Trehalose synthesis by sequential reactions of recombinant maltooligosyltrehalose synthase and maltooligosyltrehalose trehalohydrolase from Brevibacterium helvolum

A DNA fragment encoding two enzymes leading to trehalose biosynthesis, maltooligosyltrehalose synthase (BvMTS) and maltooligosyltrehalose trehalohydrolase (BvMTH), was cloned from the nonpathogenic bacterium Brevibacterium helvolum. The open reading frames for the two proteins are 2,331 and 1,770 bp long, respectively, and overlap by four nucleotides. Recombinant BvMTS, BvMTH, and fusion gene BvMTSH, constructed by insertion of an adenylate in the overlapping region, were expressed in Escherichia coli. Purified BvMTS protein catalyzed conversion of maltopentaose to maltotriosyltrehalose, which was further hydrolyzed by BvMTH protein to produce trehalose and maltotriose. The enzymes shortened maltooligosaccharides by two glucose units per cycle of sequential reactions and released trehalose. Maltotriose and maltose were not catalyzed further and thus remained in the reaction mixtures depending on whether the substrates had an odd or even number of glucose units. The bifunctional in-frame fusion enzyme, BvMTSH, catalyzed the sequential reactions more efficiently than an equimolar mixture of the two individual enzymes did, presumably due to a proximity effect on the catalytic sites of the enzymes. The recombinant enzymes produced trehalose from soluble starch, an abundant natural source for trehalose production. Addition of alpha-amylase to the enzyme reaction mixture dramatically increased trehalose production by partial hydrolysis of the starch to provide more reducing ends accessible to the BvMTS catalytic sites.     

Over-expression of BvMTSH,a fusion gene for maltooligosyltrehalose synthase and maltooligosyltrehalose trehalohydrolase,enhances drought tolerance in transgenic rice

Plant abiotic stress tolerance has been modulated by engineering the trehalose synthesis pathway. However, many stress-tolerant plants that have been genetically engineered for the trehalose synthesis pathway also show abnormal development. The metabolic intermediate trehalose 6-phosphate has the potential to cause aberrations in growth. To avoid growth inhibition by trehalose 6-phosphate, we used a gene that encodes a bifunctional in-frame fusion (BvMTSH) of maltooligosyltrehalose synthase (BvMTS) and maltooligosyltrehalose trehalohydrolase (BvMTH) from the nonpathogenic bacterium Brevibacterium helvolum. BvMTS converts maltooligosaccharides into maltooligosyltrehalose and BvMTH releases trehalose. Transgenic rice plants that over-express BvMTSH under the control of the constitutive rice cytochrome c promoter (101MTSH) or the ABA-inducible Ai promoter (105MTSH) show enhanced drought tolerance without growth inhibition. Moreover, 101MTSH and 105MTSH showed an ABA-hyposensitive phenotype in the roots. Our results suggest that over-expression of BvMTSH enhances drought-stress tolerance without any abnormal growth and showes ABA hyposensitive phenotype in the roots. [BMB Reports 2014; 47(1): 27-32]     

Identification of maltooligosyltrehalose synthase and maltooligosyltrehalose trehalohydrolase enzymes catalysing trehalose biosynthesis in Anabaena 7120 exposed to NaCl stress

Anabaena 7120 cells were exposed to NaCl (25-175 mM) stress. Maximum growth was recorded in media containing 150mM NaCl. Short-term exposure (48h) of the cyanobacterial biomass to 150mM NaCl, induced highest trehalose level (37mM). Control cells lacking NaCl did not show any trace of trehalose as ascertained by NMR and HPLC analysis. Trehalose biosynthesis observed with NaCl plus high temperature (40 degrees C) indicated that its production was specifically triggered by NaCl, not temperature. The increase in trehalose level during NaCl stress was the result of overexpression of the trehalose-forming enzymes maltooligosyltrehalose synthase (MTSase), EC 5.4.99.15 (114kDa) and maltooligosyltrehalose trehalohydrolase (MTHase), EC 3.2.1.141 (68 kDa) as evidenced by SDS-PAGE analysis. To our knowledge this is the first report of induced trehalose biosynthesis in Anabaena 7120 during salt-stress, accompanied by identification of MTSase and MTHase enzymes on gel. It is suggested that Anabaena 7120 cells synthesize the osmolyte trehalose to withstand osmotic fluctuations.     

