Purification and Properties of NADP-Dependent Sorbitol-6-Phosphate Dehydrogenase from Apple Seedlings

NADP-dependent sorbitol-6-phosphate dehydrogenase (S6PDH) waspurified from apple (Malus domestica) seedlings by a purificationprocedure that included two fractionations by affinity chromatography.The purified enzyme was a homogeneous protein that migratedas a single polypeptide chain with an apparent relative massof 36,000 during SDS-polyacrylamide gel electrophoresis andthe native enzyme was a homodimer of the polypeptide. The maximumvelocity of the reduction of glucose-6-phosphate (G6P) was muchhigher than that of the oxidation of sorbitol-6-phosphate (S6P)and the enzyme had high G6P-reducing activity over the pH rangefrom 7 to 11 even though the oxidation of S6P proceeded veryslowly at neutral pH. These results are consistent with thehypothesis that S6PDH plays a major role in the biosynthesisof sorbitol in vivo. The reduction of G6P to S6P was inhibitedby the addition of nucleotide di- or triphosphates. ATP, thestrongest inhibitor, and ADP inhibited the reduction of G6Pin a competitive manner with respect to NADPH and the K_i valueswere 0.18 mM for ATP and 0.30 mM for ADP. (Received March 24, 1992; Accepted May 25, 1993)     

苹果S6PDH基因克隆与原核表达

6-磷酸山梨醇脱氢酶(sorbitol-6-phosphate dehydrogenase,S6PDH)是蔷薇科植物中合成山梨醇的重要酶。以苹果叶片为材料,利用RT-PCR法克隆到S6PDHcDNA全长,将其与大肠杆菌表达载体pET-32a( )构建原核表达载体pET-S并转化大肠杆菌BL21,经IPTG诱导表达后,SDS-PAGE检测结果表明该基因表达了1个约54kD的蛋白,为进一步研究目的蛋白的结构和功能提供了试验基础。     

The Carbohydrate Nutrition of Tomato Roots: VIII. The Mechanism of the Inhibition by D-Mannose of the Respiration of Excised Roots

A study of certain aspects of the respiratory metabolism ofexcised tomato roots has been undertaken. Mitochondria derivedfrom such roots possess an active Krebs cycle. Neither the operationof the Krebs cycle nor the glucose-6-phosphate dehydrogenaseactivity of the preparations are inhibited by mannose. Tracerexperiments using mannose-U-¹⁴C indicate that mannose, on enteringthe root, is rapidly phosphorylated to mannose-6-phosphate whichaccumulates due to lack of phosphomannose isomerase activityin the tissues. The formation of mannose-6-phosphate is dueto the activity of a hexokinase, the presence of which has alsobeen demonstratedIn vitro. The participation of mannose in thehexo-kinase reaction implies its competitive interaction withthe natural substrates of this enzyme. Accumulated mannose-6-phosphateprobably also inhibits respiration through its demonstratedcompetitive inhibition of phosphoglucose isomerase. Certainobservations suggest that it may also inhibit respiration bydepleting the intracellular level of inorganic phosphate. Glucose antagonizes the mannose-inhibition of respiration. Oneeffect of glucose is to inhibit mannose uptake. An enhancedglucose level may also promote the formation of glucose-6-phosphaterather than mannose-6-phosphate by the hexokinase system.     

Glucose-6-Phosphate Dehydrogenase from the Human Pathogen Trypanosoma cruzi Evolved Unique Structural Features to Support Efficient Product Formation

Glucose-6-phosphate dehydrogenase (G6PDH) is the key enzyme supplying reducing power (NADPH) to the cells, by oxidation of glucose-6-phosphate (G6P), and in the process providing a precursor of ribose-5-phosphate. G6PDH is also a virulence factor of pathogenic trypanosomatid parasites. To uncover the biochemical and structural features that distinguish TcG6PDH from its human homolog, we have solved and analyzed the crystal structures of the G6PDH from Trypanosoma cruzi (TcG6PDH), alone and in complex with G6P. TcG6PDH crystallized as a tetramer and enzymatic assays further indicated that the tetramer is the active form in the parasite, in contrast to human G6PDH, which displays higher activity as a dimer. This quaternary structure was shown to be particularly stable. The molecular reasons behind this disparity were unveiled by structural analyses: a TcG6PDH-specific residue, R323, is located at the dimer–dimer interface, critically contributing with two salt bridges per subunit that are absent in the human enzyme. This explains why TcG6PDH dimerization impaired enzyme activity. The parasite protein is also distinct in displaying a 37-amino-acid extension at the N-terminus, which comprises the non-conserved C8 and C34 involved in the covalent linkage of two neighboring protomers. In addition, a cysteine triad (C53, C94 and C135) specific of Kinetoplastid G6PDHs proved critical for stabilization of TcG6PDH active site. Based on the structural and biochemical data, we posit that the N-terminal region and the catalytic site are highly dynamic. The unique structural features of TcG6PDH pave the way toward the design of efficacious and highly specific anti-trypanosomal drugs.     

