Functional Analysis of Type 1 Isopentenyl Diphosphate Isomerase from Halobacterium sp. NRC-1

Here we report the characterization of the type-1 isopentenyl diphosphate isomerase derived from Halobacterium sp. NRC-1. The expressed purified enzyme showed maximum isomerase activity in the presence of 1 M NaCl at 37 °C at pH 6.0. This type-1 enzyme appears to be the first for which the Co²⁺ ion is required for activity.     

The crystal and solution studies of glucosamine-6-phosphate synthase from Candida albicans

Glucosamine 6-phosphate (GlcN-6-P) synthase is an ubiquitous enzyme that catalyses the first committed step in the reaction pathway that leads to formation of uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc), a precursor of macromolecules that contain amino sugars. Despite sequence similarities, the enzyme in eukaryotes is tetrameric, whereas in prokaryotes it is a dimer. The activity of eukaryotic GlcN-6-P synthase (known as Gfa1p) is regulated by feedback inhibition by UDP-GlcNAc, the end product of the reaction pathway, whereas in prokaryotes the GlcN-6-P synthase (known as GlmS) is not regulated at the post-translational level. In bacteria and fungi the enzyme is essential for cell wall synthesis. In human the enzyme is a mediator of insulin resistance. For these reasons, Gfa1p is a target in anti-fungal chemotherapy and in therapeutics for type-2 diabetes. The crystal structure of the Gfa1p isomerase domain from Candida albicans has been analysed in complex with the allosteric inhibitor UDP-GlcNAc and in the presence of glucose 6-phosphate, fructose 6-phosphate and an analogue of the reaction intermediate, 2-amino-2-deoxy-d-mannitol 6-phosphate (ADMP). A solution structure of the native Gfa1p has been deduced using small-angle X-ray scattering (SAXS). The tetrameric Gfa1p can be described as a dimer of dimers, with each half similar to the related enzyme from Escherichia coli. The core of the protein consists of the isomerase domains. UDP-GlcNAc binds, together with a metal cation, in a well-defined pocket on the surface of the isomerase domain. The residues responsible for tetramerisation and for binding UDP-GlcNAc are conserved only among eukaryotic sequences. Comparison with the previously studied GlmS from E. coli reveals differences as well as similarities in the isomerase active site. This study of Gfa1p focuses on the features that distinguish it from the prokaryotic homologue in terms of quaternary structure, control of the enzymatic activity and details of the isomerase active site.     

Distinct type-1 protein phosphatases are associated with hepatic glycogen and microsomes

The type-1 protein phosphatase associated with hepatic microsomes has been distinguished from the glycogen-bound enzyme in five ways. (1) The phosphorylase phosphatase/synthase phosphatase activity ratio of the microsomal enzyme (measured using muscle phosphorylase a and glycogen synthase (labelled in sites-3) as substrates) was 50-fold higher than that of the glycogen-bound enzyme. (2) The microsomal enzyme had a greater sensitivity to inhibitors-1 and 2. (3) Release of the catalytic subunit from the microsomal type-1 phosphatase by tryptic digestion was accompanied by a 2-fold increase in synthase phosphatase activity, whereas release of the catalytic subunit from the glycogen-bound enzyme decreased synthase phosphatase activity by 60%. (4) 95% of the synthase phosphatase activity was released from the microsomes with 0.3 M NaCl, whereas little activity could be released from the glycogen fraction with salt. (5) The type-1 phosphatase separated from glycogen by anion-exchange chromatography could be rebound to glycogen, whereas the microsomal enzyme (separated from the microsomes by the same procedure, or by extraction with NaCl) could not. These findings indicate that the synthase phosphatase activity of the microsomal enzyme is not explained by contamination with glycogen-bound enzyme. The microsomal and glycogen-associated enzymes may contain a common catalytic subunit complexed to microsomal and glycogen-binding subunits, respectively. Thiophosphorylase a was a potent inhibitor of the dephosphorylation of ribosomal protein S6, HMG-CoA reductase and glycogen synthase, by the glycogen-associated type-1 protein phosphatase. By contrast, thiophosphorylase a did not inhibit the dephosphorylation of S6 or HMG-CoA reductase by the microsomal enzyme, although the dephosphorylation of glycogen synthase was inhibited. The I50 for inhibition of synthase phosphatase activity by thiophosphorylase a catalysed by either the glycogen-associated or microsomal type-1 phosphatases, or for inhibition of S6 phosphatase activity catalysed by the glycogen-associated enzyme, was decreased 20-fold to 5-10 nM in the presence of glycogen. The results suggest that the physiologically relevant inhibitor of the glycogen-associated type-1 phosphatase is the phosphorylase a-glycogen complex, and that inhibition of the microsomal type-1 phosphatase by phosphorylase a is unlikely to play a role in the hormonal control of cholesterol or protein synthesis. Protein phosphatase-1 appears to be the principal S6 phosphatase in mammalian liver acting on the serine residues phosphorylated by cyclic AMP-dependent protein kinase.     

