Gen. Physiol.

Biophys (1998), 17, 193—210 193

Minireview

Malate Dehydrogenase: Distribution,

Function and Properties

R. A. M U S R A T I , M. KOLLÁROVA, N. M E R N I K AND D . MIKULÁŠOVÁ

Department of Biochemistry, Faculty of Natural Sciences, Comenius University,

Mlynská dolina CH-1, 842 15 Bratislava, Slovak Republic

A b s t r a c t . Malate dehydrogenase (MDH) (EC 1.1.1.37) catalyzes the conversion

of oxaloacetate and malate. This reaction is important in cellular metabolism, and
it is coupled with easily detectable cofactor oxidation/reduction. It is a rather ubi-
quitous enzyme, for which several isoforms have been identified, differing in their
subcellular localization and their specifity for the cofactor NAD or N A D P . T h e
nucleotide binding characteristics can be altered by a single amino acid change.
Multiple amino acid sequence alignments of MDH show t h a t there is a low degree
of primary structural similarity, a p a r t from several positions crucial for catalysis,
cofactor binding and the subunit interface. Despite the low amino acids sequence
identity their 3-dimensional structures are very similar. MDH is a group of multi-
meric enzymes consisting of identical subunits usually organized as either dimer or
tetramers with subunit molecular weights of 30-35 kDa. MDH has been isolated
from different sources including archaea, eubacteria, fungi, plant and mammals.

K e y w o r d s : Malate dehydrogenase — Isoenzymes — Protein structure

Malate dehydrogenase is a widely distributed enzyme. T h e preferred substrate

for MDH is oxaloacetate (Nicholls et al. 1992). It plays a crucial role in many impor-
t a n t metabolic pathway including the tricarboxylic acid cycle, glyoxylate bypass,
amino acid synthesis, gluconeogenesis and facilitation of exchange of metabolites
between cytoplasm and subcellular organelles.
MDH is a multimeric enzyme consisting of identical subunits usually orga-
nized as either dimers or t e t r a m e r s with subunit molecular weights of 3 0 - 3 5 kDa.
MDH from Nitzschia alba is composed of eight identical subunits. It is the only
octameric MDH so far reported (Yueh et al. 1989). Each subunit of MDH contains
a dinucleotide-binding domain which is similar in sequence and structure t o t h a t of

Correspondence to: Marta Kollárova, Department of Biochemistry, Faculty of Na-

tural Sciences, Comenius University, Mlynská dolina CH-1, 842 15 Bratislava, Slovakia
E-mail: kollaurmQfns.uniba.sk
194 Musrati et al.

other nicotinamide-nucleotide dependent enzymes. X-ray structures of MDH show

the bound cofactor to be in an extended conformation (Hill et al. 1972; Birktoft et
al. 1989b), and similar to the conformation of cofactor bound to lactate dehydro-
genase (LDH) (Piontek et al. 1990).
Malate dehydrogenase isoenzymes can serve as a model system for studying
the evolution, protein sorting to different cell compartments, and for comparison
of MDH isoenzymes.
Eukaryotic tissue contains multiple forms of MDH involved in different meta-
bolic pathways and located in different subcellular compartments. MDH in eukary-
otic organisms is found in microbodies such as glyoxysomes and peroxisomes, in the
mitochondria, in the cytoplasm and in chloroplasts. All MDHs are NAD-dependent
except the chloroplastic enzyme which requires NADP as a cofactor.
Escherichia coli has only one form of MDH (Hall et al. 1992). Its MDH is
most similar to eukaryotic mitochondrial MDH which has 59% sequence identity
and a similar tertiary structure. Two isoenzymes of MDH, mitochondrial and cy-
toplasmic, are present in mammalian cells. In higher plants, there are three NAD-
dependent forms located in the cytoplasm, the mitochondria and the microbodies,
and one NADP-dependent form present in chloroplasts (Poeydomenge et al. 1995).
The different isoenzymes are encoded in genes of the nucleus, and they are synthe-
sized on cytoplasmic ribosomes and imported to their respective organelles.
In principle, different isoenzymes can be derived from unrelated genes, from
genes in the same family sharing a common ancestral gene or by alternative RNA
splicing and posttranslational modification from a single gene.
The MDH amino acid sequences show divergence into two main phylogenetic
groups of closely related enzymes - cytoplasmic and mitochondrial MDHs. Some
of the most distanly related isoenzymes of MDH are found compartmentalized in
different subcellular organelles of the same cell types (Goward and Nicholls 1994).
The archaebacterial sequence of Haloarcula morismortui (Cendrin et al. 1993) sug-
gests a link between the evolution of MDHs and lactate dehydrogenases (LDHs).
An explanation of the difference between MDHs from organelles and those from
the cytosol is that a common ancestral mdh gene may have been duplicated before
invasion of primordial eukaryotes by bacteria to produce mitochondria according to
the probable endosymbiotic origin of these organelles (McAlister-Henn et al. 1987).

