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Copyright 2003 by the American Chemical Society Volume 42, Number 44 NoVember 11, 2003 Current Topics Structure and Function of Malic Enzymes, A New Class of Oxidative Gu-Gang Chang*,‡ and Liang Tong§ Faculty of Life Sciences, Institute of Biochemistry, Proteome Research Center, National Yang-Ming UniVersity, Taipei 112, Taiwan, and Department of Biological Sciences, Columbia UniVersity, New York, New York 10027 ReceiVed July 16, 2003; ReVised Manuscript ReceiVed September 24, 2003 ABSTRACT: Malic enzyme is a tetrameric protein with double dimer structure in which the dimer interfaceis more intimately contacted than the tetramer interface. Each monomeric unit of the enzyme is composedof four structural domains, which show a different folding topology from those of the other oxidativedecarboxylases. The active center is located at the interface between domains B and C. For humanmitochondrial malic enzyme, there is an exo nucleotide-binding site for the inhibitor ATP and an allostericsite for the activator fumarate, located at the tetramer and dimer interfaces, respectively. Crystal structuresof the enzyme in various complexed forms indicate that the enzyme may exist in equilibrium among twoopen and two closed forms. Interconversion among these forms involves rigid-body movements of thefour structural domains. Substrate binding at the active site shifts the open form to the closed form thatrepresents an active site closure. Fumarate binding at the allosteric site induces the interconversion betweenforms I and II, which is mediated by the movements of domains A and D. Structures of malic enzymefrom different sources are compared with an emphasis on the differences and their implications to structure-function relationships. The binding modes of the substrate, product, cofactors, and transition-state analogueat the active site, as well as ATP and fumarate at the exo site and allosteric site, respectively, provide aclear account for the catalytic mechanism, nucleotide specificities, allosteric regulation, and functionalroles of the quaternary structure. The proposed catalytic mechanism involves tyrosine-112 and lysine-183as the general acid and base, respectively. In addition, a divalent metal ion (Mn2+ or Mg2+) is essentialin helping the catalysis. Binding of the metal ion also plays an important role in stabilizing the quaternarystructural integrity of the enzyme.
In a reflections article (1), Kornberg vividly described the liver malic enzyme (ME)1 (2). Later, this enzyme was found way leading to the discovery and characterization of pigeon to be widely distributed in nature, in bacteria, plants, andanimals. In mammals, malic enzymes have three identifiable † Supported by the National Science Council, ROC (Frontiers in isoforms: cytosolic NADP+-dependent (c-NADP-ME) (EC Sciences Program, NSC 91-2321-B-010-002 and International Coopera-, mitochondrial NADP+-dependent (m-NADP-ME) tive Program, NSC 92-2321-B-012-066 to G.-.G.C.) and the NationalScience Foundation, USA (MCB-99-74700 to L.T.).
* Corresponding author. E-mail: [email protected] and tong@ 1 Abbreviations: ME, malic enzyme; c-NADP-ME, cytosolic NADP+- dependent malic enzyme; m-NAD-ME, mitochondrial NAD(P)+- ‡ National Yang-Ming University.
dependent malic enzyme; m-NADP-ME, mitochondrial NADP+- § Columbia University.
dependent malic enzyme.
10.1021/bi035251+ CCC: $25.00 2003 American Chemical Society Published on Web 10/14/2003 12722 Biochemistry, Vol. 42, No. 44, 2003 (EC, and mitochondrial NAD(P)+-dependent malic in a binary complex with NAD+ or quaternary complex with enzymes (m-NAD-ME) (EC m-NAD-ME can use NADH, tartronate, and Mg2+ (24, 25), have also been either NAD+ or NADP+ as the cofactor but prefers NAD+ reported (Table 1, Supporting Information).
under physiological conditions (3). Distinct from c-NADP- New Class of OxidatiVe Decarboxylases. The crystal ME and m-NADP-ME, m-NAD-ME is an allosteric enzyme structures of human m-NAD-ME, pigeon c-NADP-ME, and with fumarate as an activator and ATP as an inhibitor (3).
A. suum m-NAD-ME show that the polypeptide backbone Malic enzyme catalyzes a reversible oxidative decarboxy- of malic enzymes has a different topology from that of the lation of L-malate to give carbon dioxide and pyruvate in other oxidative decarboxylases (Figure 1A) (12, 18, 23, 24).
the concomitant reduction of NAD(P)+ to NAD(P)H. The Therefore, the structure information establishes malic en- enzyme thus has a systematic name of (S)-malate:NAD(P)+ zymes as a new class of oxidative decarboxylases.
oxidoreductase (oxaloacetate-decarboxylating). The reaction Malic enzyme has an R/â structure. It belongs to an amino also needs an essential divalent metal ion (Mn2+ or Mg2+) acid dehydrogenase-like family and a superfamily that for the catalysis.
contains the NAD(P)-binding Rossmann-fold domain. Thecore structure includes three layers of R/â/R type, a parallel â-sheet of six strands in the order of 321456. The ME monomer was divided into four domains (A, B, C, and D) CO + pyruvate + NAD(P)H + H (Figure 1A) (12), and these domains behave mostly as rigid-bodies in the conformational transition between open and c-NADP-ME was grouped as a lipogenic enzyme because closed forms of the enzyme (Figure 1E and following text).
of its involvement in providing NADPH for the biosynthesis Domain A contains residues 23-1302 and is mostly helical of long-chain fatty acids or steroids (3-5). In C4 plants, (RA1 through RA6) (Figure 1A). Domain B consists of two ME is involved in the anaplerotic replenish of the tricar- segments of the polypeptide chain, residues 131-277 and boxylic acid-cycle intermediate (6, 7). Human m-NAD-ME 467-538 (Figure 1B), with domain C (residues 278-466) has received much attention because of its involvement in as an inserted cassette. Domain D, residues 539-573, the energy metabolism in neuron or neoplasia tissues (8- contains one helix followed by a long extended structure that 11). Mitochondrial malic enzyme could associate with the protrudes away from the rest of the monomer (Figure 1A).
