MCC

A Brief Introduction to Malate Dehydrogenase

by Ellis & Jessica Bell 

Malate Dehydrogenases catalyze the reaction:

The reaction is thought to involve a base catalyzed abstraction of the proton from the Malate O-H group involving a conserved histidine in the active site of the enzyme. In all known MDHs there is a conserved Aspartate adjacent to the histidine which is thought to increase the basicity of the N: of the histidine ring, enhancing its ability to abstract the proton from the Malate O-H.(2) The malate/oxaloacetate substrate is held in place by the presence of three conserved arginine residues, whose positive charges interact with the negative charges of the malate/oxaloacetate. One of them, (R153 in e Coli) has been the subject of mutagenesis and rescue using chemical biology to probe its involvement in binding and catalysis (3).

This reaction plays a number of important roles in metabolism, illustrated by a reaction in the mitochondrion in the Tricarboxylic acid cycle, a reaction playing a role in the shuttling of reducing equivalents from the cytosol to the mitochondria, in peroxisomes, and in plants a reaction in the Glyoxysome:(4)

It is clear that there must exist Malate Dehydrogenase in at least two different locations within the cell and in fact there are distinct cytoplasmic MDH [cMDH] (5) and mitochondrial MDH [mMDH] (6) isoenzymes in higher eukaryotes which have different amino acid sequences and slightly different three dimensional structures. Peroxisomes [pMDH] (7) and in plants, glyoxysomes [gMDH] (8), also have distinct isoenzymes. The organelle forms (mitochondria, glyoxysomes) are synthesized as precursors coded for by nuclear genes, synthesized in the cytosol and transported to the appropriate organelle, guided by a “pre-sequence” of about 40 amino acids that is removed upon import to the organelle (9).

Pig cytoplasmic and mitochondrial malate dehydrogenases were among the first enzyme structures to be determined by X ray crystallography (10,11). Since then the structure of Malate Dehydrogenase from a number of sources have also been determined by X Ray crystallography. In addition to the details of the amino acids that play a role in substrate binding and catalysis, indicated in figure 2, the structure has several notable features. As with many enzymes that bind NADH there is a clear “Rossman fold” (12) associated with cofactor binding consisting of a β−α−β−α−β−β−α−β−α−β secondary structure motif that in enzymes with specificity for NAD(H) has an aspartate at the C-terminal end of the β2 strand that interacts with the Adenine ribose of the cofactor, figure 3.

For substrate (Malate/Oxaloacetate) binding there is a “flexible loop” (13) that contains two of the three arginine residues involved in malate/oxaloacetate binding, and swings in and out of the active site to complete the active complex of the enzyme (figure 4).

The enzyme has quaternary structure and is in most instances a dimer of two identical (in terms of amino acid sequence) polypeptide chains and displays a clear interface between the two subunits. The quaternary structure undergoes a pH dependent dissociation (14), and monomeric forms have been engineered by mutation of specific amino acids at the subunit interface (15). Monomeric forms show little if any activity.

MDH is thought to form loose multienzyme complexes with several other enzymes sharing substrates, so called “metabolons” (16). In particular Aspartate AminoTransferases (which catalyze the transamination of Glutamate and Oxaloacetate to give Aspartate and 2-Oxoglutarate, a key reaction in the the aspartate-malate shuttle, the Glyoxylate Cycle and Gluconeogenesis) (17), Citrate synthase, (which catalyzes the next step in the Tricarboxylic Acid (Krebs) Cycle) (18), and Fumarase, (which catalyzes the preceding reaction in the Krebs cycle)(19) have been suggested to form metabolons with malate dehydrogenase in certain organisms. Such metabolons are thought to either increase the effectiveness of consecutive reactions by “channeling” products from one enzyme to the next one and or allow for coordinated regulation of a pathway.

Given its central role in a number of metabolic pathways, some forms of the enzyme are subject to allosteric regulation by citrate (20), by post-translational modifications such as by lysine acetylation (21) and by transcriptional control mediated through miR-743a (22).