Structure/function relationships responsible for the kinetic differences between human type 1 and type 2 3beta-hydroxysteroid dehydrogenase and for the catalysis of the type 1 activity

Two distinct genes encode the 93% homologous type 1 (placenta, peripheral tissues) and type 2 (adrenals, gonads) 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD/isomerase) in humans. Mutagenesis studies using the type 1 enzyme have produced the Y154F and K158Q mutant enzymes in the Y(154)-P-H(156)-S-K(158) motif as well as the Y269S and K273Q mutants from a second motif, Y(269)-T-L-S-K(273), both of which are present in the primary structure of the human type 1 3beta-HSD/isomerase. In addition, the H156Y mutant of the type 1 enzyme has created a chimera of the type 2 enzyme motif (Y(154)-P-Y(156)-S-K(158)) in the type 1 enzyme. The mutant and wild-type enzymes have been expressed and purified. The K(m) value of dehydroepiandrosterone is 13-fold greater, and the maximal turnover rate (K(cat)) is 2-fold greater for wild-type 2 3beta-HSD compared with the wild-type 1 3beta-HSD activity. The H156Y mutant of the type 1 enzyme has substrate kinetic constants for 3beta-HSD activity that are very similar to those of the wild-type 2 enzyme. Dixon analysis shows that epostane inhibits the 3beta-HSD activity of the wild-type 1 enzyme with 14-17-fold greater affinity compared with the wild-type 2 and H156Y enzymes. The Y154F and K158Q mutants exhibit no 3beta-HSD activity, have substantial isomerase activity, and utilize substrate with K(m) values similar to those of wild-type 1 isomerase. The Y269S and K273Q mutants have low, pH-dependent 3beta-HSD activity, exhibit only 5% of the maximal isomerase activity, and utilize the isomerase substrate very poorly. From these studies, a structural basis for the profound differences in the substrate and inhibition kinetics of the wild-type 1 and 2 3beta-HSD, plus a catalytic role for the Tyr(154) and Lys(158) residues in the 3beta-HSD reaction have been identified. These advances in our understanding of the structure/function of human type 1 and 2 3beta-HSD/isomerase may lead to the design of selective inhibitors of the type 1 enzyme not only in placenta to control the onset of labor but also in hormone-sensitive breast, prostate, and choriocarcinoma tumors to slow their growth.     

Structure/function relationships responsible for coenzyme specificity and the isomerase activity of human type 1 3 beta-hydroxysteroid dehydrogenase/isomerase

Human type 1 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD/isomerase) catalyzes the two sequential enzyme reactions on a single protein that converts dehydroepiandrosterone or pregnenolone to androstenedione or progesterone, respectively, in placenta, mammary gland, breast tumors, prostate, prostate tumors, and other peripheral tissues. Our earlier studies show that the two enzyme reactions are linked by the coenzyme product, NADH, of the 3 beta-HSD activity. NADH activates the isomerase activity by inducing a time-dependent conformational change in the enzyme protein. The current study tested the hypothesis that the 3 beta-HSD and isomerase activities shared a common coenzyme domain, and it characterized key amino acids that participated in coenzyme binding and the isomerase reaction. Homology modeling with UDP-galactose-4-epimerase predicts that Asp36 is responsible for the NAD(H) specificity of human 3 beta-HSD/isomerase and identifies the Rossmann-fold coenzyme domain at the amino terminus. The D36A/K37R mutant in the potential coenzyme domain and the D241N, D257L, D258L, and D265N mutants in the potential isomerase domain (previously identified by affinity labeling) were created, expressed, and purified. The D36A/K37R mutant shifts the cofactor preference of both 3 beta-HSD and isomerase from NAD(H) to NADP(H), which shows that the two activities utilize a common coenzyme domain. The D257L and D258L mutations eliminate isomerase activity, whereas the D241N and D265N mutants have nearly full isomerase activity. Kinetic analyses and pH dependence studies showed that either Asp257 or Asp258 plays a catalytic role in the isomerization reaction. These observations further characterize the structure/function relationships of human 3 beta-HSD/isomerase and bring us closer to the goal of selectively inhibiting the type 1 enzyme in placenta (to control the timing of labor) or in hormone-sensitive breast tumors (to slow their growth).     