pyruvate dehydrogenase complex in the inner mitochondrial Domain B contains a central, parallel five-stranded â-sheet membrane. This could localize m-NAD-ME in the vicinity (âB1 through âB5), which is surrounded by helices on both of the inner mitochondrial membrane, making m-NAD-ME sides (RB1 through RB8) (Figure 1B). This â-sheet represents able to intercept exogenous malate from malate dehydroge- a new backbone-fold for a five-stranded parallel â-sheet.
nase as malate passing through the inner mitochondrial There is a short â-hairpin structure (âB2′-âB3′) between membrane (10). This may be the reason that glutamine strand âB2 and helix RB2 in this domain (Figure 1B).
instead of glucose is the major energy source of some tumor Residues in this hairpin structure are highly conserved among cells (9). Mitochondrial malic enzyme is thus a potential malic enzymes, including the first phosphate-binding target in cancer chemotherapy. A structure-based rational GXGXXG motif in malic enzymes, 168-GLGDLG-173.
drug design for ME could lead to potential anti-cancer drugs.
However, the residues in this motif are not involved in NAD+ In this perspective, solving the first crystal structure of ME binding, although they are located near the active site of the in 1999 (12) had a special impact in this important field.
There are a total of 13 crystal structures of ME available Domain C has the dinucleotide-binding Rossmann fold, in the Protein Data Bank (Table 1, Supporting Information).
with the exception that strand three is replaced by a short All of these ME have similar overall tertiary structure albeit antiparallel strand (âC2′, Figure 1C). In addition, there is with small local differences, which have important structural an extra â-strand (âC7) at the C-terminal end of the domain, implications on the catalytic and regulatory mechanisms. This together with a â-hairpin insertion between âC6 and âC7.
review summarizes the structural features and the functional The NAD+ cofactor in the active site is associated with this implications of this new class of oxidative decarboxylases.
domain. The second dinucleotide-binding signature motif, Structural Studies of Malic Enzymes. Crystals of pigeon 311-GAGEAA-316, is located between âC1 and RC1 in this c-NADP-ME were first reported more than 30 years ago (13).
domain and mediates the binding of the phosphates of the Since then, crystals of other malic enzymes have also been cofactor as in other Rossmann folds (Figure 1C). However, obtained, including rat c-NADP-ME (14), Ascaris suum the amino acid conservation between this and other Ross- m-NAD-ME (15), human m-NAD-ME (16), and new crystal mann-fold domains is very low, in the 15% range.
forms of pigeon c-NADP-ME (17). The first crystal structure Open and Closed Forms of Malic Enzymes. The first of any malic enzyme is that of human m-NAD-ME, in a structure of human m-NAD-ME, in a binary complex with binary complex with NAD+ (Table 1, Supporting Informa- NAD+, is in an open form, as the active site region is fully tion) (12, 18). This is followed by the structures of thisenzyme in a ternary complex with NAD+ and Lu3+ (19), quaternary complexes with NAD+, substrate analogue inhibi- To facilitate the comparisons of the sequences and structures of this large family of highly conserved enzymes, all the residues in these tors, and divalent cations (20); as well as pentary complexes enzymes were numbered according to those in human m-NAD-ME, with NAD+, NADH, or the inhibitor ATP, substrate malate for which the first structural information was obtained on these enzymes.
or pyruvate, divalent cation, and the allosteric activator For pigeon c-NADP-ME, 21 should be subtracted from the correspond- fumarate (21, 22). In addition, the structures of pigeon ing numbers. For Ascaris suum m-NAD-ME, 14 should be addedinstead. When residues from other subunits are discussed, small letters c-NADP-ME in a quaternary complex with NADP+, oxalate, are used to indicate the origin of the subunit (e.g., Trp572d denotes and a divalent cation (23), as well as A. suum m-NAD-ME Trp572 from subunit d).
Biochemistry, Vol. 42, No. 44, 2003 12723 FIGURE 1: Structures of monomers of human m-NAD-ME. (A) The structure of m-NAD-ME in a binary complex with NAD+, in openform I. The â-strands are shown in cyan, R-helices in yellow, and the connecting loops in purple. The four domains of the structure arelabeled. The active site is indicated by the red star. Only the ADP portion of the NAD+ molecule in the exo site is shown. (B) The structureof domain B, with a central parallel five-stranded â-sheet. (C) The structure of domain C, with a bound NAD+ molecule. (D) The structurem-NAD-ME, in a pentary complex with NADH, malate (labeled as M), Mn2+ (pink sphere), and fumarate, in closed form II. (E) Overlapof the CR traces of the binary, open form (in cyan) and pentary, closed form (in yellow) of m-NAD-ME, showing the large rigid-bodymovements of the domains. (F) Molecular surface of the binary complex, in open form I, near the active site, colored according to electrostaticpotential. (G) Molecular surface of the pentary complex, in closed form II, near the active site, colored by the three domains.
exposed to the solvent (Figure 1F) (12). Upon binding the domains (Figure 1E) (20). Interestingly, residues in domains divalent cation and the substrate (malate or pyruvate) or A and D also undergo a rigid-body shift, with a rotation of substrate-analogue inhibitors (oxalate, tartronate, or keto- 9.2° (Figure 1E). This causes a reorganization of the tetramer malonate), the enzyme undergoes a large conformational of human m-NAD-ME. The movements of domain C and change (20-22). In this closed form of the enzyme, the domains A and D are independent of each other. In the divalent cation and the substrate or inhibitor are shielded structure of human m-NAD-ME in a ternary complex with from the solvent (Figure 1G). The closed form of the enzyme NAD+ and Lu3+ (19), the movement in domains A and D is is likely the catalytically competent conformation, while the observed, but the active site remains open. Therefore, it open form may be required for substrate binding and product appears that human m-NAD-ME can exist in two open forms release. Therefore, it is possible that most malic enzymes and possibly two closed forms as well.
can undergo the open-closed transition during catalysis.