Catalysis

The active site of MDH consists of a predominantly "hydrophobic vacuole", which

contains binding sites for the substrate and the nicotinamide ring of the coenzyme.
Upon formation of the enzyme:coenzyme:substrate ternary complex there is a pro-
tein conformational change in which an external loop closes over the active-site
vacuole. In addition, other functionally important residues are brought into close
Malate Dehydrogenase 195

Figure 1. Active-site vacuole of MDH. The schematic drawing summarizes the function
of active-site residues with the substrate oxaloacetate and coenzyme NADH. The arrow
indicates the position of phosphate in the case of NADP + (Goward and Nicholls 1994).

proximity of the substrate (Grau et al. 1981; Clarke et al. 1986; Wigley et al. 1992).
The loop is highly conserved among MDHs (residues 98-110), reflecting its crucial
role in catalysis (Fig. 1). X-ray structures of MDHs crystallized in the presence
and absence of substrate analogues, which bind to the active site, have identified 2
conformationally distinct forms of the enzyme with external loop in either the up
or down position. The active site of this enzyme contains invariant and interacting
H195 and D168 residues. His-Asp pair linked by a hydrogen bond may function as a
proton relay system during catalysis and allow the imidazole ring of the histidine to
act as both an acid and a base. This pair could also provide an explanation for the
relatively stronger binding by cytoplasmic MDH of NADH versus NAD (Birktoft
and Banaszak 1983).
196 Musrati et al.

The side chain of residue D53 is important for coenzyme binding and specificity
by hydrogen bonding with the adenosine ribose hydroxyl groups. It is chemically
conserved with an acidic side chain in all NAD-MDHs. G53 was found in the
chloroplastic NADP-MDH of maize and sorghum (Birktoft et al. 1989a; Hall et al.
1992; Wigley et al. 1992; Kelly et al. 1993). There are 3 arginine residues (R102,
R109 and R171) which are important for substrate binding and catalysis. R102
and R109 are on the underside of the mobile loop and interact with substrate in
ternary complex. The quanidium side chains of R102 and R171 form counterions
for the substrate carboxylate groups, which contribute to binding and orientation
of the substrate in the active site (Clarke et al. 1986; Goward and Nicholls 1994).

