J. Mol. Biol. (1991) 220, 551-553
Purification and Crystallization of Recombinant Escherichia coli Malate Dehydrogenase Michael D. Hall’, David G. Levitt2, Lee McAllister-Henn3 and Leonard J. Banaszak’t ‘Department
University
of Biochemistry
‘Department of Physiology Minneapolis, MN
of Minnesota,
‘Department University
and 55455, U.S.A.
of Biological Chemistry, College of Medicine of California, Irvine, CA 95217, U.S.A.
(Received 15 April
1991; accepted 7 May
1991)
Malate dehydrogenase from Escherichia coli has been crystallized with polyethylene glycol and citrate buffer at pH 57. The enzyme was obtained from an E. coli strain in which the chromosomal malate dehydrogenase gene was contained on a pBR322 vector. Two types of crystals have been observed; a monoclinic C2 form and an orthorhombic C222, form, which is found infrequently. Monoclinic crystals were used as seeds in several rounds of crystallization until large crystals suitable for diffraction analysis were available. A complete X-ray data set to 20 A has been collected. Keywords: malate dehydrogenase;
MDHase; citric acid cycle; dehydrogenase:
Malate dehydrogenase catalyzes the reversible conversion of L-malate to oxaloacetate by reduction of NAD to NADH. In eukaryotes, three major forms of MDHaseS have been identified. There is a cytosolic: a mitochondrial and a glyoxysomal form; MDHase functions as a member enzyme in the malate shuttle, the citric acid cycle and the glyoxylate cycle respectively. In prokaryotes, a single form of MDHase is present and most often it is homologous with the mitochondrial MDHase. However, in a few cases such as MDHase from Thermusjlavus, the prokaryotic form is homologous with the cytosolic MDHase (Honka et al., 1990). All known forms of MDHase are homodimers in solution. The component polypeptide chain ranges from about 300 to 340 amino acids. The crystal structures of both the cytosolic and mitochondrial forms of MDHase from porcine heart have been determined (Birktoft et al., 1989; Roderick & Banaszak, 1986). They show a high degree of structural homology despite rather low amino acid sequence homologies of about 20 to 25 o/o (Birktoft et al., 1989). Escherichia coli MDHase contains 312 residues, and there is 46% identity with yeast mitochondrial MDHase and 58o/o with porcine mitot Author to whom all correspondence should be addressed. 1 Abbreviations used: MDHase, malate dehpdrogenase; PEG, polyethylene glycol; DTT. dithiothreitol. 0022-2836/91/l
NJ.551 -03 $03.00/O
MDH
chondrial MDHase. Neither the eukaryotic mitochondrial MDHase nor the E. co& MDHase exhibit much homology with the cytoplasmic forms. There is only approximately 100/b sequence identity between the E. coli enzyme and porcine cytoplasmic MDHase and only 20% between the mitochondrial and cytoplasmic porcine forms. Unlike the mitochondrial MDHases, the E. coli enzyme has no translocation sequence on the NH, terminus. When various domains of E. coli MDHase and mitochondrial MDHases are compared, the homology varies. There is 68% identity in the NAD/NADH binding region and 7’4% in residues involved in subunit interactions (Thompson et al., 1988). As noted above, E. coli MDHase shows little homology with another bacterial MDHase isolated from T. jlavus. Interestingly enough the T. Jlauus enzyme shares approximately 50% identity with the cytoplasmic MDHase from pig. Apart from this it appears as if most prokaryotic exception, MDHase are homologous with eukaryotic mitochondrial MDHase. Many of these MDHase homology relationships contrast markedly to those of citrate synthase in which the two eukaryotic isozymes are quite similar but differ considerably from prokaryotic forms. Crystallographically, neither the cytosolic nor the mitochondrial MDHase has provided a particularly useful system for detailed studies on mechanism or for site-specific mutations. The cytosolic MDHase has 666 amino acids in the asymmetric unit and the 551 0
