Purification, Characterization, and Cloning of Cinnamyl Alcohol Dehydrogenase in Loblolly Pine (Pinus taeda L.)

Cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1. 195) has been purified to homogeneity from differentiating xylem tissue and developing seeds of loblolly pine (Pinus taeda L.). The enzyme is a dimer with a native molecular weight of 82,000 and a subunit molecular weight of 44,000, and is the only form of CAD involved in lignification in differentiating xylem. High levels of loblolly pine CAD enzyme were found in nonlignifying seed tissue. Characterization of the enzyme from both seeds and xylem demonstrated that the enzyme is the same in both tissues. The enzyme has a high affinity for coniferaldehyde (K_m = 1.7 micromolar) compared with sinapaldehyde (K_m in excess of 100 micromolar). Kinetic data strongly suggest that coniferin is a noncompetitive inhibitor of CAD enzyme activity. Protein sequences were obtained for the N-terminus (28 amino acids) and for two other peptides. Degenerate oligonucleotide primers based on the protein sequences were used to amplify by polymerase chain reaction a 1050 base pair DNA fragment from xylem cDNA. Nucleotide sequence from the cloned DNA fragment coded for the N-terminal protein sequence and an internal peptide of CAD. The N-terminal protein sequence has little similarity with the λCAD4 clone isolated from bean (MH Walter, J Grima-Pettenati, C Grand, AM Boudet, CJ Lamb [1988] Proc Natl Acad Sci USA 86:5546-5550), which has homology with malic enzyme.     

Partial Purification and Characterization of a 3'- Phosphoadenosine 5' -Phosphosulfate: Desulfoglucosinolate Sulfotransferase from Cress (Lepidium sativum)

A 3′ -phosphoadenosine 5′ -phosphosulfate (PAPS):desulfoglucosinolate sulfotransferase (EC 2.8.2-) was extensively purified from light-grown cress (Lepidium sativum L.) seedlings by gel filtration and concanavalin A-Sepharose 4B, Matrex Gel Green A, and Mono Q fast protein liquid chromatography. The purified enzyme, which required bovine serum albumin for stabilization, had a native molecular weight of 31,000 ± 5,000 and an apparent isoelectric point of 5.2. Using PAPS (K_m 60 micromolar) as sulfur donor, it catalyzed the sulfation of desulfobenzylglucosinolate (K_m 82 micromolar), desulfo-p-hydroxybenzylglucosinolate (K_m 670 micromolar), and desulfoallylglucosinolate (K_m 6.5 millimolar) at an optimal pH of 9.0. All other potential substrates tested, including flavonoids, flavonoid glycosides, cinnamic acids, and phenylacetaldoxime, were not sulfated. Sulfotransferase activity was stimulated by MgCl₂, MnCl₂ and reducing agents and inhibited by ZnCl₂, PbNO₃ NiCl₂ and the reaction product PAP. The thiol reagents N-ethylmaleimide, p-chloromercuriphenylsulfonic acid, and 5,5′ -dithio-bis-(2-nitrobenzoic acid) were also potent inhibitors, but the enzyme was protected from covalent modification by β-mercaptoethanol. The kinetics of desulfobenzylglucosinolate sulfation were consistent with a rapid equilibrium ordered mechanism with desulfobenzylglucosinolate binding first and PAPS second.     

Substrate Specificity of Purified Recombinant Human β-Carotene 15,15′-Oxygenase (BCO1)

Humans cannot synthesize vitamin A and thus must obtain it from their diet. β-Carotene 15,15′-oxygenase (BCO1) catalyzes the oxidative cleavage of provitamin A carotenoids at the central 15–15′ double bond to yield retinal (vitamin A). In this work, we quantitatively describe the substrate specificity of purified recombinant human BCO1 in terms of catalytic efficiency values (k_cat/K_m). The full-length open reading frame of human BCO1 was cloned into the pET-28b expression vector with a C-terminal polyhistidine tag, and the protein was expressed in the Escherichia coli strain BL21-Gold(DE3). The enzyme was purified using cobalt ion affinity chromatography. The purified enzyme preparation catalyzed the oxidative cleavage of β-carotene with a V_max = 197.2 nmol retinal/mg BCO1 × h, K_m = 17.2 μm and catalytic efficiency k_cat/K_m = 6098 m⁻¹ min⁻¹. The enzyme also catalyzed the oxidative cleavage of α-carotene, β-cryptoxanthin, and β-apo-8′-carotenal to yield retinal. The catalytic efficiency values of these substrates are lower than that of β-carotene. Surprisingly, BCO1 catalyzed the oxidative cleavage of lycopene to yield acycloretinal with a catalytic efficiency similar to that of β-carotene. The shorter β-apocarotenals (β-apo-10′-carotenal, β-apo-12′-carotenal, β-apo-14′-carotenal) do not show Michaelis-Menten behavior under the conditions tested. We did not detect any activity with lutein, zeaxanthin, and 9-cis-β-carotene. Our results show that BCO1 favors full-length provitamin A carotenoids as substrates, with the notable exception of lycopene. Lycopene has previously been reported to be unreactive with BCO1, and our findings warrant a fresh look at acycloretinal and its alcohol and acid forms as metabolites of lycopene in future studies.     