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The malate dehydrogenase structure and lactate dehydrogenase structures have many similarities and lactate dehydrogenases are thought to have evolved from malate dehydrogenases (23), a step requiring alteration of substrate specificity to bind lactate/pyruvate in place of malate/oxaloacetate. While many malate dehydrogenases are mesophilic, psychrophilic and thermophilic forms have also been characterized and are often studied to explore mechanisms of adaptation (24). As discussed below in the bioinformatics section the existence of cytosolic and organelle forms of malate dehydrogenase is thought to have arisen from a symbiotic relationship between bacteria (precursors of the organelle forms) and eukaryotes which contained a cytosolic form of malate dehydrogenase

Malate Dehydrogenase has also been used as a model system to examine a variety of aspects of protein folding including tertiary structure, quaternary structure (25,26) and the impact of a precursor sequence on structure (27).

Finally, as with many essential enzymes, malate dehydrogenase is a potential target for drug design in pathogenic organisms such as Mycobacterium tuberculosis (28) and Plasmodium falciparum (29), or tumor tissues depending upon enhanced metabolism (30, 31). Such drug design depends upon exploiting often subtle differences in structure function relationships or developing so called “allosteric drugs” that target flexible regions of the protein required for activity (32).

There remain many unanswered fundamental questions about MDH to be investigated.

Some examples are:

i) Folding, Stability & Oligomeric Structure: most MDHs are dimeric, some are monomers and others form tetramers. Folding and acquisition of oligomeric structure in α/β proteins such as MDH is also little studied. The role of protein dynamics and stability in biological activity is another aspect ripe for further investigation

ii) Substrate specificity and catalytic mechanism. (there exist MDH isoforms with LDH like activity, or NADPH (vs. NADH) affinity). Although the roles of the active site His-Asp diad, and the flexible loop containing 2 of the three active site arginines are frequently assumed, detailed information about their roles in catalysis and substrate binding is lacking. Little is known about the roles of so-called “second sphere” residues

iii) Allosteric regulation (some forms are regulated by citrate inhibition/activation and/or substrate inhibition) and pH, together with the role of Subunit Interactions and the subunit interface . Work on the roles of interface residues H90, E256, S266 and L269 is underway in the Bell lab and nearing publication)

Relevant Literature:

1. Guynn, RW., Gelberg, HJ.,Veech, RL., The Equilibrium Constants of the Malate Dehydrogenase, Citrate Synthase, Citrate Lyase and Acetyl CoA Hydrolysis reactions under physiological conditions, J. Biol. Chem 248, 6957-6965, 1973

2. Birktoft JJ, Banaszak LJ. 1983. The presence of a histidine-aspartic acid pair in the active site of 2-hydroxyacid dehydrogenases. J Biol Chem, 258:472-482

3. Jessica K. Bell, Hemant P. Yennawar, S. Kirk Wright, James R. Thompson,

Ronald E. Viola, and Leonard J. Banaszak, Structural Analyses of a Malate Dehydrogenase with a Variable Active Site, J. Biol Chem Vol. 276, No. 33, Issue of August 17, pp. 31156–31162, 2001

4. Musrati RA, Kollárová M, Mernik N, Mikulásová D (September 1998). "Malate dehydrogenase: distribution, function and properties". General Physiology and Biophysics. 17 (3): 193–210

5. Ding Y, Ma QH., Characterization of a cytosolic malate dehydrogenase cDNA which encodes an isozyme toward oxaloacetate reduction in wheat., Biochimie. 2004 Aug;86(8):509-18

6. Gietl C, Lehnerer M, Olsen 0. 1990. Mitochondrial malate dehydrogenase from watermelon sequence of complementary DNA clones and primary structure of the higher-plant precursor protein. Plant Mol Biol, 14:1019-1030.

7. Asaph B. Cousins, Itsara Pracharoenwattana, Wenxu Zhou, Steven M. Smith, and Murray R. Badge, Peroxisomal Malate Dehydrogenase Is Not Essential for Photorespiration in Arabidopsis But Its Absence Causes an Increase in the Stoichiometry of Photorespiratory CO2 Release, Plant Physiol. Vol. 148, 200, 786-795

8. McAlister-Henn L. 1988. Evolutionary relationships among the malate dehydrogenases.

Trends Biochem Sci 13:178-181.

9. CHRISTINE GIETL AND BERTOLD HOCK, Organelle-Bound Malate Dehydrogenase Isoenzymes Are Synthesized as Higher Molecular Weight Precursors, Plant Physiol. (1982) 70, 483-487

10. Birktoft JJ, Rhodes G, Banaszak LJ. 1989b. Refined crystal structure of cytoplasmic malate dehydrogenase at 2.5-A resolution. Biochemistry, 28:6065-6089.