1991 Academic
Press Limited
552
M. D. Hall
crystal form of the dimer does not have exact 2-fold rotational symmetry. Crystalline mitochondrial MDHase has 1256 amino acids in the asymmetric unit. The pseudo tetramer, found only in the crystalline state, has nearly perfect 222 point symmetry. However, because of the large asymmetric unit, crystallographic refinement is difficult and slow. We have therefore begun structural studies on both E. coli MDHase and yeast mitochondrial MDHase although no crystals have yet been obtained from the latter. The enzyme used in these studies comes from E. coli strain HBlOl containing the MDHase chromosomal gene on a pBR322 vector (Sutherland & McAlister-Henn, 1985). Cells were grown in Luria broth and 50 ,ug ampicillin/ml in a 24 1 fermentor. At late log phase the cells were harvested by centrifugation, frozen in liquid nitrogen, and stored at -70°C. Cells were thawed and resuspended to -1 g/ml in cracking buffer containing 50 mbr-Tris, 10% (w/v) sucrose, 0*05% (w/v) NaN3, 1 mMEDTA, 1 m&r-fi-mercaptoethanol (pH 7.9) and 0.2 o/o PMSF. Cells were disrupted at 4 “C by sonication for five one-minute intervals. The cell lysate was then centrifuged for one hour at 18,000 revs/min to remove the debris. A 35% ammonium sulfate precipitation was done to remove unwanted protein followed by a 65% precipitation to concentrate MDHase. The pellet was resuspended in a buffer containing 10 mM-Hepes, 1 m&r-EDTA, 1 m&r-fl-mercaptoethanol (pH 7.4) and dialyzed overnight with four to five buffer changes. The dialysate was applied to a Blue Sepharose (Sigma) column. The column was thoroughly washed, then a 0 mM to 500 mu-NaCl gradient over 500 ml was used to elute MDHase. Fractions were assayed for MDHase activity and pooled. The pooled sample was concentrated by dialysis against buffer consisting of 10 mw-Tris, 1 mM-EDTA, 1 mM-/?-mercaptoethanol (pH 8.5). A Mono& type anion exchange column was used. MDHase was eluted in a 0 mM to 150 mM/150 ml NaCl gradient. The purity of the MDHase active fraction was verified with SDS/polyacrylamide gels. Protein sequencing of the amino-terminal 20 residues further demonstrated that an intact protein was purified. The enzymatic activity of MDHase is assayed by measuring the change in absorbance at 340 nm when NAD is converted to NADH during the conversion of malate to oxaloacetate at 25°C. The assay buffer is made up of 850 ~1 of 61 M-sodium glycinate (pH l@O), 100 ~1 of 1-O M-malic acid (pH 7.0), 5.0 ~1 of 5 mhr-NAD. Seed crystals were grown by hanging drop vapor diffusion methods. A 10 ~1 drop containing 5.6 mg protein/ml and 14 to 16% (w/v) PEG 8000 in crystallization buffer containing 10 mw-sodium citrate, 1 mM-EDTA, 2 mM-DTT, 005% NaN, at pH 57 was placed on a silanized coverslip. The coverslip was inverted and placed on a well containing 1 ml of crystallization buffer and 15 to I8 y. PEG 8000. Vacuum grease was used to seal the
et al.
