Crystal structure of human mitochondrial NAD(P)+-dependent malic enzyme: a new class of oxidative decarboxylases.

BACKGROUND: Malic enzymes catalyze the oxidative decarboxylation of malate to pyruvate and CO2 with the concomitant reduction of NAD(P)+ to NAD(P)H. They are widely distributed in nature and have important biological functions. Human mitochondrial NAD(P)+-dependent malic enzyme (mNAD-ME) may have a crucial role in the metabolism of glutamine for energy production in rapidly dividing cells and tumors. Moreover, this isoform is unique among malic enzymes in that it is a cooperative enzyme, and its activity is controlled allosterically. RESULTS: The crystal structure of human mNAD-ME has been determined at 2.5 A resolution by the selenomethionyl multiwavelength anomalous diffraction method and refined to 2.1 A resolution. The structure of the monomer can be divided into four domains; the active site of the enzyme is located in a deep cleft at the interface between three of the domains. Three acidic residues (Glu255, Asp256 and Asp279) were identified as ligands for the divalent cation that is required for catalysis by malic enzymes. CONCLUSIONS: The structure reveals that malic enzymes belong to a new class of oxidative decarboxylases. The tetramer of the enzyme appears to be a dimer of dimers. The active site of each monomer is located far from the tetramer interface. The structure also shows the binding of a second NAD+ molecule in a pocket 35 A away from the active site. The natural ligand for this second binding site may be ATP, an allosteric inhibitor of the enzyme.     

Atomic resolution analysis of the catalytic site of an aspartic proteinase and an unexpected mode of binding by short peptides

The X-ray structures of native endothiapepsin and a complex with a hydroxyethylene transition state analog inhibitor (H261) have been determined at atomic resolution. Unrestrained refinement of the carboxyl groups of the enzyme by using the atomic resolution data indicates that both catalytic aspartates in the native enzyme share a single negative charge equally; that is, in the crystal, one half of the active sites have Asp 32 ionized and the other half have Asp 215 ionized. The electron density map of the native enzyme refined at 0.9 A resolution demonstrates that there is a short peptide (probably Ser-Thr) bound noncovalently in the active site cleft. The N-terminal nitrogen of the dipeptide interacts with the aspartate diad of the enzyme by hydrogen bonds involving the carboxyl of Asp 215 and the catalytic water molecule. This is consistent with classical findings that the aspartic proteinases can be inhibited weakly by short peptides and that these enzymes can catalyze transpeptidation reactions. The dipeptide may originate from autolysis of the N-terminal Ser-Thr sequence of the enzyme during crystallization.     

Crystal structures of substrate complexes of malic enzyme and insights into the catalytic mechanism

Malic enzymes catalyze the oxidative decarboxylation of L-malate to pyruvate and CO(2) with the reduction of the NAD(P)(+) cofactor in the presence of divalent cations. We report the crystal structures at up to 2.1 A resolution of human mitochondrial NAD(P)(+)-dependent malic enzyme in different pentary complexes with the natural substrate malate or pyruvate, the dinucleotide cofactor NAD(+) or NADH, the divalent cation Mn(2+), and the allosteric activator fumarate. Malate is bound deep in the active site, providing two ligands for the cation, and its C4 carboxylate group is out of plane with the C1-C2-C3 atoms, facilitating decarboxylation. The divalent cation is positioned optimally to catalyze the entire reaction. Lys183 is the general base for the oxidation step, extracting the proton from the C2 hydroxyl of malate. Tyr112-Lys183 functions as the general acid-base pair to catalyze the tautomerization of the enolpyruvate product from decarboxylation to pyruvate.     