1H nuclear magnetic resonance assay of erythrocyte triosephosphate isomerase

A direct method for measuring the activity of erythrocyte triosephosphate isomerase using 1H NMR spectroscopy was developed. NMR spectroscopy allows the simultaneous monitoring of the substrate and the product of the reaction by virtue of the differences in the NMR spectrum of each chemical species. The assay conditions were based on a modification of a conventional spectrophotometric method. The enzymatic activity measured using NMR gave results comparable to those obtained in a standard assay. The results were used in the kinetic characterization of triosephosphate isomerase in hemolysates from subjects with homozygous or heterozygous deficiency of the enzyme. In general, NMR spectroscopy has the potential for wide application in the rapid development of new enzyme assays.     

Affinity alkylation of human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase by 2 alpha-bromoacetoxyprogesterone: evidence for separate dehydrogenase and isomerase sites on one protein

We have copurified human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase, which synthesize progesterone from pregnenolone and androstenedione from fetal dehydroepiandrosterone sulfate, from microsomes as a homogeneous protein based on electrophoretic and NH2-terminal sequencing data. The affinity alkylator, 2 alpha-bromoacetoxyprogesterone, simultaneously inactivates the pregnene and androstene dehydrogenase activities as well as the C21 and C19 isomerase activities in a time-dependent, irreversible manner following first order kinetics. At four concentrations (50/1-20/1 steroid/enzyme M ratios), the alkylator inactivates the dehydrogenase activity (t1/2 = 1.5-3.7 min) 2-fold faster than the isomerase activity. Pregnenolone and dehydroepiandrosterone protect the dehydrogenase activity, while 5-pregnene-3,20-dione, progesterone, and androstenedione protect isomerase activity from inactivation. The protection studies and competitive kinetics of inhibition demonstrate that the affinity alkylator is active site-directed. Kitz and Wilson analyses show that 2 alpha-bromoacetoxyprogesterone inactivates the dehydrogenase activity by a bimolecular mechanism (k3' = 160.9 l/mol.s), while the alkylator inactivates isomerase by a unimolecular mechanism (Ki = 0.14 mM, k3 = 0.013 s-1). Pregnenolone completely protects the dehydrogenase activity but does not slow the rate of isomerase inactivation by 2 alpha-bromoacetoxyprogesterone at all. NADH completely protects both activities from inactivation by the alkylator, while NAD+ protects neither. From Dixon analysis, NADH competitively inhibits NAD+ reduction by dehydrogenase activity. Mixed cofactor studies show that isomerase binds NAD+ and NADH at a common site. Therefore, NADH must not protect either activity by simply binding at the cofactor site. We postulate that NADH binding as an allosteric activator of isomerase protects both the dehydrogenase and isomerase activities from affinity alkylation by inducing a conformational change in the enzyme protein. The human placental enzyme appears to express the pregnene and androstene dehydrogenase activities at one site and the C21 and C19 isomerase activities at a second site on the same protein.     

The refolding of urea-denatured ribonuclease A is catalyzed by peptidyl-prolyl cis-trans isomerase

The refolding of urea-denatured ribonuclease A was measured at 0.31-3.1 mol . l-1 urea in the presence of various concentrations of peptidyl-prolyl cis-trans isomerase isolated from pig kidney. The rate of the slow CT-phase in the refolding reaction was found to be sensitive to this enzyme. A rate enhancement proportional to the isomerase activity has been observed. The activity of the enzyme was assayed with Glt-Ala-Ala-Pro-Phe-4-nitroanilide. The catalytic activity of the isomerase against unfolded ribonuclease is suppressed after preincubation of the enzyme with 0.001 mol . l-1 Cu2+, 0.01 mol . l-1 H+ and by heat inactivation. The results indicate the involvement of the cis/trans interconversion of proline peptide bonds during the refolding of ribonuclease A.     