coverslip and well. After two to seven days at 18”C, small to moderate size crystals formed. These crystals were washed with 12% PEG in crystallization buffer at least five times. A 10 to 15 ~1 drop containing 56 mg protein/ml and 13% PEG in crystallization buffer was added to a single seed crystal and placed on a coverslip and equilibrated with a well containing 1 ml of 14% PEG in crystallization buffer. New protein solution was added by this method at least three times until large crystals for diffraction study appeared. Crystals were mounted in quartz capillaries and placed on a Siemens-Nicollet area detector. A Rigaku rotating anode X-ray generator was used to provide X-rays at 50 kV, 150 mA collimated to 0.5 mm. For each crystal, 30 to 50 frames were collected with a 4 scan for 180 or 200 seconds at 20 = 0” using a frame step size of 025”. These data were used for initial indexing. Subsequent data collection runs consisted of 90 to 100” 4 scans at 20 = 20”, 30” or 35”. This was followed by 35 to 45” co scans at x = 15”, 45” or 90”, I# = O”, 45”, 60°, 90” or 120” and 20 = 20”, 30” or 35”. Monoclinic C2 crystals of the holoenzyme were grown to 68 mm in length and 65 mm in width and height by repeated seeding in hanging drop wells. The unit cell dimensions are a = 116.9 A (1 .& = 61 nm), b = 429 A, c = 841 A, p = 1362”. They diffracted to a maximum resolution of 1.76 A and a complete dataset at 2.0 A has been obtained. Another crystal form, one with an orthorhombic C222, space group, was also found to grow under the same conditions. The unit cell dimensions are: a = 42.9 A, b = 1283 A, c = 114.2 A. The orthorhombic form was found much less often and was not used for seeding. All data processing of reflections was done with the Xengen programs. The reflections were initially processed in a primitive lattice and listed in order to observe systematic absences. General reflection conditions are for all hkl : h + E = 2n. This confirmed the existence of a C-centered lattice. The results of the data collection runs are summarized in Table 1. Note the excellent agreement between multiple measurements and symmetry-related reflections. The highest obtainable resolution also appears to he better than for mitochondrial MDHase or cytosolic MDHase. In the first trials, the native data has been measured to 1.9 A resolution. The unit cell of the monoclinic C2 crystal has a volume of 3.22 x lo5 A3. Assuming that the crystal Table 1 X-ray
data collection statistics
on E. coli MDHase
No. of cry&& Total no. of observations Total no. of unique reflections Average no. of observations per reflection R-factor between symmetry-related reflections and multiple measurements x 100 y. of possible reflections collected to 201 A O/oof possible reflections collected to 1.87 A
4 128,427 26,366 6 59 995 71.5
Communications contains 50% (v/v) mother liquor, an asymmetric unit contains one monomer of MDHase, corresponding to a subunit volume of 4.10 x 10’ A3. The physiological dimer must have 2-fold rotational symmetry with the molecular 2-fold congruent with the crystallographic symmetry axes parallel to the b-axis. The structure of pig heart mitochondrial MDHase has been solved to 2.5 A and is currently being refined (Roderick & Banaszak, 1986). As already mentioned, the porcine mitochondrial MDHase and E. coEi MDHases have considerable primary sequence homology especially in the subunitsubunit interface. The co-ordinates of the mitochondrial MDHase structure will be used with molecular replacement methods to obtain starting phases. The catalyt,ic activity of most malate dehydrogenases are modulated by the substrates oxaloacetate and malate (Bernstein et al., 1978; Mullinax et nl., 1982; Datta et al., 1985; Fahien et al., 1988). Studies with porcine mitochondrial MDHase show that high levels of malate stimulate malate oxidation while high levels of oxaloacetate and citrate increase the rate of the reverse reaction. Unlike lactate dehydrogenase, the specificity of the dicarboxylic acid substrate appears to be limited to four carbon compounds, malate and oxaloacetate. We hope to use this crystallographically simpler form of MDHase to examine the substrate binding site. In addition, site-specific mutagenesis will permit study of key residues
in the subunit-subunit
interface.
The eMDH project was supported by the National Science Foundation, U.S.A. (L.J.B., DNB 8941746) and the NTH (T,.M.-H., GM33218).