X-ray, neutron and NMR studies of the catalytic mechanism of aspartic proteinases

Current proposals for the catalytic mechanism of aspartic proteinases are largely based on X-ray structures of bound oligopeptide inhibitors possessing non-hydrolysable analogues of the scissile peptide bond. Until recent years, the positions of protons on the catalytic aspartates and the ligand in these complexes had not been determined with certainty due to the inadequate resolution of these analyses. There has been much interest in locating the catalytic protons at the active site of aspartic proteinases since this has major implications for detailed understanding of the mechanism of action and the design of improved transition state mimics for therapeutic applications. In this review we discuss the results of studies which have shed light on the locations of protons at the catalytic centre. The first direct determination of the proton positions stemmed from neutron diffraction data collected from crystals of the fungal aspartic proteinase endothiapepsin bound to a transition state analogue (H261). The neutron structure of the complex at a resolution of 2.1 A provided evidence that Asp 215 is protonated and that Asp 32 is the negatively charged residue in the transition state complex. Atomic resolution X-ray studies of inhibitor complexes have corroborated this finding. A similar study of the native enzyme established that it, unexpectedly, has a dipeptide bound at the catalytic site which is consistent with classical reports of inhibition by short peptides and the ability of pepsins to catalyse transpeptidation reactions. Studies by NMR have confirmed the findings of low-barrier and single-well hydrogen bonds in the complexes with transition state analogues.     

Malic enzymes of salmon trout heart mitochondria: separation and some physicochemical properties of NAD-preferring and NADP-specific enzymes

Mitochondria isolated from the heart of the Baltic salmon trout Salmo trutta contain two distinct malic enzymes. One of these enzymes (NAD-preferring malic enzyme) catalyses the oxidative decarboxylation of malate in the presence of either NAD or NADP. The specific activity with NAD was six times that with NADP as coenzyme. The second enzyme is specific for NADP. These two malic enzymes have been separated by: ion exchange chromatography of DEAE-Sephacel, affinity chromatography on 2',5'ADP-Sepharose 4B, gel filtration on Sephacryl S-300 and polyacrylamide gel electrophoresis. The mol. wts of the two native malic enzymes determined by gel filtration were found to be 280,000 and 190,000 for NAD-preferring and NADP-specific malic enzyme, respectively. Chromatofocusing revealed the isoelectric points of the two enzymes at pH 5.45 and 5.85 for NAD-preferring and NADP-specific malic enzyme, respectively.     

Rhodococcus L-phenylalanine dehydrogenase: kinetics, mechanism, and structural basis for catalytic specificity

Phenylalanine dehydrogenase catalyzes the reversible, pyridine nucleotide-dependent oxidative deamination of L-phenylalanine to form phenylpyruvate and ammonia. We have characterized the steady-state kinetic behavior of the enzyme from Rhodococcus sp. M4 and determined the X-ray crystal structures of the recombinant enzyme in the complexes, E.NADH.L-phenylalanine and E.NAD(+). L-3-phenyllactate, to 1.25 and 1.4 A resolution, respectively. Initial velocity, product inhibition, and dead-end inhibition studies indicate the kinetic mechanism is ordered, with NAD(+) binding prior to phenylalanine and the products' being released in the order of ammonia, phenylpyruvate, and NADH. The enzyme shows no activity with NADPH or other 2'-phosphorylated pyridine nucleotides but has broad activity with NADH analogues. Our initial structural analyses of the E.NAD(+).phenylpyruvate and E.NAD(+). 3-phenylpropionate complexes established that Lys78 and Asp118 function as the catalytic residues in the active site [Vanhooke et al. (1999) Biochemistry 38, 2326-2339]. We have studied the ionization behavior of these residues in steady-state turnover and use these findings in conjunction with the structural data described both here and in our first report to modify our previously proposed mechanism for the enzymatic reaction. The structural characterizations also illuminate the mechanism of the redox specificity that precludes alpha-amino acid dehydrogenases from functioning as alpha-hydroxy acid dehydrogenases.     

The homing endonuclease I-CreI uses three metals, one of which is shared between the two active sites

Homing endonucleases, like restriction enzymes, cleave double-stranded DNA at specific target sites. The cleavage mechanism(s) utilized by LAGLIDADG endonucleases have been difficult to elucidate; their active sites are divergent, and only one low resolution cocrystal structure has been determined. Here we report two high resolution structures of the dimeric I-CreI homing endonuclease bound to DNA: a substrate complex with calcium and a product complex with magnesium. The bound metals in both complexes are verified by manganese anomalous difference maps. The active sites are positioned close together to facilitate cleavage across the DNA minor groove; each contains one metal ion bound between a conserved aspartate (Asp 20) and a single scissile phosphate. A third metal ion bridges the two active sites. This divalent cation is bound between aspartate residues from the active site of each subunit and is in simultaneous contact with the scissile phosphates of both DNA strands. A metal-bound water molecule acts as the nucleophile and is part of an extensive network of ordered water molecules that are positioned by enzyme side chains. These structures illustrate a unique variant of a two-metal endonuclease mechanism is employed by the highly divergent LAGLIDADG enzyme family.     