A biochemical and physicochemical comparison of two recombinant enzymes used for enzyme replacement therapies of hunter syndrome

Mucopolysaccharidosis II (MPS II, Hunter syndrome; OMIM 309900) is an X-linked lysosomal storage disease caused by a deficiency in the enzyme iduronate-2-sulfatase (IDS), leading to accumulation of glycosaminoglycans (GAGs). For enzyme replacement therapy (ERT) of Hunter syndrome, two recombinant enzymes, idursulfase (Elaprase®, Shire Human Genetic Therapies, Lexington, MA) and idursulfase beta (Hunterase®, Green Cross Corporation, Yongin, Korea), are currently available in Korea. To compare the biochemical and physicochemical differences between idursulfase and idursulfase beta, we examined the formylglycine (FGly) content, specific enzyme activity, mannose-6-phosphate (M6P) content, sialic acid content, and in vitro cell uptake activity of normal human fibroblasts of these two enzymes. The FGly content, which determines the enzyme activity, of idursulfase beta was significantly higher than that of idursulfase (79.4?±?0.9 vs. 68.1?±?2.2 %, P?<?0.001). In accordance with the FGly content, the specific enzyme activity of idursulfase beta was significantly higher than that of idursulfase (42.6?±?1.1 vs. 27.8?±?0.9 nmol/min/μg protein, P?<?0.001). The levels of M6P and sialic acid were not significantly different (2.4?±?0.1 vs 2.4?±?0.3 mol/mol protein for M6P and 12.3?±?0.7 vs. 12.4?±?0.4 mol/mol protein for sialic acid). However, the cellular uptake activity of the normal human fibroblasts in vitro showed a significant difference (K_uptake, 5.09?±?0.96 vs. 6.50?±?1.28 nM protein, P?=?0.017). In conclusion, idursulfase beta exhibited significantly higher specific enzyme activity than idursulfase, resulting from higher FGly content. These biochemical differences may be partly attributed to clinical efficacy. However, long-term clinical evaluations of Hunter syndrome patients treated with these two enzymes will be needed to demonstrate the clinical implications of significant difference of the enzyme activity and the FGly content.     

Characterization of a thermostable enzyme with phosphomannomutase/phosphoglucomutase activities from the hyperthermophilic archaeon Pyrococcus horikoshii OT3

The phosphomannomutase/phosphoglucomutase (PMM/PGM) enzyme catalyzes reversibly the intra-molecular phosphoryl interconverting reaction of mannose-6-phosphate and mannose-1-phosphate or glucose-6-phosphate and glucose-1-phosphate. Glucose-6-phosphate and glucose-1-phosphate are known to be utilized for energy metabolism and cell surface construction, respectively. PMM/PGM has been isolated from many microorganisms. By performing similarity searches using existing PMM/PGM sequences, the homologous ORFs PH0923 and PH1210 were identified from the genomic data of Pyrococcus horikoshii OT3. Since PH0923 appears to be part of an operon consisting of four carbohydrate metabolic enzymes, PH0923 was selected as the first target for the investigation of PMM/PGM activity in P. horikoshii OT3. The coding region of PH0923 was cloned and the purified recombinant protein was utilized for an examination of its biochemical properties. The enzyme retained half its initial activity after treatment at 95 degrees C for 90 min. Detailed analyses of activities showed that this protein is capable of utilizing a variety of metal ions that are not utilized by previously characterized PMM/PGM proteins. A mutated protein with an alanine residue replacing the active site serine residue indicated that this residue plays an important but non-essential role in PMM/PGM activity.     

Isolation and Expression Analysis of Plastidic Glucose-6-phosphate Dehydrogenase Gene from Rice (Oryza sativa L.)