Studies on glucose isomerase from a Streptomyces species.

Production and properties of glucose isomerase from a Co2+-sensitive Streptomyces species were studied. After 4 days of shaking cultivation at 30 degrees C and 200 rpm, a maximum of 1.1 enzyme units per ml of broth was obtained. Cell-free glucose isomerase, obtained from mycelia heat-treated in the presence of 0.5 mM Co2+, showed a 3.5-fold increase in specific activity over enzyme obtained from untreated mycelia. The optimum pH and temperature for the glucose isomerase were 7 to 8 and 80 degrees C, respectively. The Michaelis constant for fructose was 0.40 M. Mg2+ was found to enhance the glucose isomerase activity, whereas the effect of Co2+ on enzyme activity depended on the manner in which the enzyme was prepared. This glucose isomerase was quite heat stable, with a half-life of 120 h at 70 degrees C.     

Characterization of cucumber ascorbate oxidase and its reaction with hexacyanoferrate (II)

The spectroscopic features of cucumber ascorbate oxidase (AOase) and its type-2 copper-depleted (T2D) derivative, and the electron pathway among the copper sites in the enzyme have been investigated. The electronic and CD spectra of native and T2D AOase in the visible region bear a striking resemblance to those of plastocyanin or azurin, which contain type-1 copper alone. The electronic absorption shoulder of the native enzyme at around 330 nm for the native enzyme which has been assigned to type-3 copper disappears with the depletion of the type-2 copper. The reduction of AOase with a large excess of hexacyanoferrate(II) results in a selective reduction of the type-2 Cu, giving rise to an additional EPR-detectable species which is considered to be originated from partly reduced type-3 copper. The type-1 copper is, however, not reduced even in the presence of excess hexacyanoferrate(II). The redox potential of type-1 Cu was determined to be +350 mV, which is distinctly lower than that of hexacyanoferrate(II-III). Type-2 copper was supposed to be a mediator of the electron transfer between type-1 and type-3 coppers in consideration of the extremely low activity of the T2D enzyme under the same condition. A comparison of the electron pathway in AOase with that in laccase is also argued.     

Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase that increases the production rate of D-tagatose

AIMS: Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase used to increase the production rate of D-tagatose. METHODS AND RESULTS: A mutated gene was obtained by an error-prone polymerase chain reaction using L-arabinose isomerase gene from G. stearothermophilus as a template and the gene was expressed in Escherichia coli. The expressed mutated L-arabinose isomerase exhibited the change of three amino acids (Met322-->Val, Ser393-->Thr, and Val408-->Ala), compared with the wild-type enzyme and was then purified to homogeneity. The mutated enzyme had a maximum galactose isomerization activity at pH 8.0, 65 degrees C, and 1.0 mM Co2+, while the wild-type enzyme had a maximum activity at pH 8.0, 60 degrees C, and 1.0-mM Mn2+. The mutated L-arabinose isomerase exhibited increases in D-galactose isomerization activity, optimum temperature, catalytic efficiency (kcat/Km) for D-galactose, and the production rate of D-tagatose from D-galactose. CONCLUSIONS: The mutated L-arabinose isomerase from G. stearothermophilus is valuable for the commercial production of D-tagatose. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes knowledge on the characterization of a mutated L-arabinose isomerase, and allows an increased production rate for D-tagatose from D-galactose using the mutated enzyme.     