553
References Bernstein, L. H., Grisham, M. B., Cole. K. D. & Everse, J. (1978). Substrate inhibition of the mitochondrial and cytoplasmic malate dehydrogenases. J. Riol. (‘hem. 253, 8697-8701. Birktoft, J. J., Rhodes, G. & Banaszak, L. ,J. (1989). Refined crystal structure of cytoplasmic malate dehydrogenase at 2.5 b resolution. Biochemistry, 28. 6065-6081. Datta, A., Merz, J. M. & Spivey, H. 0. (1985). Substrate channeling of oxaloacetate in solid-state complexes of malate dehydrogenase and &rate synthase. ,I. Biol. Chem. 260, 15008-15012. Fahien, L. A., Kmiotek, E. H., MacDonald. M. J., Fibich, B. & Milka M. (1988). Regulation of malate dehydrogenase activity by glutamate. citrate. a-ketoglutarate, and multienzyme interact’ion. J. Biol. (‘hem. 263, 10687-10697. Honka, E., Fabry. S., Niermann. T., Palm, P. & Hensel. R. (1990). Properties and primary sequence of the L-malate dehydrogenase for the extremely thermophilic archaebacterium Methanothermu.r fervidus. Eur. J. Biochem. 188, 623-632. Mullinax, T. R., Mock, J. N., McEvily, A. .J. Cy:Harrison, *J. H., (1982). Regulation of mitochondrial malate dehydrogenase. J. Biol. Chem. 257. 13233-13239. Roderick, S. L. & Banaszak, L. ,J. (1986). The threedimensional structure of porcine heart mitochondrial malate dehydrogenase at 3.0 a resolution. ,I. Mol. Biol. 261, 946 l-9464. Sutherland, P. & McAlister-Henn. I,. (1985). Isolation and expression of the Escherichin coli gene encoding malate dehydrogenase. J. Bactwiol. 163. 1074-1079. Thompson. I,. M., Sutherland, P.. Steffan. J. S. & McAlister-Henn. I,. (1988). Gene sequence and primary structure of mitochondrial malate dehydrogenase from Succharomyces cerroisiau. b’iochemi.~try, 27, 8393-8400.
Edited by A. Klug
Purification and Crystallization of Recombinant Escherichia coli Malate Dehydrogenase Michael D. Hall’, David G. Levitt2, Lee McAllister-Henn3 and Leonard J. Banaszak’t ‘Department
University
of Biochemistry
‘Department of Physiology Minneapolis, MN
of Minnesota,
‘Department University
and 55455, U.S.A.
of Biological Chemistry, College of Medicine of California, Irvine, CA 95217, U.S.A.
(Received 15 April
1991; accepted 7 May
1991)
Malate dehydrogenase from Escherichia coli has been crystallized with polyethylene glycol and citrate buffer at pH 57. The enzyme was obtained from an E. coli strain in which the chromosomal malate dehydrogenase gene was contained on a pBR322 vector. Two types of crystals have been observed; a monoclinic C2 form and an orthorhombic C222, form, which is found infrequently. Monoclinic crystals were used as seeds in several rounds of crystallization until large crystals suitable for diffraction analysis were available. A complete X-ray data set to 20 A has been collected. Keywords: malate dehydrogenase;
MDHase; citric acid cycle; dehydrogenase:
Malate dehydrogenase catalyzes the reversible conversion of L-malate to oxaloacetate by reduction of NAD to NADH. In eukaryotes, three major forms of MDHaseS have been identified. There is a cytosolic: a mitochondrial and a glyoxysomal form; MDHase functions as a member enzyme in the malate shuttle, the citric acid cycle and the glyoxylate cycle respectively. In prokaryotes, a single form of MDHase is present and most often it is homologous with the mitochondrial MDHase. However, in a few cases such as MDHase from Thermusjlavus, the prokaryotic form is homologous with the cytosolic MDHase (Honka et al., 1990). All known forms of MDHase are homodimers in solution. The component polypeptide chain ranges from about 300 to 340 amino acids. The crystal structures of both the cytosolic and mitochondrial forms of MDHase from porcine heart have been determined (Birktoft et al., 1989; Roderick & Banaszak, 1986). They show a high degree of structural homology despite rather low amino acid sequence homologies of about 20 to 25 o/o (Birktoft et al., 1989). Escherichia coli MDHase contains 312 residues, and there is 46% identity with yeast mitochondrial MDHase and 58o/o with porcine mitot Author to whom all correspondence should be addressed. 1 Abbreviations used: MDHase, malate dehpdrogenase; PEG, polyethylene glycol; DTT. dithiothreitol. 0022-2836/91/l