Crystal structure of a truncated mutant of glucose-fructose oxidoreductase shows that an N-terminal arm controls tetramer formation

N-terminal or C-terminal arms that extend from folded protein domains can play a critical role in quaternary structure and other intermolecular associations and/or in controlling biological activity. We have tested the role of an extended N-terminal arm in the structure and function of a periplasmic enzyme glucose-fructose oxidoreductase (GFOR) from Zymomonas mobilis. We have determined the crystal structure of the NAD(+) complex of a truncated form of the enzyme, GFORDelta, in which the first 22 residues of the N-terminal arm of the mature protein have been deleted. The structure, refined at 2.7 A resolution (R(cryst)=24.1%, R(free)=28.4%), shows that the truncated form of the enzyme forms a dimer and implies that the N-terminal arm is essential for tetramer formation by wild-type GFOR. Truncation of the N-terminal arm also greatly increases the solvent exposure of the cofactor; since GFOR activity is dependent on retention of the cofactor during the catalytic cycle we conclude that the absence of GFOR activity in this mutant results from dissociation of the cofactor. The N-terminal arm thus determines the quaternary structure and the retention of the cofactor for GFOR activity and during translocation into the periplasm. The structure of GFORDelta also shows how an additional mutation, Ser64Asp, converts the strict NADP(+) specificity of wild-type GFOR to a dual NADP(+)/NAD(+) specificity.     

Structural studies of the pigeon cytosolic NADP(+)-dependent malic enzyme.

Malic enzymes are widely distributed in nature, and have important biological functions. They catalyze the oxidative decarboxylation of malate to produce pyruvate and CO(2) in the presence of divalent cations (Mg(2+), Mn(2+)). Most malic enzymes have a clear selectivity for the dinucleotide cofactor, being able to use either NAD(+) or NADP(+), but not both. Structural studies of the human mitochondrial NAD(+)-dependent malic enzyme established that malic enzymes belong to a new class of oxidative decarboxylases. Here we report the crystal structure of the pigeon cytosolic NADP(+)-dependent malic enzyme, in a closed form, in a quaternary complex with NADP(+), Mn(2+), and oxalate. This represents the first structural information on an NADP(+)-dependent malic enzyme. Despite the sequence conservation, there are large differences in several regions of the pigeon enzyme structure compared to the human enzyme. One region of such differences is at the binding site for the 2'-phosphate group of the NADP(+) cofactor, which helps define the cofactor selectivity of the enzymes. Specifically, the structural information suggests Lys362 may have an important role in the NADP(+) selectivity of the pigeon enzyme, confirming our earlier kinetic observations on the K362A mutant. Our structural studies also revealed differences in the organization of the tetramer between the pigeon and the human enzymes, although the pigeon enzyme still obeys 222 symmetry.     

Coenzyme isomerization is integral to catalysis in aldehyde dehydrogenase

Crystal structures of many enzymes in the aldehyde dehydrogenase superfamily determined in the presence of bound NAD(P)(+) have exhibited conformational flexibility for the nicotinamide half of the cofactor. This has been hypothesized to be important in catalysis because one conformation would block the second half of the reaction, but no firm evidence has been put forth which shows whether the oxidized and reduced cofactors preferentially occupy the two observed conformations. We present here two structures of the wild type and two structures of a Cys302Ser mutant of human mitochondrial aldehyde dehydrogenase in binary complexes with NAD(+) and NADH. These structures, including the Cys302Ser mutant in complex with NAD(+) at 1.4 A resolution and the wild-type enzyme in complex with NADH at 1.9 A resolution, provide strong evidence that bound NAD(+) prefers an extended conformation ideal for hydride transfer and bound NADH prefers a contracted conformation ideal for acyl-enzyme hydrolysis. Unique interactions between the cofactor and the Rossmann fold make isomerization possible while allowing the remainder of the active site complex to remain intact. In addition, these structures clarify the role of magnesium in activating the human class 2 enzyme. Our data suggest that the presence of magnesium may lead to selection of particular conformations and speed isomerization of the reduced cofactor following hydride transfer.     

