Evolutionary relationships among the malate dehydrogenases

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Evolutionary relationships among the malate dehydrogenases Lee Mc

ister-Henn

The three-dimensional structures and elements essential for catalysis are conserved between mitochondrial and cytoplasmic forms o f malate dehydrogenase in eukaryotic cells even though these isozymes are only marginally related at the level of primary structure. The high degree o f similarity between amino acid sequences o f eukaryotic mitochondrial and Escherichia coli malate dehydrogenases may thus reflect evolutionary pressure to retain structural elements for functions other than catalysis. The compartmentalized isozymes of malate dehydrogenase are an example of the isozyme pairs in eukaryotic cells that provide a mechanism for efficient exchange and metabolism of small molecules on either side of the mitochondrial membrane. Both enzymes catalyse the NAD(H)-dependent interconversion of malate and oxaloacetate. This reaction plays a key part in the malate/aspartate shuttle across the mitochondrial membrane, and in the tricarboxylic acid cycle within the mitochonddal matrix. Previous and ongoing research has focused on comparison of the primary and threedimensional structures of the mitochondrial and cytoplasmic forms of malate dehydrogenase isolated from a common source, pig heart tissue 1-3. These studies have elucidated the roles of specific amino acid residues in catalysis and in the binding of substrate and pyridine nucleotide molecules. With complete amino acid sequence data now available for the compartmentalized isozymes from pig and mouse 4'5, for the mitochondrial enzymes from yeast (Thompson, L. M., Sutherland, P., Steffan, J. S. and McAlister-Henn, L., submitted) and rat 6, and for the single malate dehydrogenase of E. coil 7, it is possible to comment on the conservation of specific residues and structural elements and the general evolution of the compartmentalized eukaryotic isozymes. As described in this review, the malate dehydrogenases provide substantial support for the endosymbiotic hypothesis of mitochondrial origin.

dimers of identical subunits with molecular weights of 36 000 and 34 000, respectively. Crystallographic analyses indicate that the main chain folding of the two proteins is remarkably similar 1'2. The enzymes share a common catalytic mechanism and their kinetic properties are similar although they are distinguishable by certain properties including differential inhibition by high concentrations of oxaloacetate s. Despite these similarities, the amino acid sequences reported for the isozymes from pig and mouse show a low degree of similarity (approximately 20% and 23%, respectively) between the compartmentalized isozymes. However, the positions of identical amino acid residues in the aligned primary sequences are not random; within certain regions of the primary sequences, the porcine isozymes, for example, are quite similar, with 30-60% identical

Compartmentalized isozymes

cMHD

_The porcine cytoplasmic and mitochondrial malate dehydrogenases are

residues (see Fig. 1). These regions of the polypeptide chains, as defined by crystallographic and chemical analyses, contain the majority of residues involved in nucleotide binding, in helices forming the subunit interface, and in catalysis. In a more detailed comparison of the malate dehydrogenase isozymes, it is instructive to include the distantly related lactate dehydrogenases. The dimeric malate and tetrameric lactate dehydrogenases share a common twofold axis of symmetry. The subunit interfaces along this axis are similar but only a limited number of specific amino acid residues implicated in subunitsubunit interactions are conserved even between the compartmentalized malate dehydrogenases (see Fig. 1). Each subunit of the malate and lactate dehydrogenases contains two structurally and functionally distinct domains. The NAD(H)-binding domain, occupying the amino-terminal half of each molecule, contains a parallel p-sheet structure, an example of the so-called 'Rossman fold' motif 9. The core dinucleotide binding structure is composed of four ~-sheets and o n e a-helix (Fig. 2). Within these secondary structural elements, specific residues believed to be critical for cofactor binding including Gly7, Glyl 1, Asp33, and Gly77 (Ref. 10) (porcine mitochondrial malate dehydrogenase residue positions) are conserved at similar positions in the malate and lactate dehydrogenases as are residues 77-90 corresponding to the NAD(H)-binding

