Crystal structure of the non-regulatory A(4 )isoform of spinach chloroplast glyceraldehyde-3-phosphate dehydrogenase complexed with NADP

Here, we report the first crystal structure of a photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) complexed with NADP. The enzyme, purified from spinach chloroplasts, is constituted of a single type of subunit (A) arranged in homotetramers. It shows non-regulated NADP-dependent and NAD-dependent activities, with a preference for NADP. The structure has been solved to 3.0 A resolution by molecular replacement. The crystals belong to space group C222 with three monomers in the asymmetric unit. One of the three monomers generates a tetramer using the space group 222 point symmetry and a very similar tetramer is generated by the other two monomers, related by a non-crystallographic symmetry, using a crystallographic 2-fold axis.The protein reveals a large structural homology with known GAPDHs both in the cofactor-binding domain and in regions of the catalytic domain. Like all other GAPDHs investigated so far, the A(4)-GAPDH belongs to the Rossmann fold family of dehydrogenases. However, unlike most dehydrogenases of this family, the adenosine 2'-phosphate group of NADP does not form a salt-bridge with any positively charged residue in its surroundings, being instead set in place by hydrogen bonds with a threonine residue belonging to the Rossmann fold and a serine residue located in the S-loop of a symmetry-related monomer. While increasing our knowledge of an important photosynthetic enzyme, these results contribute to a general understanding of NADP versus NAD recognition in pyridine nucleotide-dependent enzymes.Although the overall structure of A(4)-GAPDH is similar to that of the cytosolic GAPDH from bacteria and eukaryotes, the chloroplast tetramer is peculiar, in that it can actually be considered a dimer of dimers, since monomers are bound in pairs by a disulphide bridge formed across Cys200 residues. This bridge is not found in other cytosolic or chloroplast GAPDHs from animals, bacteria, or plants other than spinach.     

Probing the coenzyme specificity of glyceraldehyde-3-phosphate dehydrogenases by site-directed mutagenesis

By combining our knowledge of the crystal structure of the glycolytic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the sequence of the photosynthetic NADP-dependent GAPDH of the chloroplast, two particular amino acid residues were predicted as the principal determinants of differing coenzyme specificity. By use of site-directed mutagenesis, the amino acids Leu 187 and Pro 188 of GAPDH from Bacillus stearothermophilus have been replaced with Ala 187 and Ser 188, which occur in the sequence from the chloroplast enzyme. The resulting mutant was shown to be catalytically active not only with its natural coenzyme NAD but also with NADP, thus confirming the initial hypothesis. This approach has not only enabled us to alter the coenzyme specificity by minimal amino acid changes but also revealed factors that control the relative affinity of the enzyme for NAD and NADP.     

Thioredoxin-dependent regulation of photosynthetic glyceraldehyde-3-phosphate dehydrogenase: autonomous vs. CP12-dependent mechanisms

Regulation of the Calvin–Benson cycle under varying light/dark conditions is a common property of oxygenic photosynthetic organisms and photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the targets of this complex regulatory system. In cyanobacteria and most algae, photosynthetic GAPDH is a homotetramer of GapA subunits which do not contain regulatory domains. In these organisms, dark-inhibition of the Calvin–Benson cycle involves the formation of a kinetically inhibited supramolecular complex between GAPDH, the regulatory peptide CP12 and phosphoribulokinase. Conditions prevailing in the dark, i.e. oxidation of thioredoxins and low NADP(H)/NAD(H) ratio promote aggregation. Although this regulatory system has been inherited in higher plants, these phototrophs contain in addition a second type of GAPDH subunits (GapB) resulting from the fusion of GapA with the C-terminal half of CP12. Heterotetrameric A₂B₂-GAPDH constitutes the major photosynthetic GAPDH isoform of higher plants chloroplasts and coexists with CP12 and A₄-GAPDH. GapB subunits of A₂B₂-GAPDH have inherited from CP12 a regulatory domain (CTE for C-terminal extension) which makes the enzyme sensitive to thioredoxins and pyridine nucleotides, resembling the GAPDH/CP12/PRK system. The two systems are similar in other respects: oxidizing conditions and low NADP(H)/NAD(H) ratios promote aggregation of A₂B₂-GAPDH into strongly inactivated A₈B₈-GAPDH hexadecamers, and both CP12 and CTE specifically affect the NADPH-dependent activity of GAPDH. The alternative, lower activity with NADH is always unaffected. Based on the crystal structure of spinach A₄-GAPDH and the analysis of site-specific mutants, a model of the autonomous (CP12-independent) regulatory mechanism of A₂B₂-GAPDH is proposed. Both CP12 and CTE seem to regulate different photosynthetic GAPDH isoforms according to a common and ancient molecular mechanism.     

Coenzyme site-directed mutants of photosynthetic A4-GAPDH show selectively reduced NADPH-dependent catalysis, similar to regulatory AB-GAPDH inhibited by oxidized thioredoxin

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of higher plants uses both NADP(H) and NAD(H) as coenzyme and consists of one (GapA) or two types of subunits (GapA, GapB). AB-GAPDH is regulated in vivo through the action of thioredoxin and metabolites, showing higher kinetic preference for NADPH in the light than in darkness due to a specific effect on kcat(NADPH). Previous crystallographic studies on spinach chloroplast A4-GAPDH complexed with NADP or NAD showed that residues Thr33 and Ser188 are involved in NADP over NAD selectivity by interacting with the 2'-phosphate group of NADP. This suggested a possible involvement of these residues in the regulatory mechanism. Mutants of recombinant spinach GapA (A4-GAPDH) with Thr33 or Ser188 replaced by Ala (T33A, S188A and double mutant T33A/S188A) were produced, expressed in Escherichia coli, and compared to wild-type recombinant A4-GAPDH, in terms of crystal structures and kinetic properties. Affinity for NADPH was decreased significantly in all mutants, and kcat(NADPH) was lowered in mutants carrying the substitution of Ser188. NADH-dependent activity was unaffected. The decrease of kcat/Km of the NADPH-dependent reaction in Ser188 mutants resembles the behaviour of AB-GAPDH inhibited by oxidized thioredoxin, as confirmed by steady-state kinetic analysis of native enzyme. A significant expansion of size of the A4-tetramer was observed in the S188A mutant compared to wild-type A4. We conclude that in the absence of interactions between Ser188 and the 2'-phosphate group of NADP, the enzyme structure relaxes to a less compact conformation, which negatively affects the complex catalytic cycle of GADPH. A model based on this concept might be developed to explain the in vivo light-regulation of the GAPDH.     

The crystal structure of d-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaeon Methanothermus fervidus in the presence of NADP(+) at 2.1 A resolution

The crystal structure of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the archaeon Methanothermus fervidus has been solved in the holo form at 2.1 A resolution by molecular replacement. Unlike bacterial and eukaryotic homologous enzymes which are strictly NAD(+)-dependent, GAPDH from this organism exhibits a dual-cofactor specificity, with a marked preference for NADP(+) over NAD(+). The present structure is the first archaeal GAPDH crystallized with NADP(+). GAPDH from M. fervidus adopts a homotetrameric quaternary structure which is topologically similar to that observed for its bacterial and eukaryotic counterparts. Within the cofactor-binding site, the positively charged side-chain of Lys33 decisively contributes to NADP(+) recognition through a tight electrostatic interaction with the adenosine 2'-phosphate group. Like other GAPDHs, GAPDH from archaeal sources binds the nicotinamide moiety of NADP(+) in a syn conformation with respect to the adjacent ribose and so belongs to the B-stereospecific class of oxidoreductases. Stabilization of the syn conformation is principally achieved through hydrogen bonding of the carboxamide group with the side-chain of Asp171, a structural feature clearly different from what is observed in all presently known GAPDHs from bacteria and eukaryotes. Within the catalytic site, the reported crystal structure definitively confirms the essential role previously assigned to Cys140 by site-directed mutagenesis studies. In conjunction with new mutation results reported in this paper, inspection of the crystal structure gives reliable evidence for the direct implication of the side-chain of His219 in the catalytic mechanism. M. fervidus grows optimally at 84 degrees C with a maximal growth temperature of 97 degrees C. The paper includes a detailed comparison of the present structure with four other homologous enzymes extracted from mesophilic as well as thermophilic organisms. Among the various phenomena related to protein thermostabilization, reinforcement of electrostatic and hydrophobic interactions as well as a more efficient molecular packing appear to be essentially promoted by the occurrence of two additional alpha-helices in the archaeal GAPDHs. The first one, named alpha4, is located in the catalytic domain and participates in the enzyme architecture at the quaternary structural level. The second one, named alphaJ, occurs at the C terminus and contributes to the molecular packing within each monomer by filling a peripherical pocket in the tetrameric assembly.     

Probing the affinity and specificity of yeast alcohol dehydrogenase I for coenzymes.

Yeast (Saccharomyces cerevisiae) alcohol dehydrogenase I (SceADH) binds NAD+ and NADH less tightly and turns over substrates more rapidly than does horse (Equus caballus) liver alcohol dehydrogenase E isoenzyme (EcaADH), and neither enzyme uses NADP efficiently. Amino acid residues in the proposed adenylate binding pocket of SceADH were substituted in attempts to improve affinity for coenzymes or reactivity with NADP. Substitutions in SceADH (Gly202Ile or Ser246Ile) with the corresponding residues in the adenine binding site of the homologous EcaADH have modest effects on coenzyme binding and other kinetic constants, but the Ser246Ile substitution decreases turnover numbers by 350-fold. The Ser176Phe substitution (also near adenine site) significantly decreases affinity for coenzymes and turnover numbers. In the consensus nucleotide-binding betaalphabeta fold sequence, SceADH has two alanine residues (177-GAAGGLG-183) instead of the Leu200 in EcaADH (199-GLGGVG-204); the Ala178-Ala179 to Leu substitution significantly decreases affinity for coenzymes and turnover numbers. Some NADP-dependent enzymes have an Ala corresponding to Gly183 in SceADH; the Gly183Ala substitution significantly decreases affinity for coenzymes and turnover numbers. NADP-dependent enzymes usually have a neutral residue instead of the Asp (Asp201 in SceADH) that interacts with the hydroxyl groups of the adenosine ribose, along with a basic residue (at position 202 or 203) to stabilize the 2'-phosphate of NADP. The Gly203Arg change in SceADH does not significantly affect the kinetics. The Gly183Ala or Gly203Arg substitutions do not enable SceADH to use NADP+ as coenzyme. SceADH with the single Asp201Gly or double Asp201Gly:Gly203Arg substitutions have similar, low activity with NADP+. The results suggest that several of the amino acid residues participate in coenzyme binding and that conversion of specificity for coenzyme requires multiple substitutions.     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

Amino acid residues that determine functional specificity of NADP- and NAD-dependent isocitrate and isopropylmalate dehydrogenases

Isocitrate and isopropylmalalte dehydrogenases are homologous enzymes important for the cell metabolism. They oxidize their substrates using NAD or NADP as cofactors. Thus, they have two specificities, towards the substrate and the cofactor, appearing in three combinations. Although many three-dimensional (3D) structures are resolved, identification of amino acids determining these specificities remains a challenge. We present computational identification and analysis of specificity-determining positions (SDPs). Besides many experimentally proven SDPs, we predict new SDPs, for example, four substrate-specific positions (103Leu, 105Thr, 337Ala, and 341Thr in IDH from E. coli) that contact the cofactor and may play a role in the recognition process.     

Computational study of glyceraldehyde-3-phosphate dehydrogenase of Entamoeba histolytica: implications for structure-based drug design

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of the pathogenic protozoa Entamoeba histolytica (Eh) is a major glycolytic enzyme and an attractive drug target since this parasite lacks a functional citric acid cycle and is dependent solely on glycolysis for its energy requirements. The three-dimensional structure of dimeric EhGAPDH in complex with cofactor NAD(+) has been generated by homology modeling based on the crystal structure of human liver GAPDH. Our refined model indicates the presence of a parasite specific disulfide bond between two cysteine residues of adjacent monomers in the EhGAPDH dimer, which may be an important target for future drug design. Flexible docking with the substrate glyceraldehyde-3-phosphate (G3P) shows that Cys151, His178, Thr210, and Arg233 are important residues in G3P binding. The inorganic phosphate-binding site of EhGAPDH has been determined by docking study. The binding mode of a natural GAPDH inhibitor, chalepin to EhGAPDH has also been predicted. In search for a better inhibitor for EhGADPH, in silico modification of chalepin has been carried out to form an additional specific polar interaction with Asp194 of EhGAPDH whose equivalent is Leu195 in human GAPDH. In the absence of a crystal structure, our study provides an early insight into the structure of major drug target EhGAPDH, thus, facilitating the inhibitor design.     

Coenzymic activity of NADP derivatives alkylated at 2'-phosphate and 6-amino groups

Coenzymic activities of the following NADP derivatives were investigated: 2'-O-(2-carboxyethyl)phosphono-NAD (I), N6-(2-carboxyethyl)-NADP (II), 2'-O-(2-carboxyethyl)phosphono-N6-(2-carboxyethyl)-NAD (III), 2'-O-[N-(2-aminoethyl)carbamoylethyl]phosphono-NAD (IV), N6-[N-(2-aminoethyl)carbamoylethyl]-NADP (Va), 2',3'-cyclic NADP, and 3'-NADP. Derivatives I and IV show the effects of modification at the 2'-phosphate group, and derivatives II and Va show those at the 6-amino group of NADP. As for enzymes, alcohol, isocitrate, 6-phosphogluconate, glucose, glucose-6-phosphate, and glutamate dehydrogenases were used. These enzymes were grouped on the basis of the ratio of the activities for NAD and NADP into NADP-specific enzymes (ratio less than 0.01), NAD(P)-specific enzymes (0.01 less than ratio less than 100), and NAD-specific enzymes (ratio greater than 100). For NADP-specific enzymes, modifications at the 2'-phosphate group of NADP caused great loss of cofactor activity. The relative cofactor activities (NADP = 100%) of derivatives I and IV for these enzymes were 0.5-20 and 0.01-0.5%, respectively. On the other hand, NAD(P)-specific enzymes showed several types of responses to the NADP derivatives. The relative cofactor activities of I and IV for Leuconostoc mesenteroides and Bacillus stearothermophilus glucose-6-phosphate dehydrogenases and beef liver glutamate dehydrogenase were 60-200%; whereas, for B. megaterium glucose dehydrogenase and L. mesenteroides alcohol dehydrogenase, the values were 0.8-8%. For NAD-specific enzymes, these values were 20-50%. The relative cofactor activities of 2',3'-cyclic NADP and 3'-NADP were very low (less than 0.2%) except for beef liver glutamate dehydrogenase, B. stearothermophilus glucose-6-phosphate dehydrogenase, and horse liver alcohol dehydrogenase. Kinetic studies showed that the losses of the cofactor activity of NADP by these modifications were mainly due to the increase of the Km value. The mechanisms of coenzyme specificity of dehydrogenases are discussed. Unlike the 2'-phosphate group, the 6-amino group is common to NAD and NADP, and the effects of modification at the 6-amino group were independent of the coenzyme specificity of enzymes used for the assay. Derivatives II and Va had high relative cofactor activities (65-130%) for most of the enzymes except for isocitrate and glucose dehydrogenases (less than 1%) and L. mesenteroides alcohol dehydrogenase (20-60%). The cofactor activity of derivative III was generally lower than those of I and II.     

Sequences of tryptic peptides containing the five cysteinyl residues of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Tryptic peptides which account for all five cysteinyl residues in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been purified and sequenced. Collectively, these peptides contain 94 of the approximately 500 amino acid residues per molecule of subunit. Due to one incomplete cleavage at a site for trypsin and two incomplete chymotryptic-like cleavages, eight major radioactive peptides (rather than five as predicted) were recovered from tryptic digests of the enzyme that had been carboxymethylated with [³H]iodoacetate. The established sequences are: GlyTyrThrAlaPheValHisCys¹Lys TyrValAspLeuAlaLeuLysGluGluAspLeuIleAla GlyGlyGluHisValLeuCys¹AlaTyr AlaGlyTyrGlyTyrValAlaThrAlaAlaHisPheAla AlaGluSerSerThrGlyThrAspValGluValCys¹ ThrThrAsxAsxPheThrArg AlaCys¹ThrProIleIleSerGlyGlyMetAsnAla LeuArg ProPheAlaGluAlaCys¹HisAlaPheTrpLeuGly GlyAsnPheIleLys In these peptides, radioactive carboxymethylcysteinyl residues are denoted with asterisks and the sites of incomplete cleavage with vertical wavy lines. None of the peptides appear homologous with either of two cysteinyl-containing, active-site peptides previously isolated from spinach ribulosebisphosphate carboxylase/oxygenase.     

Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry.

The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.     

Structure-guided engineering of the coenzyme specificity of Pseudomonas fluorescens mannitol 2-dehydrogenase to enable efficient utilization of NAD(H) and NADP(H)

The structure of Pseudomonas fluorescens mannitol 2-dehydrogenase with bound NAD+ leads to the suggestion that the carboxylate group of Asp(69) forms a bifurcated hydrogen bond with the 2' and 3' hydroxyl groups of the adenosine of NAD+ and contributes to the 400-fold preference of the enzyme for NAD+ as compared to NADP+. Accordingly, the enzyme with the Asp(69)-->Ala substitution was found to use NADP(H) almost as well as wild-type enzyme uses NAD(H). The Glu(68)-->Lys substitution was expected to enhance the electrostatic interaction of the enzyme with the 2'-phosphate of NADP+. The Glu(68)-->Lys:Asp(69)-->Ala doubly mutated enzyme showed about a 10-fold preference for NADP(H) over NAD(H), accompanied by a small decrease in catalytic efficiency for NAD(H)-dependent reactions as compared to wild-type enzyme.     

NAD-dependent malate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase isoenzymes play an important role in dark metabolism of various plastid types

Chloroplasts isolated from spinach (Spinacia oleracea L.) leaves and green sweet-pepper (Capsicum annuum L. var. grossum (L.) Sendt.) fruits contain NADP-dependent malate dehydrogenase (MDH; EC 1.1.1.82) and the bispecific NAD(P)-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; EC 1.2.1.13). The NADP-dependent MDH and GAPDH are activated in the light, and inactive in the dark. We found that chloroplasts possess additional NAD-dependent MDH activity which is, like the NAD-dependent GAPDH activity, not influenced by light. In heterotrophic chromoplasts from red sweet-pepper fruits, the NADP-dependent MDH and the NAD(P)-GAPDH isoenzymes disappear during the developmental transition and only NAD-specific isoforms are found. Spinach chloroplasts contain both NAD/H and NADP/H at significant concentrations. Measurements of the pyridine dinucleotide redox states, performed under dark and various light conditions, indicate that NAD(H) is not involved in electron flow in the light. To analyze the contribution of NAD(H)-dependent reactions during dark metabolism, plastids from spinach leaves or green and red sweet-pepper fruits were incubated with dihydroxyacetone phosphate (DHAP). Exogenously added DHAP was oxidized into 3-phosphoglycerate by all types of plastids only in the presence of oxaloacetate, but not with nitrite or in the absence of added electron acceptors. We conclude that the NAD-dependent activity of GAPDH is essential in the dark to produce the ATP required for starch metabolism; excess electrons produced during triose-phosphate oxidation can selectively be used by NAD-MDH to form malate. Thus NADPH produced independently in the oxidative pentose-phosphate pathway will remain available for reductive processes inside the plastids. Received: 2 July 1997 / Accepted: 20 October 1997     

Role of aspartic acid 38 in the cofactor specificity of Drosophila alcohol dehydrogenase.

Drosophila alcohol dehydrogenase (ADH), an NAD(+)-dependent dehydrogenase, shares little sequence similarity with horse liver ADH. However, these two enzymes do have substantial similarity in their secondary structure at the NAD(+)-binding domain [Benyajati, C., Place, A. P., Powers, D. A. & Sofer, W. (1981) Proc. Natl Acad. Sci. USA 78, 2717-2721]. Asp38, a conserved residue between Drosophila and horse liver ADH, appears to interact with the hydroxyl groups of the ribose moiety in the AMP portion of NAD+. A secondary-structure comparison between the nucleotide-binding domain of NAD(+)-dependent enzymes and that of NADP(+)-dependent enzymes also suggests that Asp38 could play an important role in cofactor specificity. Mutating Asp38 of Drosophila ADH into Asn38 decreases Km(app)NADP 62-fold and increases kcat/Km(app)NADP 590-fold at pH 9.8, when compared with wild-type ADH. These results suggest that Asp38 is in the NAD(+)-binding domain and its substituent, Asn38, allows Drosophila ADH to use both NAD+ and NADP+ as its cofactor. The observations from the experiments of thermal denaturation and kinetic measurement with pH also confirm that the repulsion between the negative charges of Asp38 and 2'-phosphate of NADP+ is the major energy barrier for NADP+ to serve as a cofactor for Drosophila ADH.     

Production of Sm37-GAPDH, a major therapeutical target in human schistosomiasis

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in the glycolytic metabolism and the production of energy. This probably explains why GAPDH was evidenced as a major therapeutical target in several parasitic diseases; either as a vaccine candidate or as a target for chemotherapeutic treatments. Schistosoma mansoni GAPDH (Sm37-GAPDH) is one of the main schistosome vaccine candidates. The production of recombinant Sm37-GAPDH is essential to evaluate the ability of this molecule to induce protective immunity in animals and possibly in humans. The cDNA encoding Sm37-GAPDH has been cloned and sequenced. In addition, five B cell (including the major B-cell epitope Sm35-5) and two T cell epitopes have been localized on the molecule. Different expression systems have been evaluated in respect with the production yield and the GAPDH enzymatic activity. Some of them have led to either a high production of insoluble material (E. coli) or to an inactive enzyme (Pischia pastoris). The present article describes the production setting of rSm37-GAPDH using the baculovirus-insect cell system. Large amounts of soluble rSm37-GAPDH with enzymatic activity were obtained. Most sera from individuals living in an area endemic for S. mansoni recognised the rSm37 molecule and inhibited its catalytic activity.     

The covalent structure of pig kidney fructose 1,6-bisphosphatase: sequence of the 60-residue NH2-terminal peptide produced by digestion with subtilisin

Digestion of the native pig kidney fructose 1,6-bisphosphatase tetramer with subtilisin cleaves each of the 35,000-molecular-weight subunits to yield two major fragments: the S-subunit (M_{r ca.} 29,000), and the S-peptide (M_r 6,500). The following amino acid sequence has been determined for the S peptide: AcThrAspGlnAlaAlaPheAspThrAsnIle Val ThrLeuThrArgPheValMetGluGlnGlyArgLysAla ArgGlyThrGlyGlu MetThrGlnLeuLeuAsnSerLeuCysThrAlaValLys AlaIleSerThrAla z.sbnd;ValArgLysAlaGlyIleAlaHisLeuTyrGlyIleAla. Comparison of this sequence with that of the NH₂-terminal 60 residues of the enzyme from rabbit liver (El-Dorry et al., 1977, Arch. Biochem. Biophys.182, 763) reveals strong homology with 52 identical positions and absolute identity in sequence from residues 26 to 60.Although subtilisin cleavage of fructose 1,6-bisphosphatase results in diminished sensitivity of the enzyme to AMP inhibition, we have found no AMP inhibition-related amino acid residues in the sequenced S-peptide. The loss of AMP sensitivity that occurs upon pyridoxal-P modification of the enzyme does not result in the modification of lysyl residues in the S-peptide. Neither photoaffinity labeling of fructose 1,6-bisphosphatase with 8-azido-AMP nor modification of the cysteinyl residue proximal to the AMP allosteric site resulted in the modification of residues located in the NH₂-terminal 60-amino acid peptide.