The conformation of adenosine diphosphoribose and 8-bromoadenosine diphosphoribose when bound to liver alcohol dehydrogenase.

8-Bromo-adenosine diphosphoribose (br8 ADP-Rib) and nicotinamide 8-bromoadenine dinucleotide (Nbr8AD+) which are analogues of the coenzyme NAD+, were prepared and their liver alcohol dehydrogenase complexes studied by crystallographic methods. Nbr8AD+ is active in alcohol dehydrogenase complexes studied by crystallographic methods. Nbr8AD+ is active in hydrogen transport and br8ADP-Rib is a coenzyme competitive inhibitor for the enzymes liver alcohol dehydrogenase and yeast alcohol dehydrogenase. X-ray data were obtained for the complex between liver alcohol dehydrogenase and br8ADP-Rib to 0.45 nm resolution and for the liver alcohol dehydrogenase-adenosine diphosphoribose complex to 0.29-nm resolution. The conformations of these analogues were determined from the X-ray data. It was found that ADP-Rib had a conformation very similar to the corresponding part of NAD+, when NAD+ is bound to lactate and malate dehydrogenase. br8ADP-Rib had the same anti conformation of the adenine ring with respect to the ribose as ADP-Rib and NAD+, in contrast to the syn conformation found in 8-bromo-adenosine. The overcrowding at the 8-position is relieved in br8ADP-Rib by having the ribose in the 2' endo condormation instead of the usual 3' endo as in ADP-Rib and NAD+.     

Crystal structure of Tritrichomonas foetus inosine monophosphate dehydrogenase in complex with the inhibitor ribavirin monophosphate reveals a catalysis-dependent ion-binding site

Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step in GMP biosynthesis. The resulting intracellular pool of guanine nucleotides is of great importance to all cells for use in DNA and RNA synthesis, metabolism, and signal transduction. The enzyme binds IMP and the cofactor NAD(+) in random order, IMP is converted to XMP, NAD(+) is reduced to NADH, and finally, NADH and then XMP are released sequentially. XMP is subsequently converted into GMP by GMP synthetase. Drugs that decrease GMP synthesis by inhibiting IMPDH have been shown to have antiproliferative as well as antiviral activity. Several drugs are in use that target the substrate- or cofactor-binding site; however, due to differences between the mammalian and microbial isoforms, most drugs are far less effective against the microbial form of the enzyme than the mammalian form. The high resolution crystal structures of the protozoan parasite Tritrichomonas foetus IMPDH complexed with the inhibitor ribavirin monophosphate as well as monophosphate together with a second inhibitor, mycophenolic acid, are presented here. These structures reveal an active site cation identified previously only in the Chinese hamster IMPDH structure with covalently bound IMP. This cation was not found previously in apo IMPDH, IMPDH in complex with XMP, or covalently bound inhibitor, indicating that the cation-binding site may be catalysis-dependent. A comparison of T. foetus IMPDH with the Chinese hamster and Streptococcus pyogenes structures reveals differences in the active site loop architecture, which contributes to differences in cation binding during the catalytic sequence and the kinetic rates between bacterial, protozoan, and mammalian enzymes. Exploitation of these differences may lead to novel inhibitors, which favor the microbial form of the enzyme.     

Effects of site-directed mutagenesis on structure and function of recombinant rat liver S-adenosylhomocysteine hydrolase. Crystal structure of D244E mutant enzyme