3 mMDH V A V L G A S G G I G Q P L S L L L KN 6 cMDH V L V T G A A G Q I A Y S L L YS I GN NN N N N N Q QQ Q Q Q Q 29 mMDH L T L Y D i A H T P - GV 37 cMDH L V L L D 1 - - T P MMG V NNN N QQ 77 mMDH G VP R K P G M T R D D L 88 cMDH G MP R R D G M E R K D L N NNNNN mMDH

148 IVRANAF 157 L DHNRAKAQ N N QC QQ QQ L D

V A E L K G L DP A R V S I A- LKL GVTS

VP V l G - G - H

DDVKNVI QQQ

IWGNH N C

Fig. 1. Comparison of localized regions of similarity between amino acid sequencesfor porcine L. McAlister-Hennis at the Departmentof Biolo- mitochondrial (mMDH) and cytoplasmic (cMDIt) malate deBydrosenases. Identical residues are gical Chemistry, California Collegeof Medicine, emboldenedandsubscriptsindicate functions attributed to specificresiduesin NAD(H) binding (N), in Universityof California, lrvine, CA 92717, USA. formation of thesubunitinterface(Q), andin catalysis(C). ~ 1988.ElsevierPublicationsCambridge 0376-5067/88/$02.00

179

TIBS13-May1988 O.B ~A

1 i,

Fig. 2. Diagram of the core dinucleotide binding structure for porcine cytoplasmic malate dehydrogenase. The bars represent [J-strands and the cylinder an a-helix. Arrows indicate the amino- to carboxy-terminal directions of the polypeptide chain and letters the positions of conserved glycine and aspartate residues implicated in the binding of NAD (shown as a stick model). Adapted, with permission, from Ref. 11.

loop, a structure thought to undergo a conformational change in lactate dehydrogenase with cofactor binding". While the NAD(H)-binding domain is conserved to some extent in other dehydrogenases, the malate and lactate dehydrogcnases as 2-hydroxy acid dehydrogenases also share some features associated with catalysis. These include invariant residues corresponding to His176 and Asp149 believed to form a proton relay system and Arg152 thought to provide a counterion for the substrate carboxylate group 12. Except for these residues at the catalytic sites, the carboxy-terminal catalytic domains of the malate and lactate dehydrogenases are not significantly related at the level of primary sequence. The essential equivalence of these enzymes, despite dissimilar primary structures, was convincingly demonstrated by Clarke et al. 13 who constructed a catalytically active malate dehydrogenase by making substitutions involving only three amino acids in the active site of a l.~ctate dehydrogenase. Thus, it is not surprising that the compartmentalized malate dehydrogenases retain similar catalytic and kinetic properties but only marginal sequence identity. Tricarboxylic acid cycle enzymes In contrast to the overall divergence of the cytoplasmic and mitochondrial dehydrogenases, the mitochondrial tricarboxylic acid cycle enzyme has been highly conserved. Fernley ef al.14 first reported the striking similarity