Bifunctional enzyme fusion of 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase

The formaldehyde-fixing enzymes, 3-Hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI), are the key enzymes catalyzing sequential reactions in the ribulose monophosphate (RuMP) pathway. In this study, we generated two fused gene constructs of the hps and phi genes (i.e., hps–phi and phi–hps) from a methylotrophic bacterium Mycobacterium gastri MB19. The gene product of hps–phi exhibited both HPS and PHI activities at room temperature and catalyzed the sequential reactions more efficiently than a simple mixture of the individual enzymes. The gene product of phi–hps failed to display any enzyme activity. Escherichia coli strains harboring the hps–phi gene consumed formaldehyde more efficiently and exhibited better growth in a formaldehyde-containing medium than the host strain. Our results demonstrate that the engineered fusion gene has the possibility to be used to establish a formaldehyde-resistance detoxification system in various organisms.     

Molecular cloning of maltooligosyltrehalose trehalohydrolase gene from Nostoc flagelliforme and trehalose-related response to stresses

A genomic DNA fragment encoding a putative maltooligosyltrehalose trehalohydrolase (NfMTH) for trehalose biosynthesis was cloned by the degenerate primer- PCR from cyanobacterium Nostoc flagelliforme. The ORF of NfMTH is 1,848 bp in length and encodes 615 amino acid residues, constituting a 70 kDa protein. The deduced amino acid sequence of NfMTH contains 4 regions highly conserved for MTHs. By expression of NfMTH in E. coli, the function of this protein was demonstrated, where the recombinant protein catalyzed the hydrolysis of maltooligosyl trehalose to trehalose. The expressions of MTH and maltooligosyltrehalose synthase in the filaments of N. flagelliforme were upregulated significantly under dehydration stress, NaCl stress, and high temperature-drought stress. The accumulations of both trehalose and sucrose in the filaments of N. flagelliforme were also improved significantly under the above stresses. Furthermore, trehalose accumulated in smaller quantities than sucrose did when under NaCl stress, but accumulated in higher quantities than sucrose did when under temperature-drought stress, indicating that both trehalose and sucrose were involved in N. flagelliforme adapted to stresses and different strategies conducted in response to various stress conditions.     

Labeling and enzyme studies of the central carbon metabolism in Metallosphaera sedula