Glucose-6-phosphate dehydrogenase is a rate-limiting enzyme of pentose phosphate pathway, existing in cytosolic and plastidic compartments of higher plants. A novel gene encoding plastidic glucose-6-phosphate dehydrogenase was isolated from rice (Oryza sativa L.) and designated OsG6PDH2 in this article. Through semiquantitative RT-PCR approach it was found that OsG6PDH2 mRNA was weakly expressed in rice leaves, stems, immature spikes or flowered spikes, and a little higher in roots. However, the expression of OsG6PDH2 in rice seedlings was significantly induced by dark treatment. The complete opening reading frame (ORF) of OsG6PDH2 was inserted into pET30a (+), and expressed in Escherichia coli strain BL21 (DE3). The enzyme activity assay of transformed bacterial cells indicated that OsG6PDH2 encoding product had a typical function of glucose-6-phosphate dehydrogenase.     

Purification and Characteristics of Sorbitol-6-phosphate Dehydrogenase from Loquat Leaves

To study the role of sorbitol-6-phosphate dehydrogenase in sorbitol synthesis in leaves of Rosaceous plants, properties of the enzyme and its presence in several plants in the family was investigated. The activity of the enzyme, which catalyzes an NADP-dependent oxidation of the substrate to glucose-6-phosphate, was detected in leaves of Prunus mume, Prunus persica, Rhaphiolepsis indica, Sorbus aucuparia, Cydonia oblonga, Photinia glabra, Sorbaria kirilowii, and Spiraea thunbergii.     

Assay of prolyl 4-hydroxylase by the chromatographic determination of [14C]succinic acid on ion-exchange minicolumns.

Hepatic glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) is subject to nutritional regulation. To assess the possible role of hormones in this regulation, the amounts of G6PDH mRNA were studied in primary cultures of rat hepatocytes treated with insulin and dexamethasone, alone or in combination. Relative concentrations of G6PDH mRNA were directly assessed by a dot-blot hybridization procedure with nick-translated cDNA probes. G6PDH sequence abundance increased when the cultures were treated with insulin or dexamethasone, but the G6PDH mRNA induced by dexamethasone was not expressed at the protein level as active enzyme. In cultures treated with insulin and dexamethasone in combination, enzyme activity and G6PDH sequence abundance were greater than those induced by insulin alone. Our results directly demonstrate that G6PDH mRNA amounts are modulated in liver by these two classes of hormones and can partially account for the dietary induction of the enzyme observed in vivo.     

13C and 31P NMR studies on sugar metabolism in Bombyx mori and Philosamia cynthia ricini larvae

Studies were made on ¹³C and ³¹P NMR in larvae of two species of silkworm, Bombyx mori and Philosamia cynthia ricini, in vivo as well as in vitro to determine the pathways of glucose utilization, especially those to amino acids as components of silk fibroin. Results showed that the ¹³C of [1-¹³C]glucose administered orally into 5th instar larvae of both species was incorporated into glucose-1-phosphate, glucose-6-phosphate and trehalose. Serine, glutamate, glutamine, citrate, malate, trehalose and sorbitol-6-phosphate were detected in the hemolymphs of these larvae as metabolites of [1-¹³C]glucose. Two days after [1-¹³C]glucose administration, labeled alanine, glycine, serine, urea, glycogen, trehalose and glycerol were clearly detected in Bombyx larvae. Starvation caused rapid consumption of administered [1-¹³C]glucose with very little accumulation of ¹³C in glycogen or trehalose. In the in vivo³¹P NMR spectra of Bombyx larvae, ATP, arginine phosphate, sorbitol-6-phosphate, uridine diphosphoglucose, phosphoenolpyruvate and inorganic phosphate were detected with some sugar phosphates, such as glucose-1-phosphate and glucose-6-phosphate. During starvation, the intensity of the signal of inorganic phosphate increased and those of sugar phosphate other than sorbitol-6-phosphate decreased, but these changes were reversed by oral administration of glucose.     

The substrate of the glucose-6-phosphate dehydrogenase of Pseudomonas aeruginosa provides structural stability