Substrate specificity of a recombinant d‐lyxose isomerase from Serratia proteamaculans that produces d‐lyxose and d‐mannose

Aims: Characterization of substrate specificity of a d ‐lyxose isomerase from Serratia proteamaculans and application of the enzyme in the production of d ‐lyxose and d ‐mannose. Methods and Results: The concentrations of monosaccharides were determined using a Bio‐LC system. The activity of the recombinant protein from Ser. proteamaculans was the highest for d ‐lyxose among aldoses, indicating that it is a d‐ lyxose isomerase. The native recombinant enzyme existed as a 54‐kDa dimer, and the maximal activity for d‐ lyxose isomerization was observed at pH 7·5 and 40°C in the presence of 1 mmol l^?1 Mn²⁺. The K_m values for d ‐lyxose, d ‐mannose, d ‐xylulose, and d ‐fructose were 13·3, 32·2, 3·83, and 19·4 mmol l^?1, respectively. In 2 ml of reaction volume at pH 7·5 and 35°C, d ‐lyxose was produced at 35% (w/v) from 50% (w/v) d ‐xylulose by the d‐ lyxose isomerase in 3 h, while d ‐mannose were produced at 10% (w/v) from 50% (w/v) d ‐fructose in 5 h. Conclusions: We identified the putative sugar isomerase from Ser. proteamaculans as a d ‐lyxose isomerase. The enzyme exhibited isomerization activity for aldose substrates with the C2 and C3 hydroxyl groups in the left‐hand configuration. High production rates of d‐ lyxose and d ‐mannose by the enzyme were obtained. Significance and Impact of the Study: A new d‐ lyxose isomerase was found, and this enzyme had higher activity for d ‐lyxose and d ‐mannose than previously reported enzymes. Thus, the enzyme can be applied in industrial production of d ‐lyxose and d ‐mannose.     

Identification of an Archaeal type II isopentenyl diphosphate isomerase in methanothermobacter thermautotrophicus

Isopentenyl diphosphate (IPP):dimethylallyl diphosphate isomerase catalyzes the interconversion of the fundamental five-carbon homoallylic and allylic diphosphate building blocks required for biosynthesis of isoprenoid compounds. Two different isomerases have been reported. The type I enzyme, first characterized in the late 1950s, is widely distributed in eukaryota and eubacteria. The type II enzyme was recently discovered in Streptomyces sp. strain CL190. Open reading frame 48 (ORF48) in the archaeon Methanothermobacter thermautotrophicus encodes a putative type II IPP isomerase. A plasmid-encoded copy of the ORF complemented IPP isomerase activity in vivo in Salmonella enterica serovar Typhimurium strain RMC29, which contains chromosomal knockouts in the genes for type I IPP isomerase (idi) and 1-deoxy-D-xylulose 5-phosphate (dxs). The dxs gene was interrupted with a synthetic operon containing the Saccharomyces cerevisiae genes erg8, erg12, and erg19 allowing for the conversion of mevalonic acid to IPP by the mevalonate pathway. His6-tagged M. thermautotrophicus type II IPP isomerase was produced in Escherichia coli and purified by Ni2+ chromatography. The purified protein was characterized by matrix-assisted laser desorption ionization mass spectrometry. The enzyme has optimal activity at 70 degrees C and pH 6.5. NADPH, flavin mononucleotide, and Mg2+ are required cofactors. The steady-state kinetic constants for the archaeal type II IPP isomerase from M. thermautotrophicus are as follows: K(m), 64 microM; specific activity, 0.476 micromol mg(-1) min(-1); and k(cat), 1.6 s(-1).     

Intrinsic enoyl-CoA isomerase activity of rat acyl-CoA oxidase I

Rat peroxisomal acyl-CoA oxidase I is a key enzyme for the beta-oxidation of fatty acids, and the deficiency of this enzyme in patient has been previously reported. It was found that rat acyl-CoA oxidase I has intrinsic enoyl-CoA isomerase activity, which was confirmed using incubation followed with HPLC analysis in this study. Various 3-enoyl-CoA substrates with cis or trans configuration were synthesized and used in the study of enzyme substrate specificity. The isomerase activity of the enzyme was characterized through studies of kinetics, pH dependence, and enzyme inhibition. Most k(cat)/K(M) values of rat peroxisomal acyl-CoA oxidase I for isomerization reaction are comparable with those of authentic rat liver peroxisomal Delta(3)-Delta(2)-enoyl-CoA isomerase and rat liver peroxisomal multifunctional enzyme 1 when hexenoyl-CoA and octenoyl-CoA with cis- or trans-configuration were used as substrate. Glu421 was found to be the catalytic residue for both oxidase and isomerase activities of the enzyme. The isomerase activity of rat peroxisomal acyl-CoA oxidase I is probably due to a spontaneous process driven by thermodynamic equilibrium with formation of a conjugated structure after deprotonation of substrate alpha-proton. The energy level of transition state may be lowered by a stable dienolate intermediate, which gain further stabilization via charge transfer with electron-deficient FAD cofactor of the enzyme.     