A site-directed mutagenesis, D244E, of S-adenosylhomocysteine hydrolase (AdoHcyase) changes drastically the nature of the protein, especially the NAD(+) binding affinity. The mutant enzyme contained NADH rather than NAD(+) (Gomi, T., Takata, Y., Date, T., Fujioka, M., Aksamit, R. R., Backlund, P. S., and Cantoni, G. L. (1990) J. Biol. Chem. 265, 16102-16107). In contrast to the site-directed mutagenesis study, the crystal structures of human and rat AdoHcyase recently determined have shown that the carboxyl group of Asp-244 points in a direction opposite to the bound NAD molecule and does not participate in any hydrogen bonds with the NAD molecule. To explain the discrepancy between the mutagenesis study and the x-ray studies, we have determined the crystal structure of the recombinant rat-liver D244E mutant enzyme to 2.8-A resolution. The D244E mutation changes the enzyme structure from the open to the closed conformation by means of a approximately 17 degrees rotation of the individual catalytic domains around the molecular hinge sections. The D244E mutation shifts the catalytic reaction from a reversible to an irreversible fashion. The large affinity difference between NAD(+) and NADH is mainly due to the enzyme conformation, but not to the binding-site geometry; an NAD(+) in the open conformation is readily released from the enzyme, whereas an NADH in the closed conformation is trapped and cannot leave the enzyme. A catalytic mechanism of AdoHcyase has been proposed on the basis of the crystal structures of the wild-type and D244E enzymes.     

Sequestration of the active site by interdomain shifting. Crystallographic and spectroscopic evidence for distinct conformations of L-3-hydroxyacyl-CoA dehydrogenase

l-3-Hydroxyacyl-CoA dehydrogenase reversibly catalyzes the conversion of l-3-hydroxyacyl-CoA to 3-ketoacyl-CoA concomitant with the reduction of NAD(+) to NADH as part of the beta-oxidation spiral. In this report, crystal structures have been solved for the apoenzyme, binary complexes of the enzyme with reduced cofactor or 3-hydroxybutyryl-CoA substrate, and an abortive ternary complex of the enzyme with NAD(+) and acetoacetyl-CoA. The models illustrate positioning of cofactor and substrate within the active site of the enzyme. Comparison of these structures with the previous model of the enzyme-NAD(+) complex reveals that although significant shifting of the NAD(+)-binding domain relative to the C-terminal domain occurs in the ternary and substrate-bound complexes, there are few differences between the apoenzyme and cofactor-bound complexes. Analysis of these models clarifies the role of key amino acids implicated in catalysis and highlights additional critical residues. Furthermore, a novel charge transfer complex has been identified in the course of abortive ternary complex formation, and its characterization provides additional insight into aspects of the catalytic mechanism of l-3-hydroxyacyl-CoA dehydrogenase.     

Enzymes of malate metabolism in Mesorhizobium ciceri CC 1192

Electrophoretic studies were performed on enzymes concerned with the oxidation of malate in free-living and bacteroid cells of Mesorhizobium ciceri CC 1192, which forms nitrogen-fixing symbioses with chickpea (Cicer arietinum L.) plants. Two malate dehydrogenases were detected in extracts from both types of cells in native polyacrylamide electrophoresis gels that were stained for enzyme activity. One band of malate dehydrogenase activity was stained only in the presence of NADP+, whereas the other band was revealed with NAD+ but not NADP+. Further evidence for the occurrence of separate NAD- and NADP-dependent malate dehydrogenases was obtained from preliminary enzyme kinetic studies with crude extracts from free-living M. ciceri CC 1192 cells. Activity staining of electrophoretic gels also indicated the presence of two malic enzymes in free-living and bacteroid cells of M. ciceri CC 1192. One malic enzyme was active with both NAD+ and NADP+, whereas the other was specific for NADP+. Possible roles of the multiple forms of malate dehydrogenase and malic enzyme in nitrogen-fixing symbioses are discussed.     

Coenzymatic properties of low molecular-weight and macromolecular N6-derivatives of NAD+ and NADP+ with dehydrogenases of interest for organic synthesis.