In general, eukaryotic glucose-6-phosphate dehydrogenases (G6PDHs) are structurally stabilized by NADP⁺. Here we show by spectrofluorometric analysis, thermal and urea denaturation, and trypsin proteolysis, that a different mechanism stabilizes the enzyme from Pseudomonas aeruginosa (PaG6PDH) (EC 1.1.1.363). The spectrofluorometric analysis of the emission of 8-anilino-1-naphthalenesulfonic acid (ANS) indicates that this stabilization is the result of a structural change in the enzyme caused by G6P. The similarity between the K_d values determined for the PaG6PDH-G6P complex (78.0 ± 7.9 μM) and the K_0.5 values determined for G6P (57.9 ± 2.5 and 104.5 ± 9.3 μM in the NADP⁺- and NAD⁺-dependent reactions, respectively) suggests that the structural changes are the result of G6P binding to the active site of PaG6PDH. Modeling of PaG6PDH indicated the residues that potentially bind the ligand. These results and a phylogenetic analysis of the amino acid sequences of forty-four G6PDHs, suggest that the stabilization observed for PaG6PDH could be a characteristic that distinguishes this and other G6PDHs that use NAD⁺ and NADP⁺ from those that use NADP⁺ only or preferentially, such as those found in eukaryotes. This characteristic could be related to the metabolic roles these enzymes play in the organisms to which they belong.     

Purification, characterization, and cDNA sequence of glucose-6-phosphate dehydrogenase from potato (Solatium tuberosum L.)

Glucose-6-phosphate dehydrogenase (G6PDH, E.C. 1.1.1.49) has been purified from potato tuber at least 850-fold to apparent homogeneity as judged by SDS-PAGE. The enzyme was characterized by K_m values of 260 μM for glucose-6-phosphate and 6 μM for NADP and a broad pH optimum between phi 7.5 and 9. NADPH, GTP, ATP, acetyl CoA and CoA inhibited G6PDH activity. Dithiothreitol (DTT) did not inactivate the enzyme. A highly specific antiserum was produced in a rabbit and used for immunodetection of G6PDH in Western blots. A cDNA library from potato leaves was screened with DNA probes produced by the polymerase chain reaction (PCR) in the presence of g6pdh-specific primers. A full-length cDNA clone was analyzed and the derived amino acid sequence compared with known G6PDH sequences from various sources. The homology of the plant sequence with G6PDH sequences from animals and yeast was found to be rather high (52%), whereas there was significantly lower homology with sequences of bacterial origin (37%). The lack of a plastidic signal sequence as well as the insensitivity of the recombinant enzyme towards reduced DTT, support the view that the cDNA sequence of a redox-independent cytosolic isoform was obtained.     

Functional characterization of GDP-mannose pyrophosphorylase from Leptospira interrogans serovar Copenhageni

Leptospira interrogans synthesizes a range of mannose-containing glycoconjugates relevant for its virulence. A prerequisite in the synthesis is the availability of the GDP-mannose, produced from mannose-1-phosphate and GTP in a reaction catalyzed by GDP-mannose pyrophosphorylase. The gene coding for a putative enzyme in L. interrogans was expressed in Escherichia coli BL21(DE3). The identity of this enzyme was confirmed by electrospray-mass spectroscopy, Edman sequencing and immunological assays. Gel filtration chromatography showed that the dimeric form of the enzyme is catalytically active and stable. The recombinant protein was characterized as a mannose-1-phosphate guanylyltransferase. S _0.5 for the substrates were determined both in GDP-mannose pyrophosphorolysis: 0.20 mM (GDP-mannose), 0.089 mM (PPi), and 0.47 mM; and in GDP-mannose synthesis: 0.24 mM (GTP), 0.063 mM (mannose-1-phosphate), and 0.45 mM (Mg²⁺). The enzyme was able to produce GDP-mannose, IDP-mannose, UDP-mannose and ADP-glucose. We obtained a structural model of the enzyme using as a template the crystal structure of mannose-1-phosphate guanylyltransferase from Thermus thermophilus HB8. Binding of substrates and cofactor in the model agree with the pyrophosphorylases reaction mechanism. Our studies provide insights into the structure of a novel molecular target, which could be useful for detection of leptospirosis and for the development of anti-leptospiral drugs.     

Analysis of glucose-6-phosphate dehydrogenase of the cyanobacterium Synechococcus sp. PCC 7942 in the zwf mutant Escherichia coli DF214 cells