Mitochondrial M D H

The majority of mitochondrial proteins are encoded in the nucleus, synthesized

in the cytoplasm and imported to one of four locations in the mitochondria: the
matrix, the intermembrane space, the outer membrane or the inner membrane.
Many mitochondrial proteins are synthesized as precursors, which are imported
post-translationally with the aid of nucleotide triphosphate hydrolysis and the elec-
trochemical potential across the inner mebrane. Many precursors of matrix proteins
such as malate dehydrogenase contain N-terminal presequences (transit sequences)
of 10-70 residues in length, which are cleaved off by specific peptidases after import
to the mitochondria. Import is dependent on the precursor protein being in an un-
folded state, and it requires membrane potential across the inner membrane (Hartl
et al. 1989). There is evidence that import is mediated by proteins in the cytosol,
receptors in the membrane and chaperons in the matrix (Schatz 1993). Transit
sequences are relatively rich in positively charged residues (mainly arginine), lack
acidic side-chain and have a high content of serine and threonine and small clusters
of adjacent hydrophobic groups (MacLachlan et al. 1994).
Comparison of watermelon mMDH with mammalian, yeast and E. coli MDH
gives 55-60% overall identity of residues (McAlister-Henn et al. 1987). On the other
hand, the amino acid sequences of mouse mMDH and cytosolic MDH (cMDH)
show only 23% overall identity (Joh et al. 1987). Surprisingly, comparisons of the
amino acid sequences among the eukaryotic and bacterial MDHs revealed that the
similarity between the mMDHs from plants, yeast, E. coli and the thermophilic
bacterium Thermus flavus exceeds the intraspecies sequence similarity between
mitochondrial and cytoplasmic MDH (Nishiyama et al. 1986).
The structure of mitochondrial MDH is well defined. In most organisms, it
is a dimer composed of identical subunits with approximate molecular weights of
34 kDa (Banaszak and Bradshow 1975), and the known amino acid sequence con-
tains 314 residues. The gene encoding the mitochondrial isoenzyme of MDH is
present in a single copy in genomic DNA of S. cerevisiae. The specific activity of
Malate Dehydrogenase 197

mMDH was elevated approximately eight-times in yeast cells transformed with a

multicopy plasmid containing the entire encoding region for the corresponding gene
(McAlister-Henn et al. 1987). Although several collections of yeast mutants with
defects in mitochondrial functions and oxidative energy metabolism have been iso-
lated, mutants with specific defects in mitochondrial MDH have not been described
(Parker and Matoon 1969; Tzagoloff et al. 1975; Ciriacy 1977).
Mitochondrial MDH is involved in three different pathways: 1. tricarboxylic
acid cycle, 2. conversion of glycine to serine to provide reducing equivalents, and
3. supply of CO2 for fixation in bundle sheath chloroplasts of higher plants.
In the tricarboxylic acid cycle mitochondrial MDH provides, together with
isocitrate dehydrogenase and a-ketoglutarate dehydrogenase, for NADH to be oxi-
dised in the respiratory chain, while succinate dehydrogenase yields FADH2. Plant
mitochondria preferentially oxidise NADH produced in the matrix space as a re-
sult of substrate oxidation. The latter two pathways suggests cooperation of the
mitochondrion with the peroxisome in photorespiration and with the chloroplast
in the concentration mechanism of CO2 for C4 photosynthesis, respectively (Gietl
1992).
In mammalian and yeast cells mitochondrial and cytosolic MDHs there are
components of the malate/aspartate shuttle; they represent an important mecha-
nism for exchange of substrates and reducing equivalents between metabolic path-
ways separated by the mitochondrial membrane. NADH is formed in the cytoplasm
during glycolysis and is required for respiration and a variety of metabolic processes.
From the cytoplasm to the mitochondria it is transported across the mitochondrial
membrane by the malate/aspartate shuttle because intact mitochondria are imper-
meable for NADH (Setoyama et al. 1990).

Cytosolic M D H

When the mitochondrial isoenzyme is synthesized as higher molecular weight pre-

cursor, the in vitro translation of the cytosolic MDH yields a product which has
the same molecular weight as the native isoenzyme.
Eukaryotic cells contain two forms of MDH enzymes - one in the mitochondria
and the other one in the cytoplasm. Both forms of MDH are synthesized in the cy-
toplasm, where cMDH remains after acetylation of the N-terminal residue. mMDH
is synthesized with an N-terminal extension residue and is subsequently imported
into the mitochondrial matrix (Chien and Freeman 1984). Cytosolic MDH is a
homodimer, each subunit having a molecular mass of 35 kDa and containing 332
amino acids of known sequence (Birktoft et al. 1987; Joh et al. 1987).
One of the more noticeable differences between cMDH and mMDH is the
difference in the overall polarity of the two enzymes. cMDH is more polar as well
as more acidic than mMDH, and this difference is due solely to the difference in the
198 Musrati et al.