gamma-Guanidinobutyraldehyde Dehydrogenase of Vicia faba Leaves

γ-Guanidinobutyraldehyde dehydrogenase was purified 27-fold in 40% yield from extracts of Vicia faba leaves. High specificity exist only for γ-guanidinobutyraldehyde and γ-aminobutyraldehyde; the K_m value was 3.4 micromolar for γ-guanidinobutyraldehyde, 25 micromolar for γ-aminobutyraldehyde, and 84 micromolar (case of γ-guanidinobutyraldehyde) for NAD, respectively. The enzyme had a molecular weight of approximately 83,000. Optimal pH and temperature for activity were 9.5 and 45°C, respectively. The enzyme was inhibited strongly by p-chloromercuribenzoate, N-ethylmaleimide, and zincon (2-carboxy-2′-hydroxy-5′-sulfoformazylbenzene).     

Metabolism of Cytokinin : DEPHOSPHORYLATION OF CYTOKININ RIBONUCLEOTIDE BY 5'-NUCLEOTIDASES FROM WHEAT GERM CYTOSOL

Two forms (F-I and F-II) of 5′-nucleotidases (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) which catalyze the dephosphorylation of N⁶-(Δ²-isopentenyl)adenosine 5′-monophosphate and AMP to form the corresponding nucleosides were partially purified from the cytosol of wheat (Triticum aestivum) germ. Both the F-I (molecular weight, 57,000) and F-II (molecular weight, 110,000) 5′-nucleotidases dephosphorylate the ribonucleotides at an optimum pH of 7. The K_m values for the cytokinin nucleotide are 3.5 micromolar (F-I enzyme) and 12.8 micromolar (F-II enzyme) in 100 millimolar Tris-maleate buffer (pH 7) at 37 C. The F-I enzyme is less rapidly inactivated by heating than is the F-II enzyme. Both nucleotidases hydrolyze purine ribonucleoside 5′-phosphates, AMP being the preferred substrate. N⁶-(Δ²-isopentenyl)Adenosine 5′-monophosphate is hydrolyzed at a rate 72 and 86% that of AMP by the F-I and F-II nucleotides, respectively. Phenylphosphate and 3′-AMP are not substrates for the enzymes. It is proposed that dephosphorylation of cytokinin nucleotide by cytosol 5′-nucleotidases may play an important role in regulating levels of “active cytokinin” in plant cells.     

Partial Purification and Some Properties of Flavonol 7-Sulfotransferase from Flaveria bidentis

A novel flavonol-specific sulfotransferase was partially purified from the shoot tips of Flaveria bidentis var. Angustifolia O.K. (Asteraceae) by chromatography on 3′-phosphoadenosine 5′-phosphate-agarose affinity column and chromatofocusing on Mono P. The latter step resulted in the separation of two isoforms, both of which exhibited expressed specificity for position 7 of quercetin 3,3′- and quercetin 3,4′-disulfate. The 7-sulfotransferase isoforms I and II had a pH optimum of 7.5 in phosphate buffer, apparent pl values of 6.5 and 6.3, and an M_r of 35,000. They had no requirement for divalent cations and were not inhibited by EDTA or SH group reagents. Their K_m values for both the sulfate donor and flavonol acceptor were of the same order of magnitude (0.20-0.46 micromolar). This enzyme, together with the recently reported flavonol 3-, 3′-, and 4′-sulfotransferases from F. chloraefolia (L Varin, RK Ibrahim [1989] Plant Physiol 90: 977-981) form the complement involved in the biosynthesis of polysulfated flavonols in this genus. A proposed sequential order for the enzymatic sulfation in both species is described.     