The aim of this study was to express the zwf gene of Synechococcus sp. PCC 7942 in zwf mutant Escherichia coli DF214 cells and to analyse glucose-6-phosphate dehydrogenase (G6PDH) activity. Initially, mutant cells were transformed with plasmid pNUT1 containing a Synechococcus sp. PCC 7942 zwf gene with a 1 kb upstream region that is expected to contain promoter elements. Transformant DF214 cells were not complemented by this fragment in a glucose minimal medium, nor did they exhibit statistically meaningful G6PDH activity. Therefore, the zwf gene was cloned in the lac operon to express the Zwf as a fusion protein; this yielded the construct pSG162. The pSG162 transformant E. coli DF214 cells were complemented in a glucose minimal medium, indicating that cyanobacterial Zwf protein fused with the part of LacZ′ polypeptide, enabling the cells to utilize glucose via the oxidative pentose phosphate pathway. Compared with wild-type E. coli cells, approximately ten times more G6PDH activity was measured in transformant cells. This indicated that the Synechococcus sp. PCC 7942 zwf gene was expressed under the control of the E. coli lac promoter as a fusion protein and the zwf product was converted into an active G6PDH form. Analyses was also carried out to determine whether dithiothreitol (DTT) was an in vitro reducing agent affected the enzyme activity, as was previously reported for this cyanobacterial strain. The results showed no variation in enzyme activity in the reduced assay conditions. Therefore, the zwf mutant E. coli strain DF214 was found to provide a rapid system for analysis of cyanobacterial G6PDH enzymes, but not for the redox state analysis of this enzyme.     

Sorbitol-6-phosphate dehydrogenase from loquat fruit

Sorbitol-6-phosphate dehydrogenase was found in flesh tissue of mature fruit of the loquat (Eriobotrya japonica Lindl. var. Tanaka). The enzyme was purified about 30-fold from the crude extract of the fruit, and was demonstrated to catalyze sorbitol-6-phosphate + NADP glucose-6-phosphate + NADPH. The optimal pH values for sorbitol 6-phosphate oxidation and glucose 6-phosphate reduction were 9.8 and 9.1, respectively.     

Modification of a method for detecting the post-phoretic activity of mannose (Mpi, EC 5.3.1.8)- and glucose (Gpi, EC5.3.1.9)-6-phosphate isomerase]

A method for detecting activities of mannose- and glucose-6-phosphate isomerases based on enzyme production of the substrates is described. The results obtained for several animal taxa are illustrated by photographs.     

A Modified Method for Detection of Post-Phoretic Activities in Mannose- (Mpi,EC 5.3.1.8) and Glucose- (Gpi,EC 5.3.1.9) 6-Phosphate Isomerases

A method for detecting activities of mannose- and glucose-6-phosphate isomerases based on enzyme production of the substrates is described. The results obtained for several animal taxa are illustrated by photographs.     

Catalytic properties of glucose-6-phosphate dehydrogenase from pea leaves.

A homogeneous preparation of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) with a specific activity of 3.88 U/mg protein was isolated from pea (Pisum sativum L.) leaves. The molecular mass of the G6PDH is 79 +/- 2 kD. According to SDS-PAGE, the molecular mass of the enzyme subunit is 40 +/- 3 kD. The Km values for glucose-6-phosphate and NADP are 2 and 0.5 mM, respectively. The enzyme has a pH optimum of 8.0. Mg2+, Mn2+, and Ca2+ activate the enzyme at concentrations above 1 mM. Galactose-6-phosphate and fructose-6-phosphate inhibit the G6PDH from pea leaves. Fructose-1, 6-bisphosphate and galactose-1-phosphate are enzyme activators. NADPH is a competitive inhibitor of the G6PDH with respect to glucose-6-phosphate (Ki = 0.027 mM). ATP, ADP, AMP, UTP, NAD, and NADH have no effect on the activity of the enzyme.     

Intersubunit Disulfide Interactions Play a Critical Role in Maintaining the Thermostability of Glucose-6-phosphate Dehydrogenase from the Hyperthermophilic Bacterium Aquifex aeolicus

Proteins from thermophilic microorganisms are stabilized by various mechanisms to preserve their native folded states at higher temperatures. A thermostable glucose-6-phosphate dehydrogenase (tG6PDH) from the hyperthermophilic bacterium Aquifex aeolicus was expressed as a recombinant protein in Escherichia coli. The A. aeolicus G6PDH is a homodimer exhibiting remarkable thermostability (t_1/2=24 hr at 90°C). Based on homology modeling and upon comparison of its structure with human G6PDH, it was predicted that cysteine 184 of one subunit could form a disulfide bond with cysteine 352 of the other subunit resulting in reinforced intersubunit interactions that hold the dimer together. Site-directed mutagenesis was performed on tG6PDH to convert C184 and C352 to serines. The tG6PDH double mutant exhibited a dramatic decrease in the half-life from 24 hr to 3 hr at 90°C. The same decrease in half-life was also found when either C184 or C352 was mutated to serine. The result indicates that C184 and C352 may play a crucial role in strengthening the dimer interface through disulfide bond formation, thereby contributing to the thermal stability of the enzyme.