number of charged residues. There are 41 basis residues (31 lysines + 10 arginines)
and 43 acidic residues (25 aspartates + 18 glutamates) in cMDH. In mMDH there
are 25 lysines and 8 arginines for total of 33 basic residues, and 13 aspartates and
16 glutamates for a total of 29 acidic groups (Birktoft et al. 1989a).
The sequence identity between cMDH and mMDH is relatively low, being of
the order of about 20-25%. The best fit of the molecular structure of cMDH to
that of lactate dehydrogenase has been obtained by the least square method. This
similarity between the dimeric cMDH and the tetrameric LDH reported earlier by
Rao and Rossmann (1973), particularly in the nucleotide binding domain, has been
confirmed. The active sites of these two enzymes contain similarly oriented His-Asp
pairs linked by a hydrogen bond which may function as a proton relay system during
catalysis. This pair could also provide an explanation for the relatively stronger
binding by cMDH and LDH of NADH versus NAD.

Peroxisomal and glyoxysomal M D H

Peroxisomes are organelles present in almost all eukaryotic cells. Like the mitochon-
drion, the peroxisome is a major site of oxygen utilization. In fact, the peroxisome
is thought by some to represent the vestige of an ancient organelle that carried
out all of the oxygen metabolism of primitive preeukaryotic cells when oxygen en-
tered the atmosphere. Most peroxisomes catalyze the breakdown of fatty acids to
acetyl-CoA using a special H2 02-producing enzyme. The acetyl-CoA produced can
be transported via the cytosol to the mitochondria to feed the citric acid cycle, or
it can be used for biosynthetic reactions elsewhere.
Two very different types of peroxisomes have been extensively studied in
plants. One type is present in leaves, where it catalyzes the oxidation of a side
product of the reaction that fixes CO2 in carbohydrate (photorespiration). A very
different type of peroxisome is present in germinating seeds, where it serves to con-
vert the fatty acids stored in seed lipids into sugars needed for the production of the
materials of the young plant. Because this is accomplished by a series of reactions
known as the glyoxylate cycle, these peroxisomes are also called glyoxysomes.
Metabolism of glycolate carbon occurs sequentially in three organelles, the per-
oxisomes, the mitochondria and the chloroplasts (Lorimer and Andrews 1980). In
the peroxisome glycolate is oxidised to glyoxylate and then transaminated to glycine
with either glutamate or serine (Rehfeld and Tolbert 1972). Glycine then stays in
the peroxisome and is oxidised to ammonia, CO2 and serine in the mitochondrion.
Serine is converted to glycerate by serine:glyoxylate aminotransferase and hydrox-
ypyruvate reductase in the peroxisome, and in this form photorespiratory carbon
returns to the chloroplast. Glycerate is phosphorylated to phosphoglycerate by
glycerate kinase, and can reenter the photosynthetic cycle.
Photorespiration is an example for the tight cooperation of metabolic pathways
Malate Dehydrogenase 199

located in different cell compartments. A malate/oxaloacetate/aspartate shuttle

has been proposed for plant microbodies similar to that for mitochondria (Dry et
al. 1987). Leaf peroxisomes as well as seed glyoxysomes contain a large amount
of activity of an isoenzyme of NAD-malate dehydrogenase (Yamazaki and Tolbert
1969; Curry and Ting 1973). Besides catalase (an enzyme that constitutes up to 40
percent of the total peroxisomal protein), malate dehydrogenase is the most active
enzyme in plant microbodies. It is therefore not surprising that enzymes present
in both glyoxysomes and peroxisomes are identical. Peroxisomal and glyoxysomal
MDHs are serologically indistinguishable and have the same isoelectric points and
function as dimers (Hock and Gietl 1982).
The transport of glyoxysomal MDH from the site of synthesis in the cytoplasm
to the site of function in the organelle involves translocation of the protein across
the single membrane of the organelle. Glyoxysomal MDH is very similar to mito-
chondrial and cytoplasmic MDHs and also lactate dehydrogenases. Especially the
amino acids Arg-87, Gly-185 and Gly-228 which are crucial for selecting malate as
substrate, are rigorously conserved in MDH enzymes, while in LDH enzymes an
equally strong conservation is observed for Gin, Asp and Thr in the same positions
(Wilks et al. 1988).
Glyoxysomes lack DNA, they are surrounded by a single membrane and share
at least two biochemical capabilities with peroxisomes of animals and fungi: O2
processing (based on the conversion of H2O2 by catalase) and fatty acid oxida-
tion. In glyoxysomes they convert the long-chain fatty acids to their CoA esters
(Cooper 1971). Their membranes contain an alkaline lipase that hydrolyses tria-
cylglycerols (Muto and Beevers 1974; Huang 1987). They possess a complete set
of enzymes for the /3-oxidation of fatty acids, and enzymes of the glyoxylate cy-
cle. The /3-oxidation of fatty acids ends up with the production of acetyl-CoA.
The enzymes of the glyoxylate cycle catalyze the conversion of two molecules of
acetyl-CoA to succinate, and comprise citrate synthase, aconitase, isocitrate lyase,
malate synthase and malate dehydrogenase enzymes (Beevers 1969). For the fate
of NADH synthesized within the glyoxysomes which do not contain an electron
transport system linking NADH to O2, two possibilities are disscused: 1. oxidation
of NADH in glyoxysomes by a malate-aspartate shuttle (Mettler and Beevers 1980)
would involve transport of malate from the glyoxysomes to mitochondria, and 2.
the alternative possibility is coupling of /3-oxidation of fatty acids and glyoxylate
cycles to NADHxytochrome c and ferricyanide reductases in glyoxysomes which
may allow /3-oxidation and the glyoxylate cycle to be partially uncoupled from
mitochondrial oxidative phosphorylation (Donaldson and Fang 1987).