Cloning, Expression in Escherichia coli, and Characterization of Arabidopsis thaliana UMP/CMP Kinase

A cDNA encoding the Arabidopsis thaliana uridine 5′-monophosphate (UMP)/cytidine 5′-monophosphate (CMP) kinase was isolated by complementation of a Saccharomyces cerevisiae ura6 mutant. The deduced amino acid sequence of the plant UMP/CMP kinase has 50% identity with other eukaryotic UMP/CMP kinase proteins. The cDNA was subcloned into pGEX-4T-3 and expressed as a glutathione S-transferase fusion protein in Escherichia coli. Following proteolytic digestion, the plant UMP/CMP kinase was purified and analyzed for its structural and kinetic properties. The mass, N-terminal sequence, and total amino acid composition agreed with the sequence and composition predicted from the cDNA sequence. Kinetic analysis revealed that the UMP/CMP kinase preferentially uses ATP (Michaelis constant [K_m] = 29 μm when UMP is the other substrate and K_m = 292 μm when CMP is the other substrate) as a phosphate donor. However, both UMP (K_m = 153 μm) and CMP (K_m = 266 μm) were equally acceptable as the phosphate acceptor. The optimal pH for the enzyme is 6.5. P¹, P⁵-di(adenosine-5′) pentaphosphate was found to be a competitive inhibitor of both ATP and UMP.     

Biosynthesis of Cytidine 5'-Diphosphate-diacylglycerol in Endoplasmic Reticulum and Mitochondria of Castor Bean Endosperm

Cytidine 5′-triphosphate (CTP):phosphatidate cytidyltransferase from the endoplasmic reticulum and mitochondria of Ricinus communis L. var Hale was characterized. The endoplasmic reticulum enzyme has a pH optimum of 6.5 and a divalent cation is required, Mn²⁺ being preferred and giving maximum activity at 2.5 millimolar. The estimated K_m for CTP is 16.7 micromolar, but that for phosphatidate could not be determined accurately. The activity was inhibited by both deoxycholate and Triton X-100 at concentrations as low as 0.01% (w/w).

The mitochondrial enzyme has a pH optimum of 6.0 and a divalent cation requirement similar to that of the endoplasmic reticulum. Maximum stimulation of the reaction by substrates occurred with 1.5 millimolar phosphatidate (from egg phosphatidylcholine) and about 400 micromolar CTP. The apparent K_m for phosphatidate could not be estimated accurately since activity was obtained in the absence of added lipid, apparently utilizing endogenous substrate. The K_m estimated for CTP was altered by the presence of the detergent Triton X-100; in its absence the value was 33.3 micromolar, but in its presence the value was 66.7 micromolar. Inclusion of 0.6% (w/w) Triton X-100 in the assay mixture stimulated the activity about 2.5-fold.

     

Subcellular localization of hexose kinases in pea stems: mitochondrial hexokinase

The subcellular localization of hexose phosphorylating activity in extracts of pea stems has been studied by differential centrifugation and sucrose density gradient centrifugation. The hexokinase (EC 2.7.1.1) was associated with the mitochondria, whereas fructokinase (EC 2.7.1.4) was in the cytosolic fraction. Some properties of the mitochondrial hexokinase were studied. The enzyme had a high affinity for glucose (K_m 76 micromolar) and mannose (K_m 71 micromolar) and a relatively low affinity for fructose (K_m 15.7 millimolar). The K_m for MgATP was 180 micromolar. The addition of salts stimulated the activity of the hexokinase. Al³⁺ was a strong inhibitor at pH 7 but not at the optimum pH (8.2). The enzyme was not readily solubilized but, in experiments with intact mitochondria, was susceptible to proteolysis. A location on the outer mitochondrial membrane is suggested for the hexokinase of pea stems.     

Isolation, purification, and partial characterization of formate dehydrogenase from soybean seed

Soybean (Glycine soja var Beeson) formate dehydrogenase has been isolated, purified, and partially characterized by affinity chromatography. The enzyme is a dimer having a total molecular weight of 100,000 and a subunit weight of 47,000. It has activity over a broad pH range, is stable for months at 4°C, and has K_m values of 0.6 millimolar and 5.7 micromolar for formate and NAD, respectively.     