Kinetically, Lu3+ is a competitive inhibitor with respect The closure of the active site is mediated by the rigid- to Mn2+ (19). The dynamic quenching constants of the body movement of domain C with respect to domain B, intrinsic fluorescence for the metal-free and Lu3+-containing including a relative rotation of 10.6° between the two enzymes are quite different, indicating the conformational 12724 Biochemistry, Vol. 42, No. 44, 2003 differences between the two enzyme forms. The secondary absent in c-NADP-ME, are found in m-NAD-ME between structure of these two enzyme forms, on the other hand, Arg484d and Tyr543a. Other interchain cation-π inter- remains unchanged. Replacement of the catalytically essential actions greater than 3.5 Å but within 6 Å include Arg128a/ Mn2+ by other metal ions (Zn2+, Cu2+, or Fe2+) leads to a Tyr84b and Lys26a/Trp150b pairs in the A-B dimer slow conformational change of the enzyme and consequently alters the geometry of the active site (26). The transformed The tetramer interface is due to interactions between enzyme conformation, however, is unfavorable for catalysis.
subunits A and D as well as A and C. The former (A/D) Both the chemical nature of the metal ion and its correct interaction can be divided into three groups (Figure 3C). The coordination in the active site are essential for catalysis.
first is due to the contact of subunits A and D at the exo The Lu3+-induced isomerization was completely reversible.
site, which involves the protruding of Tyr543a into subunit The Lu3+-inhibited activity can be fully reactivated and the D forming a cation-π interaction between Tyr543a-CD2 and dynamic quenching constant completely returned to that of Arg484d-CZ. c-NADP-ME lacks intimate interaction in this the open form by Mn2+ (27). This finding indicates that the area. Tyr543a in c-NADP-ME points to subunit A itself, tertiary structure of the E-NAD+-Mn2+ complex is indistin- almost 180° different in direction as compared to Tyr543a guishable from the E-NAD+ open form by the fluorescence in the m-NAD-ME. The other two areas involve interactions quenching analysis. The open form transformed to the closed in the dimer interface area and the extension of the form only after substrate binding. Lu3+, on the other hand, C-terminus domain D into the other dimer (Table 2, transformed the open form into a catalytically inactive form, which may not be a physiologically relevant structure. Excess There are large differences in the organization of the Mn2+ could replace Lu3+ in the metal-binding site and tetramer of human m-NAD-ME between the binary complex convert the inactive form back to the open form and may be with NAD+ (an open form, Figure 2A) and the pentary an open form II, which is not obviously distinct from the complex with NADH/malate/Mn2+/fumarate (a closed form, open form I in the routine biophysical probe fluorescence Figure 2D) (12, 20, 22). This reorganization is mediated by quenching analysis (27).
rigid-body movements of residues in domains A and D, as Tetramer of the Enzyme. The tetramer of malic enzymes described in the previous section (Figure 1E). Both the dimer obeys a 222 point-group symmetry, with each monomer and the tetramer interfaces are affected by this reorganization.
having essentially the same environment (Figures 2A and On the basis of current structural information on the human 3A). The four monomers are positioned at the four corners m-NAD-ME, its tetramers may exist in one of the four states of a square, an arrangement first observed in the electron (and additional states may also be possible). These four states microscope images of pigeon liver malic enzyme (28). The include open forms I and II and closed forms I and II (Figure dimensions of the tetramer are about 110 × 110 × 55 Å, 2A-D). The closure of the active site, mediated by the consistent with electron microscopy and solution light movement of domain C, distinguishes the open and closed scattering studies (16, 28).
forms. The reorganization of the tetramer, mediated mostly The tetramer is a dimer of dimers, with intimate contacts by the rigid-body movements in domains A and D, distin- at the dimer interface, whereas the association of the two guishes the two open or two closed forms. The structure of dimers is weaker (Figures 2A and 3A). This is in agreement human m-NAD-ME in the NAD+ binary complex represents with the biochemical studies showing that pigeon c-NADP- open form I (Figure 2A), whereas the structure of the ternary ME exists in a monomer-dimer-tetramer equilibrium in NAD+/Lu3+ complex may be open form II (Figure 2B). The solution (29). Mutation at some positions near the N- structures of the quaternary and pentary complexes of human terminus, located at the dimer interface, affects the quaternary m-NAD-ME all correspond to closed form II (Figure 2D).
structure (30, 31). The dimer interface involves residues from The closed form I (Figure 2C) has yet to be observed domains A and B of the monomer. Helices RA3 and RA4, experimentally, and it might be unstable.
and their 2-fold symmetry mates, form a four-helical bundleat this interface (Figure 2A). Interactions at the tetramer It is not known whether other malic enzymes can also interface are primarily mediated by the long, extended assume two open and two closed forms. The quaternary segment at the C-terminus of the malic enzyme monomer complex of pigeon c-NADP-ME is in closed form II (23), (domain D) (Figure 1A). It latches onto the other dimer of and it would be of interest to determine the conformation of the tetramer and interacts with both of its monomers (Figures the open form(s) of this enzyme. The binary complex of A. 2A and 3A). The side chain of residue Trp572 is completely suum m-NAD-ME with NAD+ is in open form I (24). The buried at this tetramer interface. Mutation of this Trp to Phe closed forms of the ascaris ME are yet to be determined.
has a tremendous effect on the quaternary structure of the The two open forms for human m-NAD-ME might be enzyme (32). Besides the C-terminal segment, residues 541- related to its allosteric activation by fumarate (21). Fumarate 544 also make a contribution to the formation of the tetramer is bound at the dimer interface, but the open form I structure in human ME (Figure 2C) (Table 2, Supporting Information).
is not compatible with fumarate binding (see next). The A. There are substantial differences between pigeon c-NADP- suum m-NAD-ME can also be activated by fumarate (33).