Metallosphaera sedula (Sulfolobales, Crenarchaeota) uses the 3-hydroxypropionate/4-hydroxybutyrate cycle for autotrophic carbon fixation. In this pathway, acetyl-coenzyme A (CoA) and succinyl-CoA are the only intermediates that can be considered common to the central carbon metabolism. We addressed the question of which intermediate of the cycle most biosynthetic routes branch off. We labeled autotrophically growing cells by using 4-hydroxy[1-¹⁴C]butyrate and [1,4-¹³C₁]succinate, respectively, as precursors for biosynthesis. The labeling patterns of protein-derived amino acids verified the operation of the proposed carbon fixation cycle, in which 4-hydroxybutyrate is converted to two molecules of acetyl-CoA. The results also showed that major biosynthetic flux does not occur via acetyl-CoA, except for the formation of building blocks that are directly derived from acetyl-CoA. Notably, acetyl-CoA is not assimilated via reductive carboxylation to pyruvate. Rather, our data suggest that the majority of anabolic precursors are derived from succinyl-CoA, which is removed from the cycle via oxidation to malate and oxaloacetate. These C₄ intermediates yield pyruvate and phosphoenolpyruvate (PEP). Enzyme activities that are required for forming intermediates from succinyl-CoA were detected, including enzymes catalyzing gluconeogenesis from PEP. This study completes the picture of the central carbon metabolism in autotrophic Sulfolobales by connecting the autotrophic carbon fixation cycle to the formation of central carbon precursor metabolites.Sulfolobales (Crenarchaeota) comprise extreme thermoacidophiles from volcanic areas that grow best at a pH of around 2 and a temperature of 60 to 90°C (32, 33). Most Sulfolobales can grow chemoautotrophically on sulfur, pyrite, or H₂ under microaerobic conditions, which also applies to Metallosphaera sedula (31), the organism studied here. Its genome has been sequenced (2). Some species of the Sulfolobales secondarily returned to a facultative anaerobic or even strictly anaerobic life style (33), and some laboratory strains appear to have lost their ability to grow autotrophically (8). Autotrophic representatives of the Sulfolobales use a 3-hydroxypropionate/4-hydroxybutyrate cycle (in short, hydroxypropionate/hydroxybutyrate cycle) for autotrophic carbon fixation (Fig. ​(Fig.1)1) (6-8, 38). The enzymes of this cycle are oxygen tolerant, which predestines the cycle for the lifestyle of the aerobic Crenarchaeota (8). The presence of genes coding for key enzymes of the hydroxypropionate/hydroxybutyrate cycle in the mesophilic aerobic “marine group I” Crenarchaeota suggests that these abundant marine archaea use a similar autotrophic carbon fixation mechanism (6, 24, 68) (for a review of autotrophic carbon fixation in Archaea, see reference 7).Open in a separate windowFIG. 1.Proposed 3-hydroxypropionate/4-hydroxybutyrate cycle functioning in autotrophic carbon fixation in Sulfolobales and its relation to the central carbon metabolism, as studied in this work for Metallosphaera sedula. The situation may be similar in other Sulfolobales and possibly in autotrophic marine Crenarchaeota. Enzymes: 1, acetyl-CoA/propionyl-CoA carboxylase; 2, malonyl-CoA reductase (NADPH); 3, malonic semialdehyde reductase (NADPH); 4, 3-hydroxypropionate-CoA ligase (AMP forming); 5, 3-hydroxypropionyl-CoA dehydratase; 6, acryloyl-CoA reductase (NADPH); 7, acetyl-CoA/propionyl-CoA carboxylase; 8, methylmalonyl-CoA epimerase; 9, methylmalonyl-CoA mutase; 10, succinyl-CoA reductase (NADPH); 11, succinic semialdehyde reductase (NADPH); 12, 4-hydroxybutyrate-CoA ligase (AMP forming); 13, 4-hydroxybutyryl-CoA dehydratase; 14 and 15, crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase (NAD⁺); 16, acetoacetyl-CoA β-ketothiolase; 17, succinyl-CoA synthetase (ADP forming); 18, succinic semialdehyde dehydrogenase; 19, succinate dehydrogenase (natural electron acceptor unknown); 20, fumarate hydratase; 21, malate dehydrogenase; 22, malic enzyme; 23, PEP carboxykinase (GTP); 24, pyruvate:water dikinase (ATP); 25, enolase; 26, phosphoglycerate mutase; 27, phosphoglycerate kinase; 28, glyceraldehyde 3-phosphate dehydrogenase; 29, triosephosphate isomerase; 30, fructose 1,6-bisphosphate aldolase/phosphatase; 31, (si)-citrate synthase; 32, aconitase; 33, isocitrate dehydrogenase.In the cycle, one molecule of acetyl-coenzyme A (CoA) is formed from two molecules of bicarbonate. The key carboxylating enzyme is a bifunctional biotin-dependent acetyl-CoA/propionyl-CoA carboxylase (10, 11, 36, 38, 48, 49). In Bacteria and Eukarya, acetyl-CoA carboxylase catalyzes the first step in fatty acid biosynthesis. However, archaea do not contain fatty acids, and therefore acetyl-CoA carboxylase obviously plays a different metabolic role. The hydroxypropionate/hydroxybutyrate cycle can be divided into two parts. The first transforms acetyl-CoA and two bicarbonate molecules via 3-hydroxypropionate to succinyl-CoA, and the second converts succinyl-CoA via 4-hydroxybutyrate to two acetyl-CoA molecules. In brief, the product of the acetyl-CoA carboxylase reaction, malonyl-CoA, is reduced via malonic semialdehyde to 3-hydroxypropionate, which is further reductively converted to propionyl-CoA. Propionyl-CoA is carboxylated to (S)-methylmalonyl-CoA by the same carboxylase as that that carboxylates acetyl-CoA (11, 36). (S)-Methylmalonyl-CoA is isomerized to (R)-methylmalonyl-CoA, followed by carbon rearrangement to succinyl-CoA catalyzed by coenzyme B₁₂-dependent methylmalonyl-CoA mutase.Succinyl-CoA then is converted into two molecules of acetyl-CoA via succinic semialdehyde, 4-hydroxybutyrate, 4-hydroxybutyryl-CoA, crotonyl-CoA, 3-hydroxyacetyl-CoA, and acetoacetyl-CoA. This reaction sequence apparently is common to the autotrophic Crenarchaeota, as it also is used by autotrophic Crenarchaeota of the orders Thermoproteales and Desulfurococcales, which use a dicarboxylate/4-hydroxybutyrate cycle for autotrophic carbon fixation (8, 34, 55, 56) (also see the accompanying work [57]).From the list of intermediates of the hydroxypropionate/hydroxybutyrate cycle, acetyl-CoA and succinyl-CoA are the only intermediates considered common to the central carbon metabolism. In this work, we addressed the question of which intermediate of the cycle most biosynthetic routes branch off, and we came to the conclusion that succinyl-CoA serves as the main precursor for cellular carbon. This requires one turn of the cycle to regenerate the CO₂ acceptor and to generate one extra molecule of acetyl-CoA from two molecules of bicarbonate. Acetyl-CoA plus another two bicarbonate molecules are converted by an additional half turn of the cycle to succinyl-CoA. This strategy differs from that of the anaerobic pathways, in which acetyl-CoA is reductively carboxylated to pyruvate, and from there the other precursors for building blocks ultimately are derived (discussed in reference 7).     

