C*
(b) 0. 1 8” precession photographs of a single crystal of isocitrate dehydrogenas ;e from A. ? ii. r 8 39 . d., Kriegcr. M., Chambers, J. L., (!tlristopll, G. (:. & Stroud. It. M. (l!)i4). Acta Crystallogr. sect. A, 30, 74% 748. Lenhert, P. G. (1975). J. BppZ. Crystdogr. 8, 568-570. Low, B. IV. Sr Richards, F. M. (1952). J. Amer. Chem. Sot. 74, 1660&1666. Matthews, B. W. (1968). J. Mol. BioZ. 33, 491-497. Matt,hr\vs, 13. W. (1974). J. ivlol. Biol. 82, 513-526. Moylv, .I. & Dixon, M. (1955). Biochim. Biophys. Acta, 16, 434hK~5. Ocltoa, S. (1948). J. Biol. Chem. 174, 133-157. Plaut, (:. IV. E. (1963). The Enzymes, 7, 105-126. 1Coss1r~ittrr1, M. G. & Blow, D. M. (1962). Acta Crystullogr. 15, Z&31. I~oss~nntm. M. U.. Ford, G. C., \Vatson, H. C. k Bsnaxzak, I,. J. (1972). b. Mol. Bio7. 64, 237 249. Rtosstr~at~t~, M. C.. Maras, D. & Olsen, D. D. (1974). X&we (London), 250, 194-199. Siebert. (k., Dubuc~ . ,I., V’arner. R. C. & Plaut, G. \V. E:. (19570,). ,J. BioZ. Chem. 226, 465455. Siebwt. G., Car&&is, M. & Plaut, G. W. E. (19576). J. Biol. chewi. 226, 977-991. Suzuki. .I.. Allc\vt~ll, N. M., Kelly, D. M. & Richards, F. M. (1967). J. &loZ. Biol. 27, 563.55x.
A Preliminary Crystallographic Study of Isocitrate Dehydrogenase from Azotobacter vinelandii Large single crystals of isocitrate dehydrogenase from Azotobacter vinelandii have been grown by vapor diffusion from ammonium sulfate and phosphate solutions. The crystals are tetragonal, space group P4,2,2 with cell dimensions a = 122.1 A, c = 163.9 a. There are two molecules of 80,000 molecular weight, per asymmetric unit. Native data to 5.5 A resolution have been collected on a diffractometer. A rotation function using data between 10 A and 6 A resolution ilrdicates three possible orientations of thr non-crystallographic S-fold axis relating the two molecules.
The NADP + -specific
isocitrate
dehydrogenase
(threo-u,-isocitrate
: NADP + oxidore-
(decarboxylating), EC 1.1.1.42) catalyzes the transfer of hydrogen atoms from isocitrate to NADP+ and decarboxylates the newly formed oxalosuccinate to produce CO, and a-ketoglutarate (Ochoa, 1948; Moyle & Dixon, 1955; Siebert et al., 1957a,b). NADP+-specific IDHasef has been isolated from various sources (Plaut, 1963). The enzyme isolated from pig heart has been the most studied among the eukaryotic sources (see Ehrlich & Colman, 1975 and the references therein). Among the prokaryotic sources the NADP+-specific IDHase from Axotobacter vinelandii is the most thoroughly characterized (Chung & Franzen, 1969; Edwards et al., 1974). We wish to report here the crystallization and preliminary X-ray crystallographic data for the enzyme from A. vinelundii. The structural study of this particular IDHase was initiated for several reasons. First, a knowledge of the three-dimensional structure of the enzyme will be vital in determining the mechanism of action at an atomic level. Second, it will be of interest, to determine the differences and the similarities between the binding of NADP+ and NAD + to proteins. There are at present four NAD +-requiring dehydrogenases from mammalian sources whose structures are known, malate dehydrogenase (Hill et aI.. 1972), lactate dehydrogenase (Adams et al., 1970)? alcohol dehydrogenase (BriindBn et al.. 1973), and glyceraldehyde 3-phosphate dehydrogenase (Buehner et al., 1974). The low-resolution structure of the NADP + -specific 6-phosphogluconate dehydrogenase has recently been reported (Adams et al., 1975). This particular dehydrogenase is of a mammalian source and functions as a dimer, whereas A. vinelundii IDHase funct)ions as a monomer. A direct’ comparison of the structures of these two NADP + requiring enzymes will also be of interest from an evolutionary standpoint. Finally, it should be noted that this is only the third crystalline nicotinamide-dependent dehydrogenasc from a prokaryotic source which has been reported to be suitable for a crystallographic structural investigation, the others being glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus (Suzuki & Harris, 1971) and Lhistidinol dehydrogenase from Salmonellu typhimurium (Yang et al., 1973). A knowledge of the structure of bacterial dehydrogenases will serve as a test for the universality of the nucleotide-binding fold observed in the mammalian NAD + -specific dehydrogenasses (Rossmann et aZ., 1974). ductase
t Al~tweviation
used:
IDHase,
ixocitfrate
dchydrogenaxo. 181
182
E. W.
C’%ERb’INSKI
I000 molecular
C*
(b) 0. 1 8” precession photographs of a single crystal of isocitrate dehydrogenas ;e from A. ? ii. r 8 39 . d., Kriegcr. M., Chambers, J. L., (!tlristopll, G. (:. & Stroud. It. M. (l!)i4). Acta Crystallogr. sect. A, 30, 74% 748. Lenhert, P. G. (1975). J. BppZ. Crystdogr. 8, 568-570. Low, B. IV. Sr Richards, F. M. (1952). J. Amer. Chem. Sot. 74, 1660&1666. Matthews, B. W. (1968). J. Mol. BioZ. 33, 491-497. Matt,hr\vs, 13. W. (1974). J. ivlol. Biol. 82, 513-526. Moylv, .I. & Dixon, M. (1955). Biochim. Biophys. Acta, 16, 434hK~5. Ocltoa, S. (1948). J. Biol. Chem. 174, 133-157. Plaut, (:. IV. E. (1963). The Enzymes, 7, 105-126. 1Coss1r~ittrr1, M. G. & Blow, D. M. (1962). Acta Crystullogr. 15, Z&31. I~oss~nntm. M. U.. Ford, G. C., \Vatson, H. C. k Bsnaxzak, I,. J. (1972). b. Mol. Bio7. 64, 237 249. Rtosstr~at~t~, M. C.. Maras, D. & Olsen, D. D. (1974). X&we (London), 250, 194-199. Siebert. (k., Dubuc~ . ,I., V’arner. R. C. & Plaut, G. \V. E:. (19570,). ,J. BioZ. Chem. 226, 465455. Siebwt. G., Car&&is, M. & Plaut, G. W. E. (19576). J. Biol. chewi. 226, 977-991. Suzuki. .I.. Allc\vt~ll, N. M., Kelly, D. M. & Richards, F. M. (1967). J. &loZ. Biol. 27, 563.55x.