Purification, properties and heteromeric association of type-1 and type-2 lepidopteran farnesyl diphosphate synthases

Two forms of farnesyl diphosphate synthase (FPPS) from the spruce budworm, Choristoneura fumiferana, and one from the armyworm Pseudaletia unipuncta, have been cloned and their catalytic properties assessed. The type-2 FPPS of C. fumiferana (CfFPPS2) was efficient in the prenyl coupling of DMAPP or GPP with [(14)C]IPP, producing FPP as its final product; however, type-1 FPPSs (CfFPPS1, PuFPPS1, as well as Agrotis ipsilon FPPS1) were essentially inactive. A variety of purification methods was employed to purify the type-1 enzymes. Under mild chromatographic conditions, the isolated type-1 enzyme showed modest activity, but was apparently contaminated with endogenous prenyltransferase derived from the Escherichia coli host cells. Similarly, unpurified extracts of PuFPPS1 expressed in an E. coli FPPS-null mutant, had low FPPS activity. When equimolar amounts of homogenous CfFPPS1 and CfFPP2 were combined, a sharp synergistic enhancement of activity was observed, and the coupling of several homologous substrates, which are precursors to ethyl-branched JHs, was enhanced. Association between CfFPPS1 and CfFPPS2 was confirmed by both protein interaction chromatography and competitive ELISA. These data suggest that type-1 and type-2 FPPSs can form a heteromer, which may play a role in sesquiterpene biosynthesis, such as JH homologue formation, in moths.     

Intrinsic isomerase activity of medium-chain acyl-CoA dehydrogenase

Mitochondrial medium-chain acyl-CoA dehydrogenase is a key enzyme for the beta oxidation of fatty acids, and the deficiency of this enzyme in patients has been previously reported. We found that the enzyme has intrinsic isomerase activity, which was confirmed using incubation followed with HPLC analysis. The isomerase activity of the enzyme was thoroughly characterized through studies of kinetics, substrate specificity, pH dependence, and enzyme inhibition. E376 mutants were constructed, and mutant enzymes were purified and characterized. It was shown that E376 is the catalytic residue for both dehydrogenase and isomerase activities of the enzyme. The isomerase activity of medium-chain acyl-CoA dehydrogenase is probably a spontaneous process driven by thermodynamic equilibrium with the formation of a conjugated structure after deprotonation of substrate alpha proton. The energy level of the transition state may be lowered by a stable dienolate intermediate, which gains further stabilization via charge transfer with the electron-deficient FAD cofactor of the enzyme. This raises the question as to whether the dehydrogenase might function as an isomerase in vivo in conditions in which the activity of the isomerase is decreased.     

Purification and characterisation of glucose (xylose) isomerase from Chainia sp. (NCL 82-5-1)

Glucose (xylose) isomerase is an important enzyme in high fructose syrup industry. The enzyme generally occurs intracellularly and is specific for both glucose and xylose. A rare actinomycete Chainia sp. (NCL 82-5-1) produces extracellular specific glucose and xylose isomerases and an intracellular glucose (xylose) isomerase. The intracellular enzyme is isolated by cell autolysis and purified by preparative polyacrylamide gel electrophoresis. Its properties are studied and compared with those of extracellular specific xylose isomerase. The intracellular enzyme has a molecular weight of 1,58,000 daltons with four equal subunits of 40,700 daltons. The N-terminal amino acid sequence analysis shows Arg at the N-terminal. Diethylpyrocarbonate inhibited the enzyme and the inhibition kinetics study shows the presence of at least 2 essential His residues. The amino acid analysis shows the absence of Cys and a high proportion of hydrophobic and acidic amino acids.     