11. Birktoft JJ, Fu Z, Carnahan GE, Rhodes G, Roderick SL, Banaszak LJ. Comparison of the molecular structures of cytoplasmic and mitochondrial malate dehydrogenase. Biochem SOC Trans 17:301-304

12. Buehner M, Ford GC, Moras D, Olsen KW, Rossman MG., D-glyceraldehyde-3-phosphate dehydrogenase: three-dimensional structure and evolutionary significance., Proc Natl Acad Sci U S A. 1973 Nov;70(11):3052-4

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13. CHRISTOPHER R. GOWARD AND DAVID J. NICHOLLS, Malate dehydrogenase:

A model for structure, evolution, and catalysis, Protein Science (1994), 3:1883-1888

14. Chem J and Smith DL., Amide hydrogen exchange shows that malate dehydrogenase is

a folded monomer at pH 5, Protein Science (2001), 10:1079–1083.

15. Breiter DR, Resnik E, Banaszak LJ., Engineering the quaternary structure of an enzyme: construction and analysis of a monomeric form of malate dehydrogenase from Escherichia coli., Protein Sci. 1994 Nov;3(11):2023-32

16. Wu F, Minteer S., Krebs cycle metabolon: structural evidence of substrate channeling revealed by cross-linking and mass spectrometry., Angew Chem Int Ed Engl. 2015 Feb 2;54(6):1851-4

17. Leonard A. Fahien$, Edward H. Kmiotek, Michael J. MacDonaldg, Barbara Fibich, and

Milka Mandic, Regulation of Malate Dehydrogenase Activity by Glutamate, Citrate,

&-Ketoglutarate, and Multienzyme Interaction, J. Biol. Chem Vol. 263, No. 22, Issue of August 5, pp. 10687-10697,1988

18. Igor Morgunov and Paul A. Srere, Interaction between Citrate Synthase and Malate Dehydrogenase: SUBSTRATE CHANNELING OF OXALOACETATE, J. Biol. Chem., Vol. 273, No. 45, Issue of November 6, pp. 29540–29544, 1998

19. Sonia BEECKMANS, Edilbert VAN DRIESSCHE and Louis KANAREK, The visualization by affinity electrophoresis of a specific association between the consecutive citric acid cycle enzymes fumarase and malate dehydrogenase, Eur. J. Biochem 183,449 -454 (1 989)

20. Gelpi JL, Dordal A, Montserrat J, Mazo A, Cortes A. 1992. Kinetic studies of the regulation of mitochondrial malate dehydrogenase by citrate. Biochem J 283:289-297.

21. Shimin Zhao, Wei Xu, Wenqing Jiang, Wei Yu, Yan Lin, Tengfei Zhang, Jun Yao, Li Zhou, Yaxue Zeng, Hong Li, Yixue Li, Jiong Shi, Wenlin An, Susan M. Hancock, Fuchu He, Lunxiu Qin, Jason Chin, Pengyuan Yang, Xian Chen, Qunying Lei, Yue Xiong, Kun-Liang Guan, Regulation of Cellular Metabolism by Protein Lysine Acetylation, SCIENCE , 19 FEBRUARY 2010 VOL 327 , 1000-1004

22. Qingli Shi and Gary E. Gibson, Up-regulation of the mitochondrial malate dehydrogenase by oxidative stress is mediated by miR-743a, J Neurochem. 2011 August ; 118(3): 440–448.

23. Madern D (June 2002). "Molecular evolution within the L-malate and L-lactate dehydrogenase super-family". Journal of Molecular Evolution. 54(6): 825–40

24. Sundaram TK, Wright IP, Wilkinson AE. 1980. Malate dehydrogenase from thermophilic and mesophilic bacteria. Molecular size, subunit structure, amino acid composition, immunochemical homology, and catalytic activity. Biochemistry 19:2017-2022.

25. Gietl, C, Seidel, C., & Svendsen, I., Plant Glyoxysomal but not mitochondrial malate dehydrogenase can fold without chaperone assistance. Biochimica et Biophysica Acta 1274 (1996) 48-58

26. Bjørk A, Dalhus B, Mantzilas D, Sirevåg R, Eijsink VG., Large improvement in the thermal stability of a tetrameric malate dehydrogenase by single point mutations at the dimer-dimer interface., J Mol Biol. 2004 Aug 27;341(5):1215-26