Chloroplastic M D H

Besides the NAD-dependent isoforms of malate dehydrogenase located in microbod-

200 Musrati et al.

ies, the mitochondria and the cytoplasm, an NADP-dependent form of the enzyme
is found in the chloroplasts in higher plants. NADP-dependent MDH (NADP-
MDH) (EC. 1.1.1.82) is involved in the C 4 dicarboxylic acid cycle responsible for
the primary fixation and transfer of CO2 in C4 plants. It is also present in C3 type
plants where it is implicated in chloroplast shuttle mechanisms which might help
export reducing power.
NADPH-MDH is a model enzyme in that its activity is strictly controlled by
light (Johnson and Hatch 1970). Indeed, NADP-MDH is inactive in the dark and
gets activated by light via the ferredoxin - thioredoxin system (Wolosiuk et al.
1980).
NADP-MDH resembles the non-regulatory NAD-MDH, except for two se-
quence additions, one N-terminal and one C-terminal. Due to the presence of both
N- and C-terminal extensions chloroplastic MDHs exhibit larger molecular masses,
and they represent crucial parts of the protein for redox regulation.
Several cysteine residues are located in the NADP-MDH polypeptide, and
all are specific to this chloroplastic redox regulatory isoform. Two of these are
located in the N-terminal extension sequence and one in the C-terminal extension.
Chemical derivatization followed by sequence analysis in sorghum (Decottignies
et al. 1988) has shown that there is a light-dependent reducible disulphide bridge
present in the N-terminal extension. Site-directed mutagenesis has indeed confirmed
that there is a thioredoxin-reducible disulphide bridge involving these two residues
(Issakidis et al. 1992), but has also shown that there is a second disulphide needed
for regulation. Additional site-directed mutagenesis experiments have succeeded in
creating a redox insensitive NADP-MDH, by mutation of the N-terminal disulphide,
together with either or both of the most C-terminal cysteines (C377 and/or C365
in sorghum), indicating that these cysteines could constitute the second regulatory
disulphide (Issakidis et al. 1993; 1994). There is experimental evidence that the
disulfide bridge involving the C-terminal residues is shielding access to the catalytic
residues, and that the N-terminal end is involved in the slow conformational change
of the active site needed for activation (Issakidis et al. 1996).
On the basis of the results of mutagenesis experiments and three-dimensional
structure modeling of the chloroplastic isoenzyme, Issakidis et al. (1994) proposed
a model for the mechanism of activation of NADP-MDH (Fig. 2).
Chloroplast NADP-MDH possesses a His-Asp pair at the active site which
probably forms a proton relay system. The involvement of such a His/Asp pair in
catalysis has already been described for LDH and NAD-MDH. It is acting as an acid
in the reduction of the keto-acid and as a base in the oxidation of the hydroxy-acid
(Birktoft and Banaszak 1983). So, His-229 and Asp-201 play a crucial role in the
catalytic mechanism of chloroplastic NADP-MDHs (Lemaire et al. 1996). Sequence
data of NADP-MDH from pea (Reng et al. 1993), sorghum (Cretin et al. 1990),
maize (Metzler et al. 1989) and ice-plant are available.
Malate Dehydrogenase 201

C - terminal mutants

Activation
Conformational \j SH <]
change
(slow)

Weak activity
Low affinity for OAA

N - terminal mutants

, ^___jP^^, Reduction V ^Hjj^SH y

•^^^ Activation \-_3B&_J-""^

No activity
( active site locked in a
highly active conformation)

Wild type NADP-MDH

No activity
( active site locked in a Weak activity Fully active
weakly active conformation Low affinity for OAA High affinity for OAA

Figure 2. Schematic model of activation mechanism of NADP-MDH (either wild type

or mutant). Only one subunit is shown. The coenzyme binding site is symbolized by the
diamond, as it is accessible from the surface. The triangles represent oxaloacetate. The
N-terminal extention is to the left of the subunit and the C-terminal is to the right, close
to the active site. Trx, thioredoxin (Issakidis et al. 1994).

N A D P - M D H is an essential component of the malate/oxaloacetate shuttle,

which balances reducing equivalents between the chloroplast and the cytosol. In
C 4 plants, N A D P - M D H activity is 10-times higher and acts to convert oxaloac-
etate t o malate in chloroplasts of mesophyll cellsfor transport to bundle sheat cells
(Hatch and Slack 1969). During C 4 photosynthesis, atmospheric CO2 is fixed by
carboxylation of phosphoenolpyruvate in mesophyll cells (by P E P carboxylase),
giving C4 dicarboxylic acids which are decarboxylated in bundle sheath cells by
one of three different decarboxylases: NADP-malic enzyme, NAD-malic enzyme or
P E P carboxykinase. When malate is transported to bundle sheath cells and decar-
202 Musrati et al.

boxylated via NADP-malic enzyme, it acts as a carrier of reducing power as well

as CO2. The NADPH formed is directly utilised for phosphoglycerate reduction.
The encoded amino acid sequence of maize predicts that NADP-MDH is syn-
thesized as a preprotein of 432 amino acids and processed into a mature protein of
375 amino acids with removal of a 57 amino acids long transit peptide. The sorghum
enzyme is synthesized as a precursor of 429 amino acids and gets imported into
the chloroplast where it is processed to a mature subunit of 389 amino acids. De-
spite the lack of sequence similarities to other chloroplast transit peptides the extra
sequence shows the common features : it is rich in the hydroxylated amino acids
serine and threonine (14%), it is also rich in small hydrophobic amino acids such
as alanine and valine (28%), it shows net positive charge (8 arginines, 1 lysine),
and is generally deficient in acidic amino acids (2 aspartates). The maize enzyme is
similar to other MDHs in regions related to enzymatic function (Wilks et al. 1988).
The similarity of the C3 and C4 forms of NADP-MDH suggests that genes
for C4 enzymes may have been recruited from existing genes encoding C3 enzymes
(Gietl 1992).