NJ.551 -03 $03.00/O
MDH
chondrial MDHase. Neither the eukaryotic mitochondrial MDHase nor the E. co& MDHase exhibit much homology with the cytoplasmic forms. There is only approximately 100/b sequence identity between the E. coli enzyme and porcine cytoplasmic MDHase and only 20% between the mitochondrial and cytoplasmic porcine forms. Unlike the mitochondrial MDHases, the E. coli enzyme has no translocation sequence on the NH, terminus. When various domains of E. coli MDHase and mitochondrial MDHases are compared, the homology varies. There is 68% identity in the NAD/NADH binding region and 7’4% in residues involved in subunit interactions (Thompson et al., 1988). As noted above, E. coli MDHase shows little homology with another bacterial MDHase isolated from T. jlavus. Interestingly enough the T. Jlauus enzyme shares approximately 50% identity with the cytoplasmic MDHase from pig. Apart from this it appears as if most prokaryotic exception, MDHase are homologous with eukaryotic mitochondrial MDHase. Many of these MDHase homology relationships contrast markedly to those of citrate synthase in which the two eukaryotic isozymes are quite similar but differ considerably from prokaryotic forms. Crystallographically, neither the cytosolic nor the mitochondrial MDHase has provided a particularly useful system for detailed studies on mechanism or for site-specific mutations. The cytosolic MDHase has 666 amino acids in the asymmetric unit and the 551 0
1991 Academic
Press Limited
552
M. D. Hall
crystal form of the dimer does not have exact 2-fold rotational symmetry. Crystalline mitochondrial MDHase has 1256 amino acids in the asymmetric unit. The pseudo tetramer, found only in the crystalline state, has nearly perfect 222 point symmetry. However, because of the large asymmetric unit, crystallographic refinement is difficult and slow. We have therefore begun structural studies on both E. coli MDHase and yeast mitochondrial MDHase although no crystals have yet been obtained from the latter. The enzyme used in these studies comes from E. coli strain HBlOl containing the MDHase chromosomal gene on a pBR322 vector (Sutherland & McAlister-Henn, 1985). Cells were grown in Luria broth and 50 ,ug ampicillin/ml in a 24 1 fermentor. At late log phase the cells were harvested by centrifugation, frozen in liquid nitrogen, and stored at -70°C. Cells were thawed and resuspended to -1 g/ml in cracking buffer containing 50 mbr-Tris, 10% (w/v) sucrose, 0*05% (w/v) NaN3, 1 mMEDTA, 1 m&r-fi-mercaptoethanol (pH 7.9) and 0.2 o/o PMSF. Cells were disrupted at 4 “C by sonication for five one-minute intervals. The cell lysate was then centrifuged for one hour at 18,000 revs/min to remove the debris. A 35% ammonium sulfate precipitation was done to remove unwanted protein followed by a 65% precipitation to concentrate MDHase. The pellet was resuspended in a buffer containing 10 mM-Hepes, 1 m&r-EDTA, 1 m&r-fl-mercaptoethanol (pH 7.4) and dialyzed overnight with four to five buffer changes. The dialysate was applied to a Blue Sepharose (Sigma) column. The column was thoroughly washed, then a 0 mM to 500 mu-NaCl gradient over 500 ml was used to elute MDHase. Fractions were assayed for MDHase activity and pooled. The pooled sample was concentrated by dialysis against buffer consisting of 10 mw-Tris, 1 mM-EDTA, 1 mM-/?-mercaptoethanol (pH 8.5). A Mono& type anion exchange column was used. MDHase was eluted in a 0 mM to 150 mM/150 ml NaCl gradient. The purity of the MDHase active fraction was verified with SDS/polyacrylamide gels. Protein sequencing of the amino-terminal 20 residues further demonstrated that an intact protein was purified. The enzymatic activity of MDHase is assayed by measuring the change in absorbance at 340 nm when NAD is converted to NADH during the conversion of malate to oxaloacetate at 25°C. The assay buffer is made up of 850 ~1 of 61 M-sodium glycinate (pH l@O), 100 ~1 of 1-O M-malic acid (pH 7.0), 5.0 ~1 of 5 mhr-NAD. Seed crystals were grown by hanging drop vapor diffusion methods. A 10 ~1 drop containing 5.6 mg protein/ml and 14 to 16% (w/v) PEG 8000 in crystallization buffer containing 10 mw-sodium citrate, 1 mM-EDTA, 2 mM-DTT, 005% NaN, at pH 57 was placed on a silanized coverslip. The coverslip was inverted and placed on a well containing 1 ml of crystallization buffer and 15 to I8 y. PEG 8000. Vacuum grease was used to seal the
et al.
coverslip and well. After two to seven days at 18”C, small to moderate size crystals formed. These crystals were washed with 12% PEG in crystallization buffer at least five times. A 10 to 15 ~1 drop containing 56 mg protein/ml and 13% PEG in crystallization buffer was added to a single seed crystal and placed on a coverslip and equilibrated with a well containing 1 ml of 14% PEG in crystallization buffer. New protein solution was added by this method at least three times until large crystals for diffraction study appeared. Crystals were mounted in quartz capillaries and placed on a Siemens-Nicollet area detector. A Rigaku rotating anode X-ray generator was used to provide X-rays at 50 kV, 150 mA collimated to 0.5 mm. For each crystal, 30 to 50 frames were collected with a 4 scan for 180 or 200 seconds at 20 = 0” using a frame step size of 025”. These data were used for initial indexing. Subsequent data collection runs consisted of 90 to 100” 4 scans at 20 = 20”, 30” or 35”. This was followed by 35 to 45” co scans at x = 15”, 45” or 90”, I# = O”, 45”, 60°, 90” or 120” and 20 = 20”, 30” or 35”. Monoclinic C2 crystals of the holoenzyme were grown to 68 mm in length and 65 mm in width and height by repeated seeding in hanging drop wells. The unit cell dimensions are a = 116.9 A (1 .& = 61 nm), b = 429 A, c = 841 A, p = 1362”. They diffracted to a maximum resolution of 1.76 A and a complete dataset at 2.0 A has been obtained. Another crystal form, one with an orthorhombic C222, space group, was also found to grow under the same conditions. The unit cell dimensions are: a = 42.9 A, b = 1283 A, c = 114.2 A. The orthorhombic form was found much less often and was not used for seeding. All data processing of reflections was done with the Xengen programs. The reflections were initially processed in a primitive lattice and listed in order to observe systematic absences. General reflection conditions are for all hkl : h + E = 2n. This confirmed the existence of a C-centered lattice. The results of the data collection runs are summarized in Table 1. Note the excellent agreement between multiple measurements and symmetry-related reflections. The highest obtainable resolution also appears to he better than for mitochondrial MDHase or cytosolic MDHase. In the first trials, the native data has been measured to 1.9 A resolution. The unit cell of the monoclinic C2 crystal has a volume of 3.22 x lo5 A3. Assuming that the crystal Table 1 X-ray
data collection statistics