between amino acid residues 1-36 of unit interface, and residue positions the porcine mitochondrial and E. coli 259 to the carboxyl termini, a region enzymes and this relatedness has now containing a long terminal u-helix. been found to extend throughout the Among all malate dehydrogenase polypeptides. Yeast and porcine mito- sequences reported to date, the latter chondrial and E. coli malate dehy, carboxy-terminal region is the most drogenases are similar in size with 317, variable containing, for.example, 4 of 314 and 312 amino acid residues, the 14 total residue differences respectively, per dimeric subunit and between the pig and rat mitochondrial only short gaps are required for optimal enzymes. alignment of the three polypeptide Regions highly conserved among the sequences (Fig. 3). (Only one mam- E. coli and eukaryotic mitochondrial malian enzyme is included in this com- malate dehydrogenases contain resiparison since the amino acid sequences dues and structural elements important of rat, mouse, and pig mitochondrial for NAD(H)-binding, for formation of enzymes are greater than 90% identi- the subunit interface, and for catalysis. cal.) The amino acid sequences shown These include all regions in the cytoin Fig. 3 are remarkably similar; 48% plasmic isozyme that are clearly related and 58% of the residues in the pro- to the mitochondrial enzyme, suggestkaryotic enzyme are identical with re- ing conservation of elements necessary spective residues in the yeast and por- for basic catalytic function. Other cine enzymes (Fig. 4). Overall, 75% of highly conserved regions in the tricarthe residue positions contain at least boxylic acid cycle enzymes, for examone match among the three polypep- ple residues 209-238, may therefore tides. The NAD(H)-binding domain include structural elements essential (residues 1-146) is more highly con- for specific pathway function. A posserved, with 80% of the residue pos- sible example of the latter is a physical itions containing at least one match, as interaction with another enzyme such compared with matches at 71% of the as citrate synthase, a relationship postulated to be necessary for direct positions in the catalytic domain. Among the tricarboxylic acid cycle transfer of oxaloacetate between the enzymes, residue positions containing two enzymes and for efficient functionnon-identical amino acids tend to be ing of the tricarboxylic acid cycle 15. clustered, permitting identification of Other potentially important intervariable secondary structural elements actions of mitochondrial malate dehyin the polypeptides. Examples of vari- drogenase with fumarase, aspartate able regions include residue positions aminotransferase, and complex I of the 23 to 31, a random coil region between electron transport chain have been ,-helices that are elements of the sub- demonstrated in vitro 16'17.

E. coli MDH: Yeast ~MDH: Pig mMDH:

25 5O M K V A V L G A A G G I G Q A L A L L L K T Q L P S G S E L S L Y D I A P V T P G V A V D L S H I PTAVKI Y T G P S -LNHKVTD R LK-GAK T NSW A S P S -NS LV R T -H A E RATV

75 i00 125 KGFSGEDA--TP-ALEGADVVLISAGVRRKPGMDRSDLFNVNAGIVKNLVQQVAKTCPKACIGIITNPVN TP EPDGLNN KDT M P P T D AI S R D A A A T ESA N A LV S Y L P E Q - - L DC K C V P P T D T T AT T A A C QH D M C S

150 175 TTVAIAAEVLKKAGVYDKNKLFGVTTLDIIRSNTFVAELKGKQPGEVEVPVIGGHSGVTILPLLSQ-VPG S P V Q NK NPK S A A R IS VENTD T Q E R N I I I -TNH SIP T F H NP I V A A LD A R S A K I I CT K

200 225 250 VSFTEQEVADLTKRIQNAGTEVVEAKAGGGSATLSMGQAAARFGLSLVRALQ~EQGVVECAYVEG---~G K L M S D D K R H E IH FG D K N A AH G K A N A V L S G F K RD I PSF DSPL}qS D PQDQLST G E K A AY G VF D M~ KE SF KS---QE

275 300 QYARFFSQPLLLGKNGVEERKSIGTLSAFEQNALEGMLDTLKKDIALGQEFV-N-K EGIE AS V T PD I KIHP E SE E E N QKCKE N EK V N ASTDCPY T K I KNLG KIP EKMIAEAIPE AS KK E K M

Fig. 3. Comparison of amino acid sequences for E. coli and for yeast and pig mitochondrial malate dehydrogenases. Only those residues not identical with corresponding residues in the prokaryotic enzyme are shown. The residue numbers representpositions within the aligned sequences.