Eubacterial M D H

Escherichia coli malate dehydrogenase (eMDH) is a homodimer and it is com-

prised of 312 amino acid residues per subunit (Sutherland and McAlister-Henn
1985; McAlister-Henn et al. 1987). The structures of cytosolic and mitochondrial
MDHs from porcine heart have been determined (Roderick and Banaszak 1986;
Birktoft et al. 1989a) and a comparative study between these enzymes and eMDH
demonstrated that eMDH has 58% sequence identity with mMDH, but only about
20% identity with cMDH (Hall et al. 1992; Gleason et al. 1994).
In the crystalline structures of 2 prokaryotic and 2 eukaryotic forms, the sub-
unit interfaces are conformationally homologous. To determine whether or not the
quaternary structure of MDH is linked to catalytic activity, mutant forms of the
enzyme from E. coli have been constructed. Utilizing the high-resolution structure
of E. coli MDH, the dimer interface was analyzed critically for side chains that were
spatially constricted and needed for electrostatic interactions. Two such residues
were found, D45 and S226. At the nearest point in the homodimer, they were found
in different subunits, hydrogen bonded across the interface, and did not interact
with any catalytic residues. Each residue was mutated to a tyrosine, which should
disrupt the interface because of its large size. All mutants were cloned and purified
to homogeneity. Gel fitration of the mutants showed that D45Y and D45Y/S226Y
were both monomers, whereas the S226Y mutant remained a dimer. The monomeric
D45Y and D45/S226Y mutants had 14,000 and 17,500-times less activity, respec-
tively, than the native enzyme. The dimeric S226Y had only 1.4-times less specific
activity.
Malate Dehydrogenase 203

Crystallographic studies have shown that the dimer interface consists mainly of
interacting a-helices that fit compactly together. The active sites in these dimeric
proteins are well separated from each other; they are not in the subunit inter-
face and the bound dicarboxylic acid substrates are about 3 nm apart. The 3-
dimensional subunit-subunit relationship is the same in all the known MDH struc-
tures and in one of the interfaces present in the tetrameric lactate dehydrogenases.

Table 1. Homology withm a portion of the a-helical region in the dimer interface In
E. coli MDH, D45 and H48 have the most extensive hydrogen bonding pattern Residue
numbers correspond to E. coli MDH (Hall et al 1992)
E coh MDH G V A V D L S H T L S M G
Yeast mMDH G V A T D L S H T L S M A
Pig/mouse mMDH G v A A D L S H T L s M A
Pig cMDH G v L M E L Q D A M s A A
tMDH G v V M E L R D A A S A A
PigLDHA, LDHB G E M M D L Q H G L S V A
41 45 48 224 226 228

Some researchers have suggested the dimer structure to be critical for en-
zymatic activity. Several highly conserved amino acid residues are found in this
interface (Table 1), with D45 and H48 having the most extensive hydrogen bond-
ing interactions (Breiter et al. 1994). The stability of the subunit-subunit interface
m eMDH is the result of direct hydrogen bonds, water-mediated hydrogen bonds
and hydrophobic contacts.
Two schools of thought arose surrounding the structure-function relationship
(Harada and Wolfe 1968). The first proposed the reciprocating compulsory order
mechanism where each subunit alternates as the "active" and the "helper" subunit,
but both are needed for activity. This mechanism predicts an inactive monomer,
and was confirmed by studies that showed a dramatic reduction of enzyme activity
on dissociation to monomers at low enzyme concentration, at pH 5.0 and in the
absence of substrate (Bleile et al. 1977; Wood et al. 1981a,b). The second mech-
anism introduces an equilibrium between two conformers of MDH, one of which
preferentially binds citrate and NAD, whereas the other binds NADH. This would
suggest an active monomer (Mullinax et al. 1982).
From termophilic organism Thermus flavus MDH (tMDH) has been purified
and its enzymatic properties have been analyzed (Iijima et al. 1980). It is a dimer
enzyme composed of two identical subunits, each of a molecular weight of 35 kDa
(Iijima and Saiki 1984). Nishiyama et al. (1986) have cloned the gene for the T.
204 Musrati et al.

flavus malate dehydrogenase (tMDH) into E. coli and reported the sequence. Inter-
estingly, the prokaryotic tMDH sequence is more identical to the eukaryotic cMDH
(55%) than to mMDH (20%). Random mutagenesis of T. flavus yielded a mutant
strain that possessed 3-times higher MDH activity than the wild type strain. The
MDH gene cloned from the mutant strain was found to encode an altered enzyme
with a single amino acid exchange of He for Thr-190 which caused an increase in
the apparent enzyme activity and loss of substrate inhibition in the presence of an
excess of oxaloacetate (Nishiyama et al. 1991).
The position of the NAD-binding loop relative to the body of the protein
in tMDH was compared to that in cMDH and LDH; it was observed that the
tMDH-NAD binary complex is more similar to that in LDH than to that in the
cMDH-NAD binary complex (Kelly et al. 1993).
The malate dehydrogenase from grampositive bacteria Streptomyces aureofa-
ciens has been purified and the molecular and catalytic properties of the enzyme
have been studied. The protein is a homodimer, with a 38 kDa subunit molecular
mass. The enzyme is very similar in many respects to other bacterial MDHs with
the notable exception of a lack of inhibitionn by excess substrate. The enzyme is
much more efficient in reducing oxaloacetate than in oxidating of malate (Mernik
et al. 1998; Mikulášová et al. 1998).