ME and human m-NAD-ME in the subunit interfaces (Table The allosteric site where tartronate is bound is assumed to 2, Supporting Information). The major interactions between be the activator site for fumarate (25). In this regard, it is subunits are hydrophobic interactions in both enzymes interesting that the binary complex of this enzyme, in the (Figure 3C). Hydrogen bonding also contributes significantly absence of fumarate, is also in open form I. Therefore, the in the subunit association. Three salt bridges in c-NADP- open form I structure might be linked to allosteric regulation ME, one in Glu27a/Lys23b, and two in Arg74a/Asp80b pairs of some of these enzymes, while most other malic enzymes are absent in the m-NAD-ME. Two cation-π interactions, can only assume open form II and closed form II.
Biochemistry, Vol. 42, No. 44, 2003 12725 FIGURE 2: Tetramer of malic enzymes. Schematic drawing of the tetramer of human m-NAD-ME in (A) open form I, (B) open form II,(C) closed form I, and (D) closed form II. The monomers are colored in green, cyan, yellow, and purple, respectively. The active site isindicated by the red star. The hypothetical closed form I has not been observed experimentally yet.
The four active sites of the tetramer are separated from Comparison of ME from Various Sources. As shown in each other by about 60 Å and from the dimer or tetramer Figure 3B, the active site of ME constitutes a major interface by about 32 Å (Figures 2A and 3A). Most malic conserved region. Some of the subunit contacting regions enzymes have simple, hyperbolic kinetics with respect to are also conserved among ME (Figure 3C). The amino acid their substrates suggesting that the four active sites are sequences around the metal-binding site are highly conserved.
functioning independently. However, human and A. suum The direct metal ligands, Glu255, Asp256, and Asp279, are m-NAD-ME exhibit cooperative behavior with respect to the identical among all malic enzymes, from bacteria to humans substrate malate, but the sigmoidal kinetics is abolished in (Figure 3D). The catalytic mechanism should be essentially the presence of the fumarate activator (33-35). The binding the same for all malic enzymes.
of either malate or fumarate may induce the transition from Figure 3E shows the superimposed crystal structures of open form I to open form II in the dimer of these enzymes, some resolved malic enzymes. The overall structure of and this transition may be the molecular basis for the human m-NAD-ME, pigeon c-NADP-ME, and A. suum observed cooperativity. An anticooperative behavior has been m-NAD-ME are similar. The alignment regions are residues reported for the pigeon c-NADP-ME (36, 37), but no 21-573 for the closed form human m-NAD-ME (1OX2), asymmetry in the tetramer is apparent from the structural 23-573 for the open form human m-NAD-ME (1QR6), 23- 578 for the pigeon c-NADP-ME (1GQ2), and 12-563 for 12726 Biochemistry, Vol. 42, No. 44, 2003 FIGURE 3: Comparison of ME structures. (A) Quaternary structural features of malic enzyme. The four domains of subunit B are shownas a worm model in green, blue, red, and yellow, respectively. (B) Functional and conserved regions of ME. The discrete conservationcolor scheme used for visualization is based on the continuous conservation scores. The conserved regions are shown in violet and variableregion in blue with a visual color key provided (61), in which color grades (1-9) are assigned. (C) Surface of the ME monomer showingthe subunit contacting regions. White or light areas denote hydrophobic contacts between subunits. Magenta areas denote putative hydrogen-bond contacts. The nearby residues are shown with a stick model in CPK color. Generated with Protein Explorer v1.982b (62). (D) Sequencelogos of ME around the metal-binding site. Amino acid sequences of ME were searched for alignment by ConSurf (61), and the result isexpressed by sequence logos with error bars shown (63). The metal-binding ligands, Glu255, Asp256, and Asp279 (red stars), are strictlyidentical among all ME. Conservation of some putative second sphere hydrophobic amino acid residues is also evident in this figure. Colorcodes for the amino acids are as follows: blue for basic residues (Lys, Arg, and His), red for acidic residues (Asp and Glu), violet foramide residues (Asn and Gln), green for other neutral/polar residues, and black for hydrophobic residues. (E) Stereo model showing thesuperimposition of the crystal structures of human m-NAD-ME (1QR6, blue and 1OX2, turquoise), pigeon c-NADP-ME (1GQ2, magenta),and A. suum m-NAD-ME (1LLQ, gray). The structures of 1OX2 and 1GQ2 are those of the closed form and those for 1QR6 and 1LLQare the open form. The yellow ball denotes the manganese ion. (F) Phylogenetic tree of ME from various sources. Thirty-seven ME sequencesfrom Swiss-Prot database (release 41.16) were analyzed by the ConSurf (61). Each entry name is followed by its accession number: Mao1Bacsu (P54572); Mao1 Ecoli (P26616); Mao1 Rhime (O30807); Mao2 Bacsu (P45868); Mao2 Ecoli (P76558); Mao2 Haein (P43837);Mao2 Rhime (O30808); Mao2 Ricpr (Q9ZDF6); Mao2 Salty (Q9ZFV8); Mao3 Bacsu (O34389); Mao4 Bacsu (O34962); Maoc Flapr(P36444); Maoc Flatr (P22178); Maoc Lyces (P37222); Maoc Maize (P16243); Maoc Orysa (P43279); Maom Amahp (P37224); MaomAscsu (P27443); Maom Human (P23368); Maom Soltu (P37221); Maon Human (Q16789); Maon Soltu (P37225); Maox Anapl (P28227);Maox Bacst (P16468); Maox Colli (P40927); Maox Human (P48163); Maox Mescr (P37223); Maox Mouse (P06801); Maox Myctu (P71880);Maox Phavu (P12628); Maox Pig (Q29558); Maox Poptr (P34105); Maox Rat (P13697); Maox Schpo (P40375); Maox Vitvi (P51615);Maox Yeast (P36013); Mles Lacla (Q48662); and Mles Oenoe (Q48796).
Biochemistry, Vol. 42, No. 44, 2003 12727 the A. suum m-NAD-ME (1LLQ) (24). The backbone traces The dissociated monomers are easily polymerized. Manga- are similar in all malic enzymes with small local conforma- nese ion provides full protection against polymerization (32, tional differences, which reflect the structural basis of the 42). The native enzyme changes to a supra-active conforma- different properties observed in the catalysis of substrate or tion at 1 M urea concentration. The sedimentation coefficient of this form is slightly decreased when compared to the Among the structural differences of ME is the length of native state. All these data support a metal ion-induced open domain D (Figure 3E). Pigeon c-NADP-ME has a C-terminus form II that has a different conformation with the open form seven residues longer than the human m-NAD-ME. The A. I. In the absence of Mn2+, the partially unfolded tetramer, suum enzyme, on the other hand, has both long C- and presumably in the open form I, quickly dissociates to a N-termini. Because of these differences, the ME from partially unfolded monomer when the urea concentration different species have various strengths in the quaternary increases to 2 M. In the presence of Mn2+, it will remain as structure. At neutral pH, c-NADP-ME exists as tetramer, a partially unfolded tetramer. When the urea concentration which sediments as a single species with a sedimentation is further increased to 3-5 M, the exposed hydrophobic site coefficient of approximately 10 S. m-NAD-ME, on the other of the partially unfolded monomer induces polymerization.
hand, exists as a mixture of tetramer and dimer under the Mn2+ can delay this polymerization up to 5 M urea.
same conditions (38). It is interesting to note that both Eventually, all aggregates will be dissolved in urea higher NAD(P)+- and NADP+-dependent ME from Rhizobium than 6 M to give the denatured monomer. However, since melitoti and other Gram-negative bacteria are chimeric the enzyme activity is not restored by dilution, the unfolding proteins with a large phosphotransacetylase-like domain of is not reversible. It seems that, under physiological concen- approximately 320 amino acid residues at the C-terminus.
tration of manganese or magnesium concentration, ME exists Mutants without this C-terminal region retain ME activity as the metal ion-containing open form II, which is resistant but are unable to oligomerize into the native state (39). In to aggregation.
human m-NAD-ME, the allosteric regulated ATP and There are a total of three to four Trp in each subunit of fumarate are bound at the tetramer and dimer interfaces, ME, distributed in different structural domains of the enzyme.
respectively. A single mutation at the exo ATP site of Arg542 These Trp are ideal intrinsic chromophores to study the local to alanine will produce dimeric mutants R542A (38).
conformational changes of ME. Several Trp mutants have From the 37 available amino acid sequences of ME, a been constructed to access different structural domains of phylogenetic tree of ME was built. Five clusters of ME are the pigeon c-NADP-ME (42). Substitution of a single grouped (Figure 5F). Between the plant kingdom (upper part tryptophanyl residue, Trp572, by phenylalanine renders a loss in Figure 5F) and the animal kingdom (lower part) are of protective ability of Mn2+ against the polymerization (42).
prokaryotic bacteria and eubacteria. Two malolactic enzymes The Trp572 residue from subunit A is located in a deep, from lactic acid bacteria (Oenococcus oeni and Lactococcus hydrophobic pocket from the neighboring subunit and is lactis) catalyzing the degradation of L-malate to CO2 and involved in the tetramer interaction. The crystal structure of lactate with concomitant reduction of NAD+ to NADH also the enzyme shows that there are some nearby histidyl belong to the malic enzyme family.
residues. Protonation of these histidyl residues at a lower Dual Functional Roles of Metal Ion in the Mechanism of pH can affect the binding of the Trp572 residue in the ME. The enzyme needs an essential divalent metal ion (Mn2+ tetramer interface, which in turn, destabilizes the tetramer or Mg2+), which plays dual functional roles in catalysis and interface (29). The tetrameric structure is essential for the in structural stability. The three metal ligands Glu255, overall structural stability of ME.
Asp256, and Asp279 are 2.43, 2.19, and 2.23 Å, respectively, ActiVe Site of the Enzyme and Substrate/Inhibitor Binding to the manganese ion, forming a reaction core. Among other Modes. The active site of malic enzyme is located in a deep amino acid residues within 7 Å from the metal ion are many cleft at the interface between domains B and C of each hydrophobic amino acid residues (i.e., Phe254, Phe257, molecule (Figure 1G) together with several residues from Ile166, Leu167, and Ile179). These amino acid residues form domain A (mostly from helix RA6, Figure 4A). The amino a second sphere for the catalytic metal ion and the other polar acid residues in the active site region are generally highly groups in the active center and ensure an optimal environ- conserved among the malic enzymes, supporting their ment for substrate binding and catalytic reactions (40).
importance in substrate binding and/or catalysis (Figure 3D).
Alteration of these residues, even indirectly, might affect the The active site residues can be roughly divided into four catalytic efficiency. For example, the mutation of Phe257 categories: (1) divalent cation-binding residues (Glu255, to leucine contributes -2.19 kcal/mol for the coupling energy Asp256, and Asp279); (2) substrate-binding residues (Thr113, between Phe257 and Asp162 on the metal binding (41).
Pro114, Val116, Arg165, Ile166, Leu167, Ile179, Asn421, The importance of hydrophobicity in the second sphere and Pro422); (3) NAD(P)+ cofactor binding residues (310- of the metal binding site is enforced by the finding that the 316, Asp345, 346, Arg354, 362, Ala393, and Asn467); and change of Phe257 to a less hydrophobic alanine (F257A) (4) catalytic residues (Tyr112 and Lys183). The structural results in loss of 2.51 kcal/mol for the metal binding energy.
roles of residues binding the divalent cation and substrate/ Substitution of Phe257 by the more hydrophobic leucine inhibitor are described here. Residues binding NAD(P)+ will (F257L), on the other hand, can restore most of the lost be described in the section on cofactor selectivity, whereas binding energy (1.41 kcal/mol) (41). The functional role of the residues Tyr112 and Lys183 will be discussed in the all these amino acid residues in the putative second sphere section on the catalytic mechanism of these enzymes.
of the metal site will, however, have to await further study.
The divalent cation is bound deep in the active site cleft The c-NADP-ME is reversibly dissociated under acidic (Figure 4A) and is octahedrally coordinated by six oxygens, environment or in the presence of a chemical denaturant.
one each from the side chain carboxylate groups of Glu255, 12728 Biochemistry, Vol. 42, No. 44, 2003 FIGURE 4: Active site of malic enzymes. (A) Residues of human m-NAD-ME near the active site of the enzyme, shown in gray forcarbons. The malate molecule is shown with the carbon atoms in cyan and the NAD+ molecule in green. The Mn2+ ion is shown as a purplesphere and the water molecules in red. (B) Schematic drawing of the polar interactions in the active site of human m-NAD-ME. (C)Close-up of the active site of human m-NAD-ME, showing the hydrogen-bonding interactions for the Lys183 side chain. The hydridetransfers between the C2 atom of malate and the C4 atom of nicotinamide, and the proton transfer between Tyr112 and C3 atom of thesubstrate, are indicated in green. (D) Comparison of the binding modes of NAD+, oxalate, and Mn2+ in the quaternary complex with thoseof NADH, malate, and Mn2+ in the pentary complex. (E) A possible catalytic mechanism for malic enzymes. The other proton on theLys183 side chain is hydrogen bonded to Asp278 throughout the reaction cycle.
Asp256, and Asp279, two from the substrate or inhibitor, late group (Figure 4A). The C2 hydroxyl and one of the C1 and one from a water molecule (Figure 4C). The identifica- carboxylate oxygen atoms are ligands to the divalent cation tion of Asp279 as a ligand to the cation is in agreement with (Figure 4C). Malate is also involved in a large network of the previous biochemical studies on the pigeon and A. suum hydrogen-bonding and ionic interactions with the enzyme malic enzymes (43-45).
(Figure 4B). The carboxylate oxygen atom (O1A) that is By studying the dead-end NADH/malate and NAD+/ ligated to the divalent cation also has ionic interactions with pyruvate complexes, the bound conformations of the malate the side chain guanidinium group of Arg165, whereas the and pyruvate substrate molecules have also been determined other carboxylate oxygen atom (O1B) is hydrogen bonded (22). Malate is bound in the active site such that the C2 to the side chain amide of Asn421 and the 2′-hydroxyl of hydroxyl is essentially in the same plane as the C1 carboxy- the nicotinamide ribose.
Biochemistry, Vol. 42, No. 44, 2003 12729 The structural information is generally consistent with the observations from previous biochemical studies on theseenzymes. Chemical modification of an Arg residue in pigeonand maize malic enzyme disrupted malate binding withoutaffecting NADP+ binding (46, 47). This is likely Arg165 inthe active site (Figure 4A). Modification of tyrosyl residue,probably Tyr112, with tetranitromethane also affects malatebinding (48). The fluorescent affinity label 3-aminopyridineadenine dinucleotide phosphate dialdehyde (oAADP) specif-ically binds the enzyme at the nucleotide-binding site andforms a Schiff's base with lysyl residue (49). The labeledposition, not proved then, very likely is the Lys362. Covalentmodification of the side chain of Cys120 by the substrate-analogue bromopyruvate can also disrupt malate binding (36,50). However, Cys120 is more than 10 Å away from theactive site, suggesting an indirect effect (12).
Possible Catalytic Mechanism. The catalysis by malic enzymes generally proceeds in three stepssdehydrogenationof malate to produce oxaloacetate (k1), decarboxylation ofoxaloacetate to produce enolpyruvate (k2), and finally tau-tomerization of enolpyruvate to produce pyruvate (k3) (Figure4E) (51). The divalent cation at the optimal position helpscatalyze all the steps of the reaction, which explains itsrequirement for catalysis by malic enzymes. Trivalentlanthanide ions can bind to the enzyme at this site andpotently inhibit the catalysis by both human and pigeon malicenzymes (19, 26, 27).
For the oxidative decarboxylation of malate, a general base FIGURE 5: Possible molecular mechanism for cofactor selectivity.
(A) Structure features of pigeon c-NADP-ME near the 2′-phosphate is needed to extract the proton from the C2 hydroxyl group of NADP+. The interactions between 2′-phosphate of NADP+ and to initiate the dehydrogenation reaction (k1) (Figure 4E). For Ser346 and Lys362 are highlighted with red lines. (B) Sequence the tautomerization reaction (k3), a general acid is needed to logos of ME around the nucleotide-binding site of NADP-ME. ME protonate the enolpyruvate intermediate at the C3 position, sequences near the binding site for the 2′-phosphate of NADP+.
and a general base is needed to extract the proton from the The amino acid residues responsible for the nucleotide specificitiesare marked with red stars. The Asp345:Arg354 ion pair is C2 hydroxyl of this intermediate (Figure 4E). It has been highlighted. (C) Sequence logos of ME around the nucleotide- proposed, based on kinetic and mutagenesis studies, that binding site of NAD-ME. In panels B and C, the color codes for Asp279 is the general base and that Lys183 is the general the amino acids are as follows: blue for basic residues (Lys, Arg, acid (45, 52). The structural information suggests, however, and His), red for acidic residues (Asp and Glu), violet for amide that this is unlikely. Asp279 is a ligand to the cation and is residues (Asn and Gln), green for other neutral/polar residues, andblack for hydrophobic residues.
not positioned correctly to function as the general base, whilethe Lys183 side chain is more than 3.6 Å from the C3 atom The C4 carboxylate group of malate is out of the plane of pyruvate (22).
defined by the C1, C2, O2, and C3 atoms (Figure 4C). It is On the basis of the structures of human m-NAD-ME in within the hydrogen-bonding distance to the side chain of complex with malate and pyruvate, Lys183 has been identi- Asn466, the water ligated to the cation, and a second water fied as the general base and Tyr112 as the general acid (22).
molecule (Figure 4B). The C3 atom of malate, on the other The Lys183 side chain is hydrogen bonded to the C2 hand, does not have close contacts with atoms in the enzyme hydroxyl (or carbonyl) of the substrate and the side chains (Figure 4A).
of Tyr112 and Asp278 (Figure 4C). The Lys183 side chain, The bound conformation of the oxalate molecule is in the neutral form, is perfectly positioned to extract the consistent with its role as an analogue of the enolpyruvate proton from the C2 hydroxyl of malate (Figure 4E). In the transition-state intermediate as well as the pyruvate product decarboxylation reaction (k2), Lys183 functions as a general (20, 22). Structural comparison between pyruvate and malate acid and donates its proton to the C2 hydroxyl to produce shows that the C2 atoms of the two molecules are separated the neutral enol. For the tautomerization of enolpyruvate (k3), by about 0.6 Å (Figure 4D), partly due to the difference in Tyr112 donates a proton to the C3 position, while Lys183 the hybridization state of this atom (sp2 vs sp3) in the two extracts the proton from the C2 hydroxyl (Figure 4E). During this process, the proton shared between the two residues The active site also contains several hydrophobic residues, changes its position to maintain both of them in the neutral and the majority of them do not have direct interactions with state (Figure 4E). Therefore, Tyr112-Lys183 functions as the substrate (Figure 4A) but instead help shield the active a general acid-base pair in this reaction.
site region from the solvent in the closed form. Interestingly, Mutation of the Lys general base has large effects on the the two prolines in the active site (Pro114a and Pro422c) catalytic activity of malic enzymes (20, 52, 53). For example, are both in the cis conformation and form a lid over the active mutation of this Lys to Ala in the A. suum malic enzyme site (Figure 4A).
produces a 130 000-fold decrease in the Vmax of the overall 12730 Biochemistry, Vol. 42, No. 44, 2003 reaction (52). In comparison, mutation of the Tyr general ion-pair interactions with Arg354 (Figure 5A,B). This may acid has smaller effects on the reaction, with the Tyr to Phe explain why some malic enzymes can use NADP+ as the mutant having about 1500-fold lower kcat for the oxidative cofactor even with a conserved Asp at this position.
decarboxylation of malate (20, 52). It is likely that the The structure of the quaternary complex of pigeon c- protonation of enolpyruvate (k3) is not a rate-determining NADP-ME suggests a possible molecular mechanism for the step of the forward reaction. It may also be possible that a NADP+ specificity of this enzyme (23). The 2′-phosphate water molecule is recruited into the active site of this mutant group of NADP+ is placed on the surface of the enzyme and partially rescues its catalytic activity. It would be and interacts with residues Ser346 and the side chain interesting to characterize the effects of Tyr112 mutation on ammonium group of Lys362 (Figure 5A). These two residues the rate of the reverse reaction, the reductive carboxylation are conserved among the NADP+-dependent malic enzymes of pyruvate (k- - (Figure 5B). Several other residues near the 2′-phosphate In the complex with malate, the proton on the C2 atom is group also have variations between NADP+- and NAD+- pointed toward the C4 atom of the nicotinamide ring of dependent malic enzymes (Figure 5B,C), although the NAD+, with a hydride transfer distance of about 2 Å. This mutation of Lys347, conserved among NADP+-dependent explains the stereospecificity of malic enzyme for L-malate, malic enzymes, has little impact on the Km for NADP+ of as D-malate cannot adopt the same binding mode (12).
c-NADP-ME (53). This side chain does not directly contact Moreover, the structure predicts a hydride transfer to the A the 2′-phosphate group (Figure 5A). Mutation of Lys362, face of the nicotinamide ring for malic enzymes, consistent on the other hand, has a tremendous effect on the nucleotide with the experimental observations (54). It would be interest- binding on c-NADP-ME. The K362A mutant of pigeon ing to compare the substrate binding modes between ME c-NADP-ME has a 70-fold increase in the Km for NADP+ and tartrate dehydrogenase, which catalyzes the oxidative (53). The (kcat/Km,NADP)/(kcat/Km,NAD) ratios are 0.51 and 31, decarboxylation of D-malate (55).
respectively, for the mutant K362D and K362Q of c-NADP- NAD(P)+ Binding and Cofactor Specificity. The NAD- ME as compared to the value of 7390 and 0.11, respectively, (P)+ cofactor in the active site is associated with domain C for the WT pigeon c-NADP-ME and human m-NAD-ME (Figure 1C), at a position similar to that of the dinucleotide (Kuo, C. C., Chang, G. G., and Chou, W. Y., unpublished in other Rossmann-fold domains. The adenine ring is on the results). Residue 362 is Gln in human m-NAD-ME. It would surface of the protein and positioned between the side chains be interesting to determine whether the Q362K mutant of of residues 346 and 393, but the N1 and N6 atoms of the human m-NAD-ME would prefer to utilize NADP+ as the adenine base are not specifically recognized by the enzyme.
The nicotinamide ring is in the anti conformation. Residue Binding Site for the Allosteric ActiVator Fumarate. The Gly446, strictly conserved among malic enzymes, is located catalytic activity of human and A. suum m-NAD-ME is close to the amide group on this ring. Mutation of this residue activated by fumarate (33-35). The binding site of fumarate to Asp in the Schizosaccharomyces pombe malic enzyme in human m-NAD-ME has been located (21). An equivalent inactivated the enzyme (56). A syn conformation for the binding site may also exist in A. suum m-NAD-ME, based nicotinamide ring was observed in the quaternary complex on the structure of this enzyme with tartronate bound at this of A. suum m-NAD-ME with NADH, tartronate, and Mg2+ site (25). The regulation of human m-NAD-ME is consistent in open form I (25).
with its role in the metabolism of glutamine for energy Malic enzymes have highly conserved amino acid se- production, as fumarate is the product of the previous step quences but have distinct specificities toward the dinucleotide of this pathway while ATP is the ultimate product of this cofactor. Some malic enzymes can only use NAD+ as the energy metabolism (12). On the other hand, the c-NADP- cofactor, while others can only use NADP+. Human m-NAD- ME isoform is mostly involved in generating NADPH for ME is among the few malic enzymes that has dual-specificity fatty acid synthesis; therefore, it is not subject to regulation and can use either NAD+ or NADP+ but prefers NAD+ under by fumarate or ATP.