Bifunctional recombinant fusion proteins for rapid detection of antibodies to both HIV-1 and HIV-2 in whole blood

Background  

Availability of accurate diagnostic tests has been helpful in curtailing the spread of HIV infection. Among these, simple, point of care, inexpensive tests which require only a drop of blood from finger-prick and give reliable results within minutes are a must for expansion of testing services and for reaching mobile and marginalised populations. Such tests will not only be a boon for the infrastructure-starved developing and underdeveloped countries but will also be extremely useful in developed countries where post-testing compliance is a major problem. Our laboratory has been involved in developing reagents for heamagglutination-based rapid detection of antibodies to HIV in whole blood using recombinant molecules specific for either HIV-1 or HIV-2. Since it is not required of a screening test to differentially detect HIV and HIV-2, it would useful to create a single molecule capable of simultaneous detection of both HIV-1 and HIV-2 in a drop of blood.     

Surface exposed amino acid differences between mesophilic and thermophilic phosphoribosyl diphosphate synthase.

The amino acid sequence of 5-phospho-alpha-D-ribosyl 1-diphosphate synthase from the thermophile Bacillus caldolyticus is 81% identical to the amino acid sequence of 5-phospho-alpha-D-ribosyl 1-diphosphate synthase from the mesophile Bacillus subtilis. Nevertheless the enzyme from the two organisms possesses very different thermal properties. The B. caldolyticus enzyme has optimal activity at 60-65 degrees C and a half-life of 26 min at 65 degrees C, compared to values of 46 degrees C and 60 s at 65 degrees C, respectively, for the B. subtilis enzyme. Chemical cross-linking shows that both enzymes are hexamers. Vmax is determined as 440 micromol.min(-1).mg protein(-1) and Km values for ATP and ribose 5-phosphate are determined as 310 and 530 microM, respectively, for the B. caldolyticus enzyme. The enzyme requires 50 mM Pi as well as free Mg2+ for maximal activity. Manganese ion substitutes for Mg2+, but only at 30% of the activity obtained with Mg2+. ADP and GDP inhibit the B. caldolyticus enzyme in a cooperative fashion with Hill coefficients of 2.9 for ADP and 2.6 for GDP. Ki values are determined as 113 and 490 microm for ADP and GDP, respectively. At low concentrations ADP inhibition is linearly competitive with respect to ATP. A predicted structure of the B. caldolyticus enzyme based on homology modelling with the structure of B. subtilis 5-phospho-alpha-D-ribosyl 1-diphosphate synthase shows 92% of the amino acid differences to be on solvent exposed surfaces in the hexameric structure.     

Geranylgeranylglyceryl phosphate synthase. Characterization of the recombinant enzyme from Methanobacterium thermoautotrophicum.

Geranylgeranylglyceryl diphosphate synthase (GGGP synthase) catalyzes alkylation of (S)-glyceryl phosphate [(S)-GP] by geranylgeranyl diphosphate (GGPP) to produce (S)-geranylgeranylglyceryl phosphate [(S)-GGGP]. This reaction is the first committed step in the biosynthesis of ether-linked membrane lipids in Archaea. The gene encoding GGGP synthase from Methanobacterium thermoautotrophicum was cloned using probes designed from the N-terminal sequence determined from the purified enzyme. The open reading frame, which encoded a protein of 245 amino acids, was inserted into a pET expression vector and expressed in Escherichia coli. The recombinant GGGP synthase was purified to homogeneity. The enzyme is active as a homopentamer, as determined by size exclusion chromatography and equilibrium sedimentation experiments. GGGP synthase has optimal activity at 55 degrees C in pH 8.0 buffer containing 1 mM MgCl(2). V(max) = 4.0 +/- 0.1 micromol min(-1) mg(-1) (k(cat) = 0.34 +/- 0.03 s(-1) for pentameric GGGP synthase assuming all subunits are fully active), K(m)((S)-GP) = 13.5 +/- 1.0 microM, and K(m)(GGPP) = 506 +/- 47 nM. These steady-state catalytic constants were identical to those for enzyme isolated from cell extracts of M. thermoautotrophicum [Chen, A., Zhang, D., and Poulter, C. D. (1993) J. Biol. Chem. 268, 21701-21705]. Alignment of seven putative archaeal GGGP synthase sequences revealed a number of highly conserved residues consisting of five aspartate/glutamates, three serine/threonines, two prolines, and five glycines, including a conserved GGG motif.     

Human 6-pyruvoyltetrahydropterin synthase: cDNA cloning and heterologous expression of the recombinant enzyme.

6-Pyruvoyl-tetrahydropterin synthase (PTPS) is involved in the biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor for enzymes such as the hepatic phenylalanine hydroxylase. BH4 deficiency causes malignant hyperphenylalaninemia. We cloned the human liver cDNA encoding PTPS. The coding region for PTPS contains 145 amino acids and predicts a polypeptide of 16'387 Da. The human amino acid sequence showed a 82% identity with the rat liver sequence. Expression of the cDNA in E. coli yielded the active enzyme and showed immunoreactivity with antibodies against the rat liver PTPS. This is the basis for the molecular understanding of BH4 deficiency in patients suffering from a defect in PTPS activity.     

Trehalose-phosphate synthase of Mycobacterium tuberculosis. Cloning, expression and properties of the recombinant enzyme.

The trehalose-phosphate synthase (TPS) of Mycobacterium smegmatis was previously purified to apparent homogeneity and several peptides from the 58 kDa protein were sequenced. Based on that sequence information, the gene for TPS was identified in the Mycobacterium tuberculosis genome, and the gene was cloned and expressed in Escherichia coli with a (His)6 tag at the amino terminus. The TPS was expressed in good yield and as active enzyme, and was purified on a metal ion column to give a single band of approximately 58 kDa on SDS/PAGE. Approximately 1.3 mg of purified TPS were obtained from a 1-L culture of E. coli ( approximately 2.3 g cell paste). The purified recombinant enzyme showed a single band of approximately 58 kDa on SDS/PAGE, but a molecular mass of approximately 220 kDa by gel filtration, indicating that the active TPS is probably a tetrameric protein. Like the enzyme originally purified from M. smegmatis, the recombinant enzyme is an unusual glycosyltransferase as it can utilize any of the nucleoside diphosphate glucose derivatives as glucosyl donors, i.e. ADP-glucose, CDP-glucose, GDP-glucose, TDP-glucose and UDP-glucose, with ADP-glucose, GDP-glucose and UDP-glucose being the preferred substrates. These studies prove conclusively that the mycobacterial TPS is indeed responsible for catalyzing the synthesis of trehalose-P from any of the nucleoside diphosphate glucose derivatives. Although the original enzyme from M. smegmatis was greatly stimulated in its utilization of UDP-glucose by polyanions such as heparin, the recombinant enzyme was stimulated only modestly by heparin. The Km for UDP-glucose as the glucosyl donor was approximately 18 mm, and that for GDP-glucose was approximately 16 mm. The enzyme was specific for glucose-6-P as the glucosyl acceptor, and the Km for this substrate was approximately 7 mm when UDP-glucose was the glucosyl donor and approximately 4 mm with GDP-glucose. TPS did not show an absolute requirement for divalent cations, but activity was increased about twofold by 10 mm Mn2+. This recombinant system will be useful for obtaining sufficient amounts of protein for structural studies. TPS should be a valuable target site for chemotherapeutic intervention in tuberculosis.     

Detection and characterization of a thermophilic biotin biosynthetic enzyme, 7-keto-8-aminopelargonic acid synthase, from various thermophiles

By detailed BLAST searches of the genome database of various thermophiles, five ORFs with similarity to the bioF gene, which encodes 7-keto-8-aminopelargonic acid synthase (BioF) involved in biotin biosynthesis, of Escherichia coli were found: AqbioF, CltbioF, GkbioF, SytbioF, and TsebioF, from Aquifex aeolicus VF5, Clostridium thermocellum ATCC27405, Geobacillus kaustophilus JCM12893, Symbiobacterium thermophilum IAM14863, and Thermosynechococcus elongatus BP-1 respectively. The five purified recombinant bioF gene products, which were overexpressed in E. coli, had the enzyme activity of BioF. The optimum temperature range and thermostability of five BioFs, AqBioF, CltBioF, GkBioF, SytBioF, and TseBioF, were higher than those of E. coli BioF. In particular, AqBioF was found to show the highest thermostability of the α-oxoamine synthase family enzymes reported to date. Substrate specificity experiments revealed that SytBioF was also able to catalyze the reaction of 2-amino-3-ketobutyrate CoA ligase, a member of the α-oxoamine synthase family, and that it used acetyl-CoA and glycine as substrates, like the TTHA1582 protein of Thermus thermophilus. The other purified BioFs, AqBioF and GkBioF, did not show any activity with acyl-CoAs and amino acids other than pimeloyl-CoA and L-alanine as substrates.     

Isolation and cultivation of a novel microorganism producing a maltopentaose-forming enzyme

Summary A novel microorganism producing a maltopentaose-forming enzyme was screened from a soil sample. The enzyme produced by the bacteria formed maltopentaose from starch during the first stage of the reaction. The bacterium was characterized asPseudomonas sp. (KO-8940) on the basis of its morphological, physiological and biological properties. Culture conditions for enzyme production were investigated and established. The medium was composed of beef extract 0.8%, ammonium sulphate 1.0% and maltose 0.8% in tap water. Optimum conditions for bacterial growth were: initial pH 8.0, temperature 45°C, and 3 days cultivation with a rotary shaker (200 rpm). The broth supernatant obtained contained the maltopentaose-forming crude enzyme, with which 40% of starch was converted to maltopentaose.     

Functional interactions between starch synthase III and isoamylase-type starch-debranching enzyme in maize endosperm

This study characterized genetic interactions between the maize (Zea mays) genes dull1 (du1), encoding starch synthase III (SSIII), and isa2, encoding a noncatalytic subunit of heteromeric isoamylase-type starch-debranching enzyme (ISA1/ISA2 heteromer). Mutants lacking ISA2 still possess the ISA1 homomeric enzyme. Eight du1(-) mutations were characterized, and structural changes in amylopectin resulting from each were measured. In every instance, the same complex pattern of alterations in discontinuous spans of chain lengths was observed, which cannot be explained solely by a discrete range of substrates preferred by SSIII. Homozygous double mutants were constructed containing the null mutation isa2-339 and either du1-Ref, encoding a truncated SSIII protein lacking the catalytic domain, or the null allele du1-R4059. In contrast to the single mutant parents, double mutant endosperms affected in both SSIII and ISA2 were starch deficient and accumulated phytoglycogen. This phenotype was previously observed only in maize sugary1 mutants impaired for the catalytic subunit ISA1. ISA1 homomeric enzyme complexes assembled in both double mutants and were enzymatically active in vitro. Thus, SSIII is required for normal starch crystallization and the prevention of phytoglycogen accumulation when the only isoamylase-type debranching activity present is ISA1 homomer, but not in the wild-type condition, when both ISA1 homomer and ISA1/ISA2 heteromer are present. Previous genetic and biochemical analyses showed that SSIII also is required for normal glucan accumulation when the only isoamylase-type debranching enzyme activity present is ISA1/ISA heteromer. These data indicate that isoamylase-type debranching enzyme and SSIII work in a coordinated fashion to repress phytoglycogen accumulation.     

Purification and characterization of recombinant human prostacyclin synthase

Prostacyclin synthase (PGIS), which catalyzes the conversion of prostaglandin (PG) H(2) to prostacyclin (PGI(2)), is a member of the cytochrome P-450 (P450) superfamily, CYP8A1. To study the enzymatic and protein characteristics of human PGIS, the enzyme was overexpressed in Spodoptera frugiperda 21 (Sf21) cells using the baculovirus expression system. PGIS was expressed in the microsomes of the infected Sf21 cells after culture in 5 microg/ml hematin-supplemented medium for 72 h. The holoenzyme was isolated from the solubilized microsomal fraction by calcium phosphate gel absorption and purified to homogeneity by DEAE-Sepharose and hydroxyapatite column chromatography. The K(m) and V(max) values of the purified human PGIS for PGH(2) were 30 microM and 15 micromol/min/mg of protein at 24 degrees C, respectively. The optical absorption and EPR spectra of the enzyme revealed the characteristics of a low-spin form of P450 in the oxidized state. The carbon monoxide-reduced difference spectrum, however, exhibited a peak at 418 nm rather than 450 nm. The addition of a PGH(2) analogue, U46619, to the enzyme produced an oxygen-ligand type of the difference spectrum with maximum absorption at 407 nm and minimum absorption at 430 nm. Treatment with another PGH(2) analogue, U44069, produced a peak at 387 nm and a trough at 432 nm in the spectrum (Type I), while treatment with tranylcypromine, a PGIS inhibitor, produced a peak at 434 nm and a trough at 412 nm (Type II). A Cys441His mutant of the enzyme possessed no heme-binding ability or enzyme activity. Thus, we succeeded in obtaining a sufficient amount of the purified recombinant human PGIS from infected insect cells for spectral analyses that has high specific activity and the characteristics of a P450, indicating substrate specificity.     

Purification and characterization of recombinant mouse thymidylate synthase

Recombinant mouse thymidylate synthase (TS) expressed at high levels in Escherichia coli was purified to homogeneity in greater than 70% yield by a rapid three-step procedure. Both 0.1% Triton X-100 and 10% glycerol were required to stabilize the enzyme whose activity remained unchanged after 1 month when stored at -20 degrees C. Thermal inactivation of the enzyme was a first-order process at 37 degrees C, with t1/2 values of 6.9, 15.6 and 3.0 min at pH 5.5, 7.0 and 8.5, respectively. The presence of saturating levels of dUMP at pH 8.5 increased the t1/2 of inactivation of 38 min. The pH profile for enzyme activity showed a narrow optimum region centered at pH 7.0, which was mirrored by the shape of the Km, dUMP/Vmax plot. The pH dependence of Kd for the covalent inhibitory ternary complex of enzyme, 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate exhibited a broad minimum between pH 5.5 and 8.5, and ranged between 3.1, 0.8 and 1.1 nM at pH 5.5, 7.0 and 8.5, respectively. The UV/VIS spectrum of the native enzyme exhibited a maximum at 280 nm (epsilon = 98,200 M-1 cm-1), while that of the inhibitory ternary complex showed an additional maximum at 320 nm. The 19F-NMR spectrum of the mouse enzyme:FdUMP binary complex revealed two new resonances at -2.8 and -34.8 ppm. The most deshielded resonance represented the noncovalent binary complex while the other resonance was assigned to the nucleotide covalently bound to the enzyme. The alteration of nucleotide binding equilibria produced by addition of H4 folate was exemplified by both an increase in intensity and a 5 ppm deshielding of the resonance attributed to the covalent FdUMP-enzyme complex. Addition of formaldehyde to the latter mixture produced the covalent ternary complex which resulted in the collapse of the resonances at -2.8 and -39.5 ppm and the appearance of a new resonance at -12.4 ppm.     

Comparing the thermodynamic stabilities of a related thermophilic and mesophilic enzyme

Several models have been proposed to explain the high temperatures required to denature enzymes from thermophilic organisms; some involve greater maximum thermodynamic stability for the thermophile, and others do not. To test these models, we reversibly melted two analogous protein domains in a two-state manner. E2cd is the isolated catalytic domain of cellulase E2 from the thermophile Thermomonospora fusca. CenAP30 is the analogous domain of the cellulase CenA from the mesophile Cellulomonas fimi. When reversibly denatured in a common buffer, the thermophilic enzyme E2cd had a temperature of melting (Tm) of 72.2 degrees C, a van't Hoff enthalpy of unfolding (DeltaHVH) of 190 kcal/mol, and an entropy of unfolding (DeltaSu) of 0.55 kcal/(mol*K); the mesophilic enzyme CenAP30 had a Tm of 56.4 degrees C, a DeltaHVH of 107 kcal/mol, and a DeltaSu of 0. 32 kcal/(mol*K). The higher DeltaHVH and DeltaSu values for E2cd suggest that its free energy of unfolding (DeltaGu) has a steeper dependence on temperature at the Tm than CenAP30. This result supports models that predict a greater maximum thermodynamic stability for thermophilic enzymes than for their mesophilic counterparts. This was further explored by urea denaturation. Under reducing conditions at 30 degrees C, E2cd had a concentration of melting (Cm) of 5.2 M and a DeltaGu of 11.2 kcal/mol; CenAP30 had a Cm of 2.6 M and a DeltaGu of 4.3 kcal/mol. Under nonreducing conditions, the Cm and DeltaGu of CenAP30 were increased to 4.5 M and 10.8 kcal/mol at 30 degrees C; the Cm for E2cd was increased to at least 7.4 M at 32 degrees C. We were unable to determine a DeltaGu value for E2cd under nonreducing conditions due to problems with reversibility. These data suggest that E2cd attains its greater thermal stability (DeltaTm = 15.8 degrees C) through a greater thermodynamic stability (DeltaDeltaGu = 6.9 kcal/mol) compared to its mesophilic analogue CenAP30.     

Heterologous expression and characterization of a recombinant thermophilic arylsulfatase from Thermotoga maritima

Production of low sulfated agar or agarose from agar or agaropectins by enzymatic hydrolysis has advantages but a high melting temperature is needed. The arylsulfatase gene from thermophilic Thermotoga maritima was cloned and expressed in Escherichia coli W3110 with pCol-MICT as the vector. The gene was comprised of 1,782 bp and encoded a protein of 593 amino acids with a molecular weight of 65 kDa. The recombinant arylsulfatase was partially purified by heat treatment (70°C, 30 min) and characterized. The enzyme was prepared with a total protein content of 2.4 mg and a specific activity of 20.63 U/mg. Optimal temperature and pH of the enzyme were 80°C and 7.0, respectively, for hydrolysis of p-nitrophenyl sulfate and sulfate content of agar was diminished to 40% after a 12 h treatment at that condition. Enhanced electrophoretic movement of DNA was observed in enzymetreated agar gel compared to that in a non-treated agar gel. These results suggest that thermophilic arylsulfatase expressed in E. coli could be useful for producing a low sulfated agar and electrophoretic grade agarose.     

Kinetics and mechanism of the citrate synthase from the thermophilic archaeon Thermoplasma acidophilum

The kinetics and mechanism of the citrate synthase from a moderate thermophile, Thermoplasma acidophilum (TpCS), are compared with those of the citrate synthase from a mesophile, pig heart (PCS). All discrete steps in the mechanistic sequence of PCS can be identified in TpCS. The catalytic strategies identified in PCS, destabilization of the oxaloacetate substrate carbonyl and stabilization of the reactive species, acetyl-CoA enolate, are present in TpCS. Conformational changes, which allow the enzyme to efficiently catalyze both condensation of acetyl-CoA thioester and subsequently hydrolysis of citryl-CoA thioester within the same active site, occur in both enzymes. However, significant differences exist between the two enzymes. PCS is a characteristically efficient enzyme: no internal step is clearly rate-limiting and the condensation step is readily reversible. TpCS is a less efficient catalyst. Over a broad temperature range, inadequate stabilization of the transition state for citryl-CoA hydrolysis renders this step nearly rate-limiting for the forward reaction of TpCS. Further, excessive stabilization of the citryl-CoA intermediate renders the condensation step nearly irreversible. Values of substrate and solvent deuterium isotope effects are consistent with the kinetic model. Near its temperature optimum (70 degrees C), there is a modest increase in the reversibility of the condensation step for TpCS, but reversibility still falls short of that shown by PCS at 37 degrees C. The root cause of the catalytic inefficiency of TpCS may lie in the lack of protein flexibility imposed by the requirement for thermal stability of the protein itself or its temperature-labile substrate, oxaloacetate.