Archaebacterial M D H

The limits of viability of organisms in their natural habitat are determined by

extremes of temperature (-5°C to 110°C), hydrostatic pressure (<120 MPa ),
pH (0-12) and water activity (< 5.5 mol/1 NaCl and KC1 ). Adaptation to these
extremes during the evolution led to development of thermophiles, barophiles, aci-
dophiles, halophiles, etc. Numerous investigations of the molecular mechanism of
adaptation at the genomic, metabolic and cellular level have not indicated a general
strategy (Jaenicke 1981, 1988; Hecht et al. 1989). Comparison of homologous en-
zymes from species adapted to widely different conditions have revealed that ligand
affinities seem to be conserved. The kinetic analog of this conservation of "corre-
sponding states" would be compensatory effects via adjustments of activation free
energies. To give an example, it has been established that cold and warm-adapted
organisms show similar metabolic rates when the comparison is based on the tem-
perature of their natural habitats (Hochachka and Somero 1973; Somero and Low
1976; Jaenicke 1981).
Halophilic eukaryotes and eubacteria overcome the extracellular osmotic pres-
sure by accumulating "osmoprotectants" (Yancey et al. 1982). On the other hand,
halophilic archaebacteria accumulate salt (particularly K + ions) at concentrations
that can reach and exceed saturation (Christian and Waltho 1962). The biochem-
ical machinery of these prokaryotes has, therefore, been adapted in the course of
Malate Dehydrogenase 205

evolution to be able to function at salt concentrations at which most biochemi-

cal systems will cease to function T h e biochemical and biophysical properties of
several halophilic enzymes were studied in great detail (Eisenberg et al 1992)
T h e enzyme malate dehydrogenase of the extremely halophilic archaebactena
Haloarcula marismortm (hMDH) has been studied most extensively using a wide
range of biochemical and biophysical methods (Pundak and Eisenberg 1981, Pun-
dak et al 1981, Zaccai et al 1989) It was found t h a t the enzyme is only stable m
highly concentrated solutions of certain salts T h e gene encoding h M D H could be
isolated and sequenced The enzyme is composed of 303 a m m o acids and its molec-
ular mass is 32,638 Da T h e deduced amino acid sequence of the enzyme was found
to be more similar to the sequence of L-lactate dehydrogenase (LDH) from various
sources than to the sequence of other MDHs The structural gene was cloned in
the E coli expression vector p E T l l a , and large amounts of a soluble but inactive
form of the enzyme were produced upon its induction Activation of the enzyme
was obtained by increasing the salt concentration to 3 mol/1 NaCl (Cendrin et al
1993) Both the halophilic and non-halophihc (mitochondrial) MDH show a close
similarity in their Km profiles On other hand, the catalytic efficiency of the two
homologous enzymes varies dramatically, halophilic malate dehydrogenase exhibits
deactivation at low salt (Hecht et al 1989)
42 residues of the N-terminal amino acid sequence of MDH from thermoaci-
dophihc archaebactenum Sulfolobus acidocaldarius have been determined In the
archaebactenal enzyme the residue Gly(7) G l y ( l l ) and Asp(33) are also present
T h e d a t a suggest t h a t in the enzyme from S acidocaldarius like in the other MDHs
the binding domain for NAD(H) is localized at the N-terminal p a r t of the polypep-
tide chain (Gorisch and Jany 1989)
MDH from the extremely thermophilic methanogen Methanothermus fervidus
has been isolated and its properties were characterized This enzyme is a homomeric
dimer with a molecular mass of 70 kDa displaying low specifity for NAD or N A D P
(Honka et al 1990)

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Final version accepted July 8, 1998