on E. coli MDHase
No. of cry&& Total no. of observations Total no. of unique reflections Average no. of observations per reflection R-factor between symmetry-related reflections and multiple measurements x 100 y. of possible reflections collected to 201 A O/oof possible reflections collected to 1.87 A
4 128,427 26,366 6 59 995 71.5
Communications contains 50% (v/v) mother liquor, an asymmetric unit contains one monomer of MDHase, corresponding to a subunit volume of 4.10 x 10’ A3. The physiological dimer must have 2-fold rotational symmetry with the molecular 2-fold congruent with the crystallographic symmetry axes parallel to the b-axis. The structure of pig heart mitochondrial MDHase has been solved to 2.5 A and is currently being refined (Roderick & Banaszak, 1986). As already mentioned, the porcine mitochondrial MDHase and E. coEi MDHases have considerable primary sequence homology especially in the subunitsubunit interface. The co-ordinates of the mitochondrial MDHase structure will be used with molecular replacement methods to obtain starting phases. The catalyt,ic activity of most malate dehydrogenases are modulated by the substrates oxaloacetate and malate (Bernstein et al., 1978; Mullinax et nl., 1982; Datta et al., 1985; Fahien et al., 1988). Studies with porcine mitochondrial MDHase show that high levels of malate stimulate malate oxidation while high levels of oxaloacetate and citrate increase the rate of the reverse reaction. Unlike lactate dehydrogenase, the specificity of the dicarboxylic acid substrate appears to be limited to four carbon compounds, malate and oxaloacetate. We hope to use this crystallographically simpler form of MDHase to examine the substrate binding site. In addition, site-specific mutagenesis will permit study of key residues
in the subunit-subunit
interface.
The eMDH project was supported by the National Science Foundation, U.S.A. (L.J.B., DNB 8941746) and the NTH (T,.M.-H., GM33218).
553
References Bernstein, L. H., Grisham, M. B., Cole. K. D. & Everse, J. (1978). Substrate inhibition of the mitochondrial and cytoplasmic malate dehydrogenases. J. Riol. (‘hem. 253, 8697-8701. Birktoft, J. J., Rhodes, G. & Banaszak, L. ,J. (1989). Refined crystal structure of cytoplasmic malate dehydrogenase at 2.5 b resolution. Biochemistry, 28. 6065-6081. Datta, A., Merz, J. M. & Spivey, H. 0. (1985). Substrate channeling of oxaloacetate in solid-state complexes of malate dehydrogenase and &rate synthase. ,I. Biol. Chem. 260, 15008-15012. Fahien, L. A., Kmiotek, E. H., MacDonald. M. J., Fibich, B. & Milka M. (1988). Regulation of malate dehydrogenase activity by glutamate. citrate. a-ketoglutarate, and multienzyme interact’ion. J. Biol. (‘hem. 263, 10687-10697. Honka, E., Fabry. S., Niermann. T., Palm, P. & Hensel. R. (1990). Properties and primary sequence of the L-malate dehydrogenase for the extremely thermophilic archaebacterium Methanothermu.r fervidus. Eur. J. Biochem. 188, 623-632. Mullinax, T. R., Mock, J. N., McEvily, A. .J. Cy:Harrison, *J. H., (1982). Regulation of mitochondrial malate dehydrogenase. J. Biol. Chem. 257. 13233-13239. Roderick, S. L. & Banaszak, L. ,J. (1986). The threedimensional structure of porcine heart mitochondrial malate dehydrogenase at 3.0 a resolution. ,I. Mol. Biol. 261, 946 l-9464. Sutherland, P. & McAlister-Henn. I,. (1985). Isolation and expression of the Escherichin coli gene encoding malate dehydrogenase. J. Bactwiol. 163. 1074-1079. Thompson. I,. M., Sutherland, P.. Steffan. J. S. & McAlister-Henn. I,. (1988). Gene sequence and primary structure of mitochondrial malate dehydrogenase from Succharomyces cerroisiau. b’iochemi.~try, 27, 8393-8400.
Edited by A. Klug