physiological conditions (57). The molecular basis for The fumarate-binding site is located at the dimer interface cofactor selectivity by these and other enzymes is still poorly (Figures 2D and 3E), about 30 Å from the active site, confirming that fumarate functions through an allosteric Earlier studies with other enzymes have revealed two mechanism (21). The binding pocket is on the surface of major determinants for cofactor specificity (58, 59). First of domain A of one monomer (Figures 1D and 6A). One all, an Asp residue near the end of the second strand of the carboxylate group of fumarate has bidentate ion-pair inter- Rossmann fold generally indicates NAD+ preference, as it actions with the side chain of Arg91, and the other carboxy- recognizes the 2′-hydroxyl group on the ribose and cannot late is in a mono-dentate ion pair with Arg67. Residues 123- tolerate the phosphate group in NADP+. Second, enzymes 130 from the other monomer of the dimer, in the linker that contain a GXGXXG dinucleotide-binding motif gener- between domain A and domain B, cover this binding pocket.
ally prefer NAD+, whereas those with a GXGXXA motif Mutagenesis studies with Arg67 and Arg91 residues in generally prefer NADP+. However, malic enzymes appear human m-NAD-ME confirm their importance in fumarate to disobey both of these rules. An Asp residue (Asp345) is binding (21). Both residues are conserved in A. suum conserved among all malic enzymes at the end of the second m-NAD-ME (Figure 6B) and show similar interactions with strand (âC2) in domain C. Moreover, most malic enzymes the bound tartronate (25). Mutation of Arg91 in A. suum from animals contain the GXGXXA motif, whereas those m-NAD-ME to alanine results in a decrease in both V/Kmalate from lower organisms contain the GXGXXG motif, irrespec- and V/KMg, and the mutant lost its fumarate activation tive of their cofactor specificity. The structures show that property (60). However, both Arg67 and Arg91 are also the Asp345 residue is pointed away from the ribose, forming conserved among many other malic enzymes (Figure 6B), Biochemistry, Vol. 42, No. 44, 2003 12731 FIGURE 6: Binding sites for ATP and fumarate in human m-NAD-ME. (A) The binding of fumarate (in yellow for carbon atoms) to theallosteric site in the dimer interface (in cyan and green for the two monomers). (B) Sequence alignment of residues in the fumarate-bindingsite. Residues that interact with fumarate are shown in cyan. (C) Comparison of the binding modes of ATP (in cyan for carbon atoms) andthe ADP portion of NAD+ (in gray) to the exo site (in cyan for carbon atoms). The phosphorus atoms of ATP are shown in green and thoseof NAD+ in yellow. (D) Sequence alignment of residues in the exo site. (E) Molecular surface of human m-NAD-ME in the active siteregion, showing the binding modes of ATP and NAD+.
which are insensitive to fumarate. Therefore, there are which are conserved only in human m-NAD-ME (Figure additional structural determinants for fumarate binding to 6D). This exo site may be unique to human m-NAD-ME.
human and A. suum m-NAD-ME.
ATP is an inhibitor of human m-NAD-ME (34). The The open form I structure of human m-NAD-ME is not structures of human m-NAD-ME in complex with ATP compatible with fumarate binding, as the side chain of Phe68 reveal that ATP can bind to the active site as well as the assumes a different conformation and blocks this binding exo site (Figure 6C,E) (21). Mutation of the three Arg site (21). Fumarate may activate the enzyme by promoting residues in this site can abolish the binding of ATP to the the transition from open form I to open form II, which may exo site, but ATP has the same inhibitory potency against be more catalytically competent as it requires fewer structural these mutants. Kinetic studies show that the inhibition by changes to go to closed form II. The presence of fumarate ATP is competitive with respect to the cofactor NAD+ or removes the cooperative behavior of the enzyme with respect malate (38). Therefore, ATP is an active site inhibitor of to the substrate malate (33-35). The cooperativity is likely human m-NAD-ME with a Ki of about 0.2 mM (21, 38).
due to a coupled transition from open form II to open form The inhibition of m-NAD-ME by ATP is consistent with its I upon malate binding. The presence of fumarate can convert putative role in energy metabolism (12). The biological the enzyme monomers to the open form II state, thereby function of the exo site is currently unclear. It may be related removing the cooperative behavior. This also suggests that to the quaternary structural integrity of m-NAD-ME because the cooperativity occurs within the dimers of the enzyme mutation at this site will result in dimeric mutants (38).
and is consistent with the Hill coefficient of about 1.5 for Future Prospects. ME has a tremendous amount of kinetic the tetramer (12).
data available (4, 51, 60). The rapid progress in structural Exo Site for ATP/NAD+ Binding. Human m-NAD-ME has studies has provided a clear picture of this enzyme never an exo site for the binding of ADP, ATP, and the ADP seen before. Combining the kinetic analyses with the portion of the NAD+ cofactor (12, 21). This exo site is about structural results allows us to deduce plausible catalytic and 35 Å from the active site (Figure 1A) and is located near allosteric regulation mechanisms of the enzyme. Most of the the N-terminal end of the parallel â-sheet in domain B and biochemical analyses results now have a structural basis for the residues from domain D (Figure 6C). The N6 amino interpretation. However, the structure of m-NADP-ME is still group and the N1 ring nitrogen of the adenine base are unknown. The ME structures from plants and bacteria are specifically recognized in this site. The phosphate groups also unknown. Furthermore, the structural basis for the half- interact with the side chains of Arg197, Arg542, and Arg556, of-the-site reactivity of the tetrameric c-NADP-ME has not 12732 Biochemistry, Vol. 42, No. 44, 2003 yet been demonstrated. The structural interconversions 19. Yang, Z., Batra, R., Floyd, D. L., Hung, H. C., Chang, G. G., and proposed in Figure 2 need further experimental verification.
Tong, L. (2000) Biochem. Biophys. Res. Commun. 274, 440-444.
Experimental evidence for a distinct structure of the ME- 20. Yang, Z., Floyd, D. L., Loeber, G., and Tong, L. (2000) Nat. Struct. NAD(P)-Mn or the ME-NAD(P)-fumarate ternary com- Biol. 7, 251-257.
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22. Tao, X., Yang, Z., and Tong, L. (2003) Structure 11, 1141-1150.
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