Kinetic and Structural Properties of NADP-Malic Enzyme from Sugarcane Leaves

Oligomeric structure and kinetic properties of NADP-malic enzyme, purified from sugarcane (Saccharam officinarum L.) leaves, were determined at either pH 7.0 and 8.0. Size exclusion chromatography showed the existence of an equilibrium between the dimeric and the tetrameric forms. At pH 7.0 the enzyme was found preferentially as a 125 kilodalton homodimer, whereas the tetramer was the major form found at pH 8.0. Although free forms of l-malate, NADP⁺, and Mg²⁺ were determined as the true substrates and cofactors for the enzyme at the two conditions, the kinetic properties of the malic enzyme were quite different depending on pH. Higher affinity for l-malate (K_m = 58 micromolar), but also inhibition by high substrate (K_i = 4.95 millimolar) were observed at pH 7.0. l-Malate saturation isotherms at pH 8.0 followed hyperbolic kinetics (K_m = 120 micromolar). At both pH conditions, activity response to NADP⁺ exhibited Michaelis-Menten behavior with K_m values of 7.1 and 4.6 micromolar at pH 7.0 and 8.0, respectively. Negative cooperativity detected in the binding of Mg²⁺ suggested the presence of at least two Mg²⁺ - binding sites with different affinity. The K_a values for Mg²⁺ obtained at pH 7.0 (9 and 750 micromolar) were significantly higher than those calculated at pH 8.0 (1 and 84 micromolar). The results suggest that changes in pH and Mg²⁺ levels could be important for the physiological regulation of NADP-malic enzyme.     

Purification, characterization, and immunological properties for two isoforms of glutathione reductase from eastern white pine needles

Glutathione reductase (EC 1.6.4.2) was purified from Eastern white pine (Pinus strobus L.) needles. The purification steps included affinity chromatography using 2′, 5′-ADP-Sepharose, FPLC-anion-exchange, FPLC-hydrophobic interaction, and FPLC-gel filtration. Separation of proteins by FPLC-anion-exchange resulted in the recovery of two distinct isoforms of glutathione reductase (GR_A and GR_B). Purified GR_A had a specific activity of 1.81 microkatals per milligram of protein and GR_B had a specific activity of 6.08 microkatals per milligram of protein. GR_A accounted for 17% of the total units of glutathione reductase recovered after anion-exchange separation and GR_B accounted for 83%. The native molecular mass for GR_A was 103 to 104 kilodaltons and for GR_B was 88 to 95 kilodaltons. Both isoforms of glutathione reductase were dimers composed of identical subunit molecular masses which were 53 to 54 kilodaltons for GR_A and 57 kilodaltons for GR_B. The pH optimum for GR_A was 7.25 to 7.75 and for GR_B was 7.25. At 25°C the K_m for GSSG was 15.3 and 39.8 micromolar for GR_A and GR_B, respectively. For NADPH, the K_m was 3.7 and 8.8 micromolar for GR_A and GR_B, respectively. Antibody produced from purified GR_B was reactive with both native and denatured GR_B, but was cross-reactive with only native GR_A.     

Purification and properties of adenylyl sulphate:ammonia adenylyltransferase from Chlorella catalysing the formation of adenosine 5′-phosphoramidate from adenosine 5′-phosphosulphate and ammonia

Extracts of Chlorella pyrenoidosa, Euglena gracilis var. bacillaris, spinach, barley, Dictyostelium discoideum and Escherichia coli form an unknown compound enzymically from adenosine 5′-phosphosulphate in the presence of ammonia. This unknown compound shares the following properties with adenosine 5′-phosphoramidate: molar proportions of constituent parts (1 adenine:1 ribose:1 phosphate:1 ammonia released at low pH), co-electrophoresis in all buffers tested including borate, formation of AMP at low pH through release of ammonia, mass and i.r. spectra and conversion into 5′-AMP by phosphodiesterase. This unknown compound therefore appears to be identical with adenosine 5′-phosphoramidate. The enzyme that catalyses the formation of adenosine 5′-phosphoramidate from ammonia and adenosine 5′-phosphosulphate was purified 1800-fold (to homogeneity) from Chlorella by using (NH₄)₂SO₄ precipitation and DEAE-cellulose, Sephadex and Reactive Blue 2–agarose chromatography. The purified enzyme shows one band of protein, coincident with activity, at a position corresponding to 60000–65000 molecular weight, on polyacrylamide-gel electrophoresis, and yields three subunits on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of 26000, 21000 and 17000 molecular weight, consistent with a molecular weight of 64000 for the native enzyme. Isoelectrofocusing yields one band of pI4.2. The pH optimum of the enzyme-catalysed reaction is 8.8. ATP, ADP or adenosine 3′-phosphate 5′-phosphosulphate will not replace adenosine 5′-phosphosulphate, and the apparent K_m for the last-mentioned compound is 0.82mm. The apparent K_m for ammonia (assuming NH₃ to be the active species) is about 10mm. A large variety of primary, secondary and tertiary amines or amides will not replace ammonia. One mol.prop. of adenosine 5′-phosphosulