An unexpected phosphate binding site in Glyceraldehyde 3-Phosphate Dehydrogenase: Crystal structures of apo, holo and ternary complex of Cryptosporidium parvum enzyme

Background  

The structure, function and reaction mechanism of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) have been extensively studied. Based on these studies, three anion binding sites have been identified, one 'Ps' site (for binding the C-3 phosphate of the substrate) and two sites, 'Pi' and 'new Pi', for inorganic phosphate. According to the original flip-flop model, the substrate phosphate group switches from the 'Pi' to the 'Ps' site during the multistep reaction. In light of the discovery of the 'new Pi' site, a modified flip-flop mechanism, in which the C-3 phosphate of the substrate binds to the 'new Pi' site and flips to the 'Ps' site before the hydride transfer, was proposed. An alternative model based on a number of structures of B. stearothermophilus GAPDH ternary complexes (non-covalent and thioacyl intermediate) proposes that in the ternary Michaelis complex the C-3 phosphate binds to the 'Ps' site and flips from the 'Ps' to the 'new Pi' site during or after the redox step.     

Glyceraldehyde 3-Phosphate Dehydrogenases and Glyoxylate Reductase: II. Far Red Light-Dependent Development of Glyceraldehyde 3-Phosphate Dehydrogenase Isozyme Activities in Sinapis Alba Cotyledons

Ammonium sulfate chromatography has been employed to separate glyceraldehyde 3-phosphate dehydrogenases (GPD) of Sinapis alba cotyledons of various developmental stages. Cotyledons of dark-grown seedlings possess one major NAD-specific enzyme designated NAD-GPD I. Irradiation with continuous far red light leads to a strong increase in NADP-GPD activity and to the formation of a second NAD activity designated NAD-GPD II. These two activities occur in a constant ratio during cotyledon development, and they are eluted together in ammonium sulfate chromatography. In a later stage of cotyledon development the light-dependent increase in NAD-GPD II is matched by an equivalent decrease in NAD-GPD I. These data suggest that the chloroplast marker enzyme NADP-GPD (EC 1.2.1.13) also has NAD activity and that the light-dependent formation of this bifunctional enzyme is correlated with activity changes of the NAD-GPD of cytoplasmic glycolysis (EC 1.2.1.12).     

Regulation of NADP-Dependent Glyceraldehyde 3-Phosphate Dehydrogenase Activity in Spinach Chloroplasts*

Activation of NAD(P)-glyceraldehyde 3-phosphate dehydrogenase (NADP-GAPDH, EC 1.2.1.13) can be achieved in isolated chloroplasts in the light, or in the dark upon addition of dithiothreitol (DTT) and/or 3-phosphoglycerate plus ATP. Activation in darkened chloroplasts is only partial with DTT or 3-phosphoglycerate plus ATP alone, but complete when both effectors are added. In the light, full activation is only achieved upon addition of ATP. The time-course of activation appears to depend upon the actual concentration of 1,3-bisphosphoglycerate (1,3bisPGA) inside the chloroplasts. The K_a values for 1,3bisPGA are in the same range as has been determined for the purified enzyme, namely around 20 μM for the dark form (in the absence of DTT) and around 1 μM for the light form or in the presence of DTT. In contrast, the K_a value for ATP is 1 to 2 mM for both the oxidized and the reduced enzyme forms. The observed activation of NADP-GAPDH is strongly paralleled by an increase of 3PGA, and consequently of 1,3bisPGA in the illuminated chloroplast, while the ATP level remains constant or declines. Activation by 1,3bisPGA is accompanied by dissociation of the 600 kDa form to the 150 kDa form, while reduction alone does not induce a shift in molecular mass as documented by fast gel filtration on Superdex 200. Thus partial activation by DTT in the dark is due to an increased activity of the 600 kDa form, while the activation state in the light is the result of a partial conversion of the 600 kDa form into the more active 150 kDa form. The principle of this activation is a fast reduction of the enzyme by the ferredoxin/thioredoxin system, resulting in a lowered K_avalue for 1,3bisPGA, and thus adjusting the properties of the enzyme to the stromal 1,3bisPGA level. The occurrence of a 300 kDa oligomer mainly during inactivation has also been observed. From these results a model is constructed that describes the reversible interconversion of various activation and aggregation states of NADP-GAPDH as observed upon light/dark transitions in isolated spinach chloroplasts.     

Purification and Immunochemical Characterization of NADP-Dependent Glyceraldehyde 3-Phosphate Dehydrogenase of Euglena gracilis

NADP-dependent glyceraldehyde 3-phosphate dehydrogenase fromEuglena gracilis (EC 1.2.1.13 [EC] ) was purified about 170-fold bya two-step procedure involving DEAE-SH cellulose chromatographyand affinity chromatography on ADP-Sepharose. The homogeneousenzyme from mildly sonicated cells contained equal amounts oftwo types of subunits with mol wts of 34,000 (A) and 38,000(B). The active enzyme had a mol wt 144,000 and is thereforean A₂B₂ tetramer. Enzyme from strongly sonicated Euglena cellscontained, in addition, a second allomer with a probable A₄structure. NADdependent glyceraldehyde 3-phosphate dehydrogenase,a tetramer with 36,000 mol wt subunits, was unrelated immunologicallyto the NADP-dependent enzyme although the latter also showedminor NAD-dependent activity. Both isoenzymes of the NADPlinkedglyceraldehyde 3-phosphate dehydrogenase, however, were immunologicallyidentical. ¹Dedicated, to Prof. Dr. O. H. Volk on his 80th birthday. (Received October 13, 1982; Accepted March 21, 1984)     

Comparative Studies of Some Kinetic Properties of Glyceraldehyde 3-Phosphate Dehydrogenase and Fructose-bisphosphatase in Chloroplasts from Wheat and Rice

Enzymatic properties of the chloroplast GAP dehydrogenase and FBPase from the wheat or the rice leaves of the main stems during grain filling period have been investigated. According to the effect of the substrate or coenzymes concentration, it is shown that the GAP dehydrogenases in freshly ruptured wheat or rice chloroplast show higher activities than FBPase, especially the activity of the wheat chloroplast GAP dehydrogenase is over ten times greater than that of FBPase. However, the activities of the above two enzymes in the rice chloroplast show only little difference, but the activity of the rice FBPase is higher than that of the wheat. The maximum initial velocities (Vmax) of the two enzymes in the above two chloroplasts are determined and are apparantly different from each other. At about 0.2 mM NAD(P)H concentration the activity of GAP dehydrogenase in the rice chloroplast is saturated. But, with the same concentration the wheat GAP dehydrogenase is still not saturated. It is noteworthy that the ratio of the activities of the two chloroplast GAP dehydrogenases for NADPH and NADH approaches equal as the coenzyme concentration is increased. Possible physiological significance of the kinetic properties of the above two enzymes during the wheat and rice grain filling period was discussed.     

The apo and ternary complex structures of a chemotherapeutic target: human glycinamide ribonucleotide transformylase

Glycinamide ribonucleotide transformylase (GART; 10-formyltetrahydrofolate:5'-phosphoribosylglycinamide formyltransferase, EC 2.1.2.2), an essential enzyme in de novo purine biosynthesis, has been a chemotherapeutic target for several decades. The three-dimensional structure of the GART domain from the human trifunctional enzyme has been solved by X-ray crystallography. Models of the apoenzyme, and a ternary complex with the 10-formyl-5,8-dideazafolate cosubstrate and a glycinamide ribonucleotide analogue, hydroxyacetamide ribonucleotide [alpha,beta-N-(hydroxyacetyl)-d-ribofuranosylamine], are reported to 2.2 and 2.07 A, respectively. The model of the apoenzyme represents the first structure of GART, from any source, with a completely unoccupied substrate and cosubstrate site, while the ternary complex is the first structure of the human GART domain that is bound at both the substrate and cosubstrate sites. A comparison of the two models therefore reveals subtle structural differences that reflect substrate and cosubstrate binding effects and implies roles for the invariant residues Gly 133, Gly 146, and His 137. Preactivation of the DDF formyl group appears to be key for catalysis, and structural flexibility of the active end of the substrate may facilitate nucleophilic attack. A change in pH, rather than folate binding, correlates with movement of the folate binding loop, whereas the phosphate binding loop position does not vary with pH. The electrostatic surface potentials of the human GART domain and Escherichia coli enzyme explain differences in the binding affinity of polyglutamylated folates, and these differences have implications to future chemotherapeutic agent design.     

人类3-磷酸甘油醛脱氢酶的原核表达、纯化和鉴定

为了研究3-磷酸甘油醛脱氧酶(GAPDH)化生物学功能,以pcDNA3.1-GAPDH质粒为模板,PCR方法扩增GAPDH基因,经BamHI和SalI双酶切后插入相同酶切的pET32a(+)载体,构建His-GAPDH融合蛋白表达质粒pET32a(+)-GAPDH;转化JM109感受态细胞,并进行阳性克隆筛选,扩增目的质粒;转化大肠杆菌BL21菌株,经IPTG诱导产生融合蛋白,亲和层析柱纯化后,用SDS-PAGE和Western blotting检测GAPDH蛋白表达情况.结果表明,构建的人GAPDH基因表达载体经序列测定证实,与GenBank数据完全一致;双酶切鉴定证实,克隆基因正确插入pET32a(+)载体;表达质粒pET32a(+)-GAPDH在大肠杆菌BL21中成功地诱导表达了可溶性His-GAPDH融合蛋白,纯化后SDS-PAGE和Western blotting检测证实融合蛋白表达成功.人GAPDH原核表达载体的成功构建,及6His-GAPDH融合蛋白的正确表达,为进一步深入研究GAPDH的生物学功能奠定了基础.     

Fates of Evolutionarily Distinct,Plastid‐type Glyceraldehyde 3‐phosphate Dehydrogenase Genes in Kareniacean Dinoflagellates

The ancestral kareniacean dinoflagellate has undergone tertiary endosymbiosis, in which the original plastid is replaced by a haptophyte endosymbiont. During this plastid replacement, the endosymbiont genes were most likely flowed into the host dinoflagellate genome (endosymbiotic gene transfer or EGT). Such EGT may have generated the redundancy of functionally homologous genes in the host genome—one has resided in the host genome prior to the haptophyte endosymbiosis, while the other transferred from the endosymbiont genome. However, it remains to be well understood how evolutionarily distinct but functionally homologous genes were dealt in the dinoflagellate genomes bearing haptophyte‐derived plastids. To model the gene evolution after EGT in plastid replacement, we here compared the characteristics of the two evolutionally distinct genes encoding plastid‐type glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) in Karenia brevis and K. mikimotoi bearing haptophyte‐derived tertiary plastids: “gapC1h” acquired from the haptophyte endosymbiont and “gapC1p” inherited from the ancestral dinoflagellate. Our experiments consistently and clearly demonstrated that, in the two species examined, the principal plastid‐type GAPDH is encoded by gapC1h rather than gapC1p. We here propose an evolutionary scheme resolving the EGT‐derived redundancy of genes involved in plastid function and maintenance in the nuclear genomes of dinoflagellates that have undergone plastid replacements. Although K. brevis and K. mikimotoi are closely related to each other, the statuses of the two evolutionarily distinct gapC1 genes in the two Karenia species correspond to different steps in the proposed scheme.     

Genetic Mapping of Loci for Glucose-6-Phosphate Dehydrogenase, Gluconate-6-Phosphate Dehydrogenase, and Gluconate-6-Phosphate Dehydrase in Escherichia coli

The loci on the Escherichia coli genome of mutations affecting the constitutive enzymes glucose-6-phosphate dehydrogenase (zwf) and gluconate-6-phosphate dehydrogenase (gnd), and the inducible enzyme gluconate-6-phosphate dehydrase (edd), were determined by conjugation and transduction experiments, chiefly by three-factor crosses. They are in the same region of the chromosome, and their order is gnd-his-(edd, zwf)-aroD; gnd and his are cotransduceable, as are zwf and edd. The position of gnd in Salmonella typhimurium was shown to be similar to that in E. coli.     

Crystal structures of Mycobacterium tuberculosis S-adenosyl-L-homocysteine hydrolase in ternary complex with substrate and inhibitors

S-adenosylhomocysteine hydrolase (SAHH) is a ubiquitous enzyme that plays a central role in methylation-based processes by maintaining the intracellular balance between S-adenosylhomocysteine (SAH) and S-adenosylmethionine. We report the first prokaryotic crystal structure of SAHH, from Mycobacterium tuberculosis (Mtb), in complex with adenosine (ADO) and nicotinamide adenine dinucleotide. Structures of complexes with three inhibitors are also reported: 3'-keto aristeromycin (ARI), 2-fluoroadenosine, and 3-deazaadenosine. The ARI complex is the first reported structure of SAHH complexed with this inhibitor, and confirms the oxidation of the 3' hydroxyl to a planar keto group, consistent with its prediction as a mechanism-based inhibitor. We demonstrate the in vivo enzyme inhibition activity of the three inhibitors and also show that 2-fluoradenosine has bactericidal activity. While most of the residues lining the ADO-binding pocket are identical between Mtb and human SAHH, less is known about the binding mode of the homocysteine (HCY) appendage of the full substrate. We report the 2.0 A resolution structure of the complex of SAHH cocrystallized with SAH. The most striking change in the structure is that binding of HCY forces a rotation of His363 around the backbone to flip out of contact with the 5' hydroxyl of the ADO and opens access to a nearby channel that leads to the surface. This complex suggests that His363 acts as a switch that opens up to permit binding of substrate, then closes down after release of the cleaved HCY. Differences in the entrance to this access channel between human and Mtb SAHH are identified.     

Erratic Evolution of Glycerol-3-Phosphate Dehydrogenase in Drosophila,Chymomyza, and Ceratitis

We have studied the evolution of Gpdh in 18 fruitfly species by sequencing 1,077 nucleotides per species on average. The region sequenced includes four exons coding for 277 amino acids and three variable-length introns. Phylogenies derived by a variety of methods confirm that the nominal genus Zaprionus belongs within the genus Drosophila, whereas Scaptodrosophila and Chymomyza are outside. The rate of GPDH evolution is erratic. The rate of amino acid replacements in a lineage appears to be 1.0 × 10⁻¹⁰/site/year when Drosophila species are considered (diverged up to 55 million years ago), but becomes 2.3 × 10⁻¹⁰ when they are compared to Chymomyza species (divergence around 60 My ago), and 4.6 × 10⁻¹⁰ when species of those two genera are compared with the medfly Ceratitis capitata (divergence around 100 My ago). In order to account for these observations, the rate of amino acid replacement must have been 15 or more times greater in some lineages and at some times than in others. At the nucleotide level, however, Gpdh evolves in a fairly clockwise fashion. Received: 13 June 1996 / Accepted: 16 August 1996     

Glyceraldehyde 3-Phosphate:NADP Reductase of Spinach Leaves : Steady State Kinetics and Effect of Inhibitors

The steady state kinetics of glyceraldehyde 3-phosphate:NADP⁺ oxidoreductase (GNR) (EC 1.2.1.9) have been investigated. The enzyme exhibits hyperbolic behavior over a wide range of substrate concentrations. Double-reciprocal plots are nearly parallel or distantly convergent with limiting K_m values of 2 to 5 micromolar for NADP⁺ and 20 to 40 micromolar for D-glyceraldehyde 3-phosphate (G3P). The velocity response to NADP⁺ as the varied substrate is however sigmoidal if G3P concentration exceeds 10 micromolar, whereas the response to G3P may show inhibition above this concentration. This `G3P-inhibited state' is alleviated by saturating amounts of NADP⁺ or NADPH. Product inhibition patterns indicate NADPH as a potent competitive inhibitor to NADP⁺ (K_i 30 micromolar) and mixed inhibitor towards G3P, and 3-phosphoglycerate (3PGA) as mixed inhibitor to both NADP⁺ and G3P (K_i 10 millimolar). The data, and those obtained with dead-end inhibitors, are consistent with a nonrapid equilibrium random mechanism with two alternative kinetic pathways. Of these, a rapid kinetic sequence (probably ordered with NADP⁺ binding first and G3P binding as second substrate) is dominant in the range of hyperbolic responses. A reverse reaction with 3PGA and NADPH as substrates is unlikely, and was not detected. Of a number of compounds tested, erythrose 4-phosphate (K_i 7 micromolar) and Pi (K_i 2.4 millimolar) act as competitive inhibitors to G3P (uncompetitive towards NADP⁺) and are likely to affect the in vivo activity. Ribose 5-phosphate, phosphoenolpyruvate, ATP, and ADP are also somewhat inhibitory. Full GNR activity in the leaf seems to be allowed only under high photosynthesis conditions, when levels of several inhibitors are low and substrate is high. We suggest that a main function of leaf GNR is to supply NADPH required for photorespiration, the reaction product 3PGA being cycled back to chloroplasts.     

Biologically important ternary coordination complex. Crystal and molecular structures of adenine-glycylglycine-copper (II) complex

The crystal and molecular structures of an adenine-glycyl-glycine-copper(II) complex have been determined by X-ray diffraction. The chelating atoms, amino and amide nitrogen atoms, the carboxyl oxygen atom of the dipeptide, N(9) of adenine and one water molecule form a square-pyramid. The hydrogen-bonded adenine base-pairs stack with a distance of 3.8Å, while the dipeptides contact each other by NHO hydrogen bond to form a dimer.     

Crystal structures of the key anaerobic enzyme pyruvate:ferredoxin oxidoreductase, free and in complex with pyruvate

Oxidative decarboxylation of pyruvate to form acetyl-coenzyme A, a crucial step in many metabolic pathways, is carried out in most aerobic organisms by the multienzyme complex pyruvate dehydrogenase. In most anaerobes, the same reaction is usually catalyzed by a single enzyme, pyruvate:ferredoxin oxidoreductase (PFOR). Thus, PFOR is a potential target for drug design against certain anaerobic pathogens. Here, we report the crystal structures of the homodimeric Desulfovibrio africanus PFOR (data to 2.3 A resolution), and of its complex with pyruvate (3.0 A resolution). The structures show that each subunit consists of seven domains, one of which affords protection against oxygen. The thiamin pyrophosphate (TPP) cofactor and the three [4Fe-4S] clusters are suitably arranged to provide a plausible electron transfer pathway. In addition, the PFOR-pyruvate complex structure shows the noncovalent fixation of the substrate before the catalytic reaction.     

Population structures and the role of genetic exchange in the zoonotic pathogen Cryptosporidium parvum

Apicomplexan protozoan parasites include some of the most globally important human and animal pathogens, all of which have obligatory sexual cycles in their definitive hosts. Despite their importance and the relevance of understanding the population genetic structure and role of genetic exchange in generating diversity, population genetic analysis has largely been restricted to Plasmodium spp. and Toxoplasma gondii. These species show a considerable diversity of population structure suggesting different strategies for transmission and survival in mammalian hosts. We have undertaken a population genetic analysis of a further apicomplexan species (Cryptosporidium parvum) to extend our understanding of the diversity of genetic structures and test whether it has a clonal population structure. Nothing is known about the population structure of this parasite. We have analyzed 180 parasite isolates from both humans and cattle derived from a single discrete geographical area, using three minisatellite and four microsatellite markers that define 38 multilocus genotypes. Analysis of linkage disequilibria between pairs of loci combined with measures of genetic distance and similarity provides evidence that the sample comprises four genetically isolated populations. One group of human isolates consists primarily of two closely related multilocus genotypes (clonal), while the major subtypes of a second group, common to both humans and animals, show a panmictic population structure. The data provide an important step in understanding the role of genetic exchange in these parasites, which is an essential prerequisite for determining the value of multilocus genotyping for the analysis of sources of human infection as well as future molecular epidemiological studies.     

Molecular dynamics simulations of apo,holo, and inactivator bound GABA‐at reveal the role of active site residues in PLP dependent enzymes

The pyridoxal 5‐phosphate (PLP) cofactor is a significant organic molecule in medicinal chemistry. It is often found covalently bound to lysine residues in proteins to form PLP dependent enzymes. An example of this family of PLP dependent enzymes is γ‐aminobutyric acid aminotransferase (GABA‐AT) which is responsible for the degradation of the neurotransmitter GABA. Its inhibition or inactivation can be used to prevent the reduction of GABA concentration in brain which is the source of several neurological disorders. As a test case for PLP dependent enzymes, we have performed molecular dynamics simulations of GABA‐AT to reveal the roles of the protein residues and its cofactor. Three different states have been considered: the apoenzyme, the holoenzyme, and the inactive state obtained after the suicide inhibition by vigabatrin. Different protonation states have also been considered for PLP and two key active site residues: Asp298 and His190. Together, 24 independent molecular dynamics trajectories have been simulated for a cumulative total of 2.88 µs. Our results indicate that, unlike in aqueous solution, the PLP pyridine moiety is protonated in GABA‐AT. This is a consequence of a pK_a shift triggered by a strong charge–charge interaction with an ionic “diad” formed by Asp298 and His190 that would help the activation of the first half‐reaction of the catalytic mechanism in GABA‐AT: the conversion of PLP to free pyridoxamine phosphate (PMP). In addition, our MD simulations exhibit additional strong hydrogen bond networks between the protein and PLP: the phosphate group is held in place by the donation of at least three hydrogen bonds while the carbonyl oxygen of the pyridine ring interacts with Gln301; Phe181 forms a π–π stacking interaction with the pyridine ring and works as a gate keeper with the assistance of Val300. All these interactions are hypothesized to help maintain free PMP in place inside the protein active site to facilitate the second half‐reaction in GABA‐AT: the regeneration of PLP‐bound GABA‐AT (i.e., the holoenzyme). Proteins 2016; 84:875–891. © 2016 Wiley Periodicals, Inc.     

An epidemiological survey on Cryptosporidium parvum infection of inhabitants in Chorwon-gun, Kangwon-do.

The present study was undertaken to know the infection status of Cryptosporidium parvum among the residents of Chorwon-gun, Kangwon-do in 1993. Total 461 fecal samples were collected from the inhabitants residing in Chorwon-gun during the period of August 12 to September 14, 1993. Fecal smears were prepared by formalin-ether sedimentation, and examined after modified acid fast staining. Of the 461 fecal samples, 9 (1.9%) were positive for C. parvum oocysts. The positive cases were limited to thirties (4) patients, forties (3), and sixties (2), and no oocyst was detected in other age groups. The oocyst positive rate for male was 1.4% and that of female was 2.6%.