The catalytic activity, expressed as Km and Vmax values, of 16 enzymes of practical interest with the macromolecular coenzymes poly(ethylene glycol)-N6-(2-aminoethyl)-NAD+ and poly(ethylene glycol)-N6-(2-aminoethyl)-NADP+ and their low molecular weight precursors N6-(2-aminoethyl)-NAD+ and N6-(2-aminoethyl)-NADP+, was investigated. The enzymes examined are of direct interest for organic synthesis (i.e. alcohol dehydrogenase from yeast, horse liver, or Thermoanaerobium brockii, lactic dehydrogenase, and several hydroxysteroid dehydrogenases) or are used for the regeneration of NAD+, NADP+, NADH, or NADPH (i.e. glutamate dehydrogenase from liver or Proteus, formate dehydrogenase, glucose dehydrogenase, and malic enzyme). The cycling efficiency of poly(ethylene glycol)-N6-(2-aminoethyl)-NADP+ was examined with coupled-enzymes or coupled-substrates systems. Poly(ethylene glycol)-N6-(2-aminoethyl)-NAD+ and, even more so, poly(ethylene glycol)-N6-(2-aminoethyl)-NADP+ were excellent coenzymes with several dehydrogenases. In addition, the coenzymatic properties of N6-(3-sulfonatopropyl)-NAD+, an NAD+ derivative carrying a strong anionic group, were compared with those of the newly synthesized N6-(2-hydroxy-3-trimethylammonium propyl)-NAD+, an NAD+ derivative carrying a strong cationic group. It was expected that the presence of the sulfonic or quaternary ammonium group would enhance the residence time of the coenzyme inside continuous-flow reactors if membranes with anionic or cationic groups, respectively, were used.     

Determination of the MurD mechanism through crystallographic analysis of enzyme complexes.

UDP -N- acetylmuramoyl- L -alanine: D -glutamate (MurD) ligase catalyses the addition of d -glutamate to the nucleotide precursor UDP -N- acetylmuramoyl- L -alanine (UMA). The crystal structures of three complexes of Escherichia coli MurD with a variety of substrates and products have been determined to high resolution. These include (1) the quaternary complex of MurD, the substrate UMA, the product ADP, and Mg2+, (2) the quaternary complex of MurD, the substrate UMA, the product ADP, and Mn2+, and (3) the binary complex of MurD with the product UDP - N- acetylmuramoyl- L -alanine- D -glutamate (UMAG). The reaction mechanism supported by these structures proceeds by the phosphorylation of the C-terminal carboxylate group of UMA by the gamma-phosphate group of ATP to form an acyl-phosphate intermediate, followed by the nucleophilic attack by the amino group of D-glutamate to produce UMAG. A key feature in the reaction intermediate is the presence of two magnesium ions bridging negatively charged groups.     

The sites for catalysis and activation of ribulosebisphosphate carboxylase share a common domain

The complexation of ribulosebiphosphate carboxylase with CO2, Mg2+, and carboxyarabinitol bisphosphate (CABP) to produce the quaternary enzyme-carbamate-Mg2+-CABP complex closely mimics the formation of the catalytically competent enzyme-carbamate-Mg2+-3-keto-CABP form during enzymatic catalysis. Quaternary complexes were prepared with various metals (Mg2+, Cd2+, Mn2+, Co2+, and Ni2+) and with specifically 13C-enriched ligands. 31P and 13C NMR studies of these complexes demonstrate that the activator CO2 site (carbamate site), the metal binding site, and the substrate binding site are contiguous. It follows that both the carboxylase and oxygenase activities of this bifunctional enzyme are influenced by the structures of the catalytic and activation sites.     

Characterization of two members of a novel malic enzyme class.

The Gram-negative bacterium Rhizobium meliloti contains two distinct malic enzymes. We report the purification of the two isozymes to homogeneity, and their in vitro characterization. Both enzymes exhibit unusually high subunit molecular weights of about 82 kDa. The NAD(P)(+) specific malic enzyme [EC 1.1.1.39] exhibits positive co-operativity with respect to malate, but Michaelis-Menten type behavior with respect to the co-factors NAD(+) or NADP(+). The enzyme is subject to substrate inhibition, and shows allosteric regulation by acetyl-CoA, an effect that has so far only been described for some NADP(+) dependent malic enzymes. Its activity is positively regulated by succinate and fumarate. In contrast to the NAD(P)(+) specific malic enzyme, the NADP(+) dependent malic enzyme [EC 1.1.1.40] shows Michaelis-Menten type behavior with respect to malate and NADP(+). Apart from product inhibition, the enzyme is not subjected to any regulatory mechanism. Neither reductive carboxylation of pyruvate, nor decarboxylation of oxaloacetate, could be detected for either malic enzyme. Our characterization of the two R. meliloti malic enzymes therefore suggests a number of features uncharacteristic for malic enzymes described so far.