Induction of chalcone isomerase in elicitor-treated bean cells. Comparison of rates of synthesis and appearance of immunodetectable enzyme

Chalcone isomerase, an enzyme involved in the formation of flavonoid-derived compounds in plants, has been purified nearly 600-fold from cell suspension cultures of dwarf French bean (Phaseolus vulgaris L.). Chromatofocussing yielded a single form of the enzyme of apparent pI 5.0. This preparation was used to raise rabbit anti-(chalcone isomerase) serum. Changes in the rate of synthesis of chalcone isomerase have been investigated by indirect immunoprecipitation of enzyme labelled in vivo with [35S]methionine in elicitor-treated cultures of P. vulgaris. Elicitor, heat-released from cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum, the causal agent of anthracnose disease of bean, causes increased synthesis of the isomerase, with maximum synthetic rate occurring 11-12 h after exposure to elicitor. Immune blotting studies indicate that the elicitor-mediated increase in extractable activity of the isomerase is associated with increased appearance of immunodetactable isomerase protein of Mr 27 000. However, the maximum level of immunodetectable isomerase was attained approximately 6 h earlier than maximum extractable activity. Furthermore, a 2.8-fold increase in enzyme activity above basal levels at 12 h after elicitor-treatment was associated with a corresponding 5.8-fold increase in immunodetectable enzyme. It is concluded that elicitor induces the synthesis of both active and inactive chalcone isomerase of Mr 27 000, and that some activation of inactive enzyme occurs during the elicitor-mediated increase in isomerase activity. The presence of a pool of inactive chalcone isomerase in bean cell cultures has recently been suggested on the basis of density labelling experiments utilising 2H from 2H2O [Dixon et al. (1983) Planta (Berl.) 159, 561-569].     

Proposal for a pathway to mediate the metabolic effects of insulin

This review seeks to assemble recent discoveries about insulin receptor/kinase, guanine nucleotide-binding proteins, phosphatidyl inositol metabolism, and protein phosphatases to provide a mechanistic pathway by which insulin would alter carbohydrate and fat metabolism. It proposes a hypothetical chain of events that leads from the insulin receptor to protein phosphatase-1. The sequence starts with insulin binding to its receptor, activating the intrinsic receptor/kinase activity. The insulin receptor phosphorylates a guanine nucleotide-binding protein, which activates a particular phospholipase C. This in turn stimulates the production of two lipid-derived messengers: inositol-phospho-glucosamine and diacylglycerol. These messengers trigger the effects of insulin. The diacylglycerol produced by insulin is thought to be analogous to the diacylglycerol produced by alpha-adrenergic stimulation, which activates protein kinase C. Activation of this kinase could account for increases in phosphorylation of certain proteins. The inositol-phospho-glucosamine is the cytosolic messenger for insulin. One of the enzymes activated by insulin is protein phosphatase type-1. It is known that the phosphatase decreases phosphorylation of certain target enzymes. In response to insulin, activation of protein phosphatase type-1 occurs with a stable conformational change that may involve rearrangement of disulfide bonds. Rearrangement is either directly in response to the cytosolic messenger or is catalyzed by an isomerase activated by the insulin messenger. Ultimately, protein phosphatase type-1 and/or the disulfide isomerase may together mediate the pleiotropic effects of insulin on carbohydrate and fat metabolism.     

xylA cloning and sequencing and biochemical characterization of xylose isomerase from Thermotoga neapolitana.

The xylA gene coding for xylose isomerase from the hyperthermophile Thermotoga neapolitana 5068 was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a polypeptide of 444 residues with a calculated molecular weight of 50,892. The native enzyme was a homotetramer with a molecular weight of 200,000. This xylose isomerase was a member of the family II enzymes (these differ from family I isomerases by the presence of approximately 50 additional residues at the amino terminus). The enzyme was extremely thermostable, with optimal activity above 95 degrees C. The xylose isomerase showed maximum activity at pH 7.1, but it had high relative activity over a broad pH range. The catalytic efficiency (kcat/Km) of the enzyme was essentially constant between 60 and 90 degrees C, and the catalytic efficiency decreased between 90 and 98 degrees C primarily because of a large increase in Km. The T. neapolitana xylose isomerase had a higher turnover number and a lower Km for glucose than other family II xylose isomerases. Comparisons with other xylose isomerases showed that the catalytic and cation binding regions were well conserved. Comparison of different xylose isomerase sequences showed that numbers of asparagine and glutamine residues decreased with increasing enzyme thermostability, presumably as a thermophilic strategy for diminishing the potential for chemical denaturation through deamidation at elevated temperatures.