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Evolutionary considerations The evolution of the malate dehydrogenases, as deduced from these amino acid sequence comparisons, fits well the pattern predicted by the endosymbiotic theory for the origin of mitochondria TM. Proponents of this theory propose that the mitochondria of present-day eukaryotes evolved from bacteria that existed as intracellular symbionts in primitive eukaryotes. Mitochondrial proteins, some the products of genes transferred during evolution from the endosymbiont to the host genome, would be predicted to be closely related to those of a common bacterial predecessor. One of the many examples cited in support of this model is a description of similar primary structures for chicken mitochondrial and E. coli superoxide dismutases ~9. In the malate dehydrogenase family, the mitochonddal enzyme is much more closely related to its prokaryotic (E. coli) counterpart than to the cytoplasmic isozyme, implying a more recent divergence from a common ancestral gene. A report that the mammalian cytoplasmic isozyme is related at the level of primary sequence with the enzyme from another prokaryote. Thermus flavus 2°, suggests that duplication of the primordial malate dehydrogenase gene preceded the origin of eukaryotes. Curiously in this respect, the malate dehydrogenase family turns out to be an exception when compared with several other compartmentalized isozyme systems. The mitochondrial isozyme of citrate synthase in yeast, for example, is much more closely related to the cytoplasmic form of that enzyme (75% identity) than to E. coli citrate synthase (25% identity) 21. Similarly,

E. coil MDH 47.9%//

~.3%

Yeast ..~ 53.6% ~ mMDH ~' Y

Pig mMDH 19.6%I Pig cMDH

Fig. 4. Extent of primary structure identity among various malate dehydrogenases (MDH). m, mitochondrial; c, cytoplasmic.

E. coil

I i

E. coil

i

MDH

I

mitochondrial (pig) CS

I

T. flavus

mitochondrial

cytoplasmic (pig)

cytoplasmic (yeast)

(yeast)

E. coli AAT

,,,

mitochondrial (mouse) cytoplasmic (mouse)

FUM

I

B. subtilis

I

mitochondrial/ cytoplasmic (yeast)

Fig. 5. General relationships based on primary sequences of various eukaryotic isozymes and their prokaryotic counterparts. Abbreviations representprimordial forms of malate dehydrogenase (MDH), citratesynthase (CS), aspartateaminotransferase (AA T), andfumarase (FUM).

the mitochondrial and cytoplasmic isozymes of mouse aspartate aminotransferase are 53% identical and both are approximately 49% identical with the E. coli enzyme 22. Several examples of individual nuclear genes encoding both mitochondrial and cytoplasmic forms of enzymes have also been reported, including a single yeast gene for the compartmentalized isozymes of fumarase ~. These yeast enzymes are 56% identical with Bacillus subtilis fumarase. In several of these examples, the significant similarity between the nuclear-encoded mitochondrial protein and its prokaryotic counterpart suggests a common ancestor (see Fig. 5). However, only in the malate dehydrogenase family does the cytoplasmic isozyme serve as an internal evolutionary control providing evidence for a period of independent evolution of genes encoding the mitochondrial and cytoplasmic isozymes as constituents of the genomes of different free-living organisms.

Future directions Clearly, the isolation of genes encoding various forms of malate dehydrogenase (particularly from genetically tractable organisms) increases the potential for detailed analysis of structural/functional relationships within this enzyme family. It will also be of interest to examine the extent of functional complementation of the compartmentalized eukaryotic isozymes and of the tricarboxylic acid cycle enzymes in heterologous environments. Finally, it should also be possible to begin to probe the role of protein-protein interactions involving

the malate deydrogenases in the regulation of metabolic pathways. For example, using the growth phenotypes associated with yeast strains containing mutations in MDH124, specific structural defects in the mitochondrial enzyme can be constructed followed by counterselection to screen for suppressor mutations in other structural genes such as those encoding citrate synthase or fumarase. This type of approach would ultimately allow not only definition of the interactive regions of the proteins but also assessment of the metabolic consequences of these interactions in vivo.

Acknowledgements I would like to thank Ralph A. Bradshaw for helpful discussions and Stuart M. Arfin for critical reading of this manuscri~,~t. References 1 Birktoft, J. J., Fernley, R. T., Bradshaw, R. A. an~ Banaszak, L. J. (1982) Proc. Natl Acad. ScL USA 79,6166--6170 2 Roderick, ~. L. and Banaszak, L. J. (1986) J. Biol. C/~m. 261,9461-9464 3 Birktoft, J. J., Bradshaw, R. A. and Banaszak, L. J. (1987) Biochemistry 26, 2722-2734 4 Joh, T., Takeshima, H., Tsuzuki, T., Setoyama, C., Shimada, K., Tanase, S., Kuramitsu, S., Kagamiyama H. and Morino, Y. (I987)J. Biol. Chem. 262,15127-15131 5 Joh, T., Takeshima, H., Tsuzuki, T., Shimada, K., Tanase, S. and Morino, Y. (1987) Biochemistry 26, 2515-2520 6 Grant, P. M., Tellam, J., May, V. L. and Strauss, A. W. (1986) Nucleic Acids Res. 14, 6053-6066 7 McAlister-Henn, L., Blaber, M., Bradshaw, R. A. and Nisco, S. J. (1987) Nucleic Acids Res. 15, 4993 8 Banaszak, L. J. and Bradshaw, R. A. (1975)

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9

10

11

12 13

in The Enzymes 3rd Ed., Vol. IIA, (Boyer, P. D., ed.), pp. 369-396, Academic Press Rossman, M. G., Liljas, A., Branden, C-I. and Banaszak, L. J. (1975) in The Enzymes 3rd Ed., Vol. IIA (Boyer, P. D., ed.), pp. 61-101, Academic Press Birktoft, J. J. and Banaszak, L. J. (1984) in Peptide and Protein Reviews Vol. 4 (Hearn, M. T. W., ed.), pp. 1-46, Marcel Dekker Holbrook, J. J., Liijas, A., Steindel, S. J. and Rossmann, M. G. (1975) in The Enzymes 3rd Ed., VoL IIA, (Boyer, P.D., ed.), pp. 191292, Academic Press Birktoft, J. J. and Banaszak, L. J. (1983) J. Biol. Chem. 258,472-482 Clarke, A. R., Smith, C. J., Hart, K. W.,

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16 17 18

Wilks, H. M., Chia, W. N., Lee, T. V., Birktoft, J. J., Banaszak, L. J., Barstow, D . A . , Atkinson, T. and Hoibrook, J. J. (1987) Biochem. Biophys. Res. Commun. 148,15-33 Fernley, R. T., Lentz, S. R. and Bradshaw, R. A. (1981) Biosci. Rep. 1,495-507 Srere, P. A. (1972) in Energy Metabolism and the Regulation of Metabolic Processes (Mehlman, M. A. and Hanson, R. E., eds), pp. 79-91, Academic Press Beeckmans, S. and Kanarek, L. (1981) Fur. J. Biochem. 117, 527-535 Sumegi, B. and Stere, P. A. (1984) J. Biol. Chem. 259,15040-15045 Margulis, L. S. (1970) Origin of Eucaryotic

Plasma membrane protein sorting in epithelial cells: Do secretory pathways hold the key? James R. Battles and Ann L. Hubbard The Madin-Darby canine kidney (MDCK) cell sorts newly synthesized apical and basolateral plasma membrane proteins intracellularly and then ships them directly to the correct plasma membrane domains, whereas the rat hepatocyte sorts apical and basolateral proteins after their arrival at the basolateral domain of the plasma membrane and then employs a transcytotic mechanism to deliver the apical proteins to the apical domain. This difference in plasma membrane protein sorting palhways may relate to the observation that the hepatocyte, unlike the MDCK cell, does not have an apically directed secretory path way. Most epithelial cells are organized into extensive arrays which form boundaries between two different environments. The epithelial cells exhibit an intrinsic polarity, presumably to mediate interactions with and between these two environments. This asymmetry is particularly evident in the case of the epithelial cell plasma membrane, which can be thought of as being divided into specialized regions called domains: (1) the apical or luminal domain, comprising the 'free' surface; (2) the lateral domain, tightly apposed to the lateral domains of adjacent epithelial cells; and (3) the basal or serosal domain, resting on a basal lamina and in contact with the circulation usually via an intervening layer of connective tissue 1. On the basis of observations of a J. R. ,,lartles is at the Department of Cell Biology and AJ~atomy, Northwestern University Medical School, 3o3 East Chicago Avenue, Chicago, IL 60611, t'ISA. A. L. Hubbard is at the Department of Cell Biology and Anatomy, Johns Hopkins Uniwtrsity School of Medicine, 725 North Wolfe Street, Bdt~imore, MD21205, USA.

number of epithelial cell systems, it is now apparent that there is a striking molecular correlate to this structural and functional polarity of the epithelial cell plasma membrane: in many cases, the different plasma membrane domains have been shown to contain distinct complements of integral membrane proteins (receptors, transporters, enzymes, adhesion molecules, etc.) whose asymmetric distribution ensures that their specific functions are performed at the different surfaces of the epithelial cell ~. Since virtually all of the domain-specific plasma membrane proteins studied to date are integral membrane glycoproteins with complex-type asparagine-linked oligosaccharides, they presumably have in common many steps in the initial stages of their life cycles, including: (1) cotranslational insertion into the membrane of the rough endoplasmic reticulum coincident with the receipt of the high mannose precursor form of the asparagine-linked oligosaccharides; and (2) vesicular transport from endoplasmic reticulum to the cis part of the Golgi complex, through the Golgi com-

Cells, Yale University Press 19 Steinman, H. M. and Hill, R. L. (1973) Proc. Natl Acad. Sci. USA 70, 3725-3729 20 Nishiyama, M., Matsubara, N., Yamamoto, K., lijima, S., Uozumi, T. and Beppu, T. (1986) J. Biol. Chem. 261,14178-14183 21 Rosenkrantz, M., Alam, T., Kim, K., Clark, B. J., Stere, P. A. and Guarente, L. P. (1986) Mol. Cell. Biol. 6, 4509-4515 22 Obaru, K., Nomiyama, H., Shimada, K., Nagashima, F. and Morino, Y. (1986)J. Biol. Chem. 261,16976--16983 23 Wu, M, and Tzagoloff, A. (1987) J. Biol. Chem. 262,12275-12282 24 McAlister-Henn, L. and Thompson, L. M. (1987)J. Bacteriol. 169, 5157-5166

plex in a cis-to-trans direction - coincident with the processing of the asparagine-linked oliogosaccharides to their complex (terminally glycosylated) forms - and eventually to the plasma membrane 2. How do epithelial cells sort these proteins to their respective plasma membrane domains? IntraceHular sorting in the MDCK cell Until now, most of our knowledge regarding the mechanisms of plasma membrane protein sorting in epithelial cells has been acquired from studies of the Madin-Darby canine kidney (MDCK) cell line. These cells exhibit all of the properties of polarized epithelial cells in culture: they grow to form a tightly sealed monolayer, and they exhibit the expected structural and functional polarity ~'3. When infected with certain enveloped RNA viruses, viral progeny bud from MDCK cells in a domain-specific fashion, e.g. influenza virions bud exclusively from the apical surface and vesicular stomatitis virions bud exclusively from the basolateral surface t'3. This polarity of virus budding reflects the domain-specific delivery of the respective viral envelope glycoproteins, HA and G protein, to the MDCK cell plasma membrane (Table I) 1'3. As a result, these viral glycoproteins have now been employed as model apical and basolateral proteins in innumerable studies of nlasma membrane protein sorting L4. One of the primary advantages for considering the behavior of these exogenous integral membrane proteins is that the absence of any pre-existing pool makes it relatively easy to monitor the progress of the initial wave of viral proteins, whether using biochemical or morphological techniques. On the basis of the data that have emerged, it is clear that HA and G travel the biosynthetic pathway together until they reach the trans-most portion of the ~) 1988.ElsevierPublicationsCambridge 0376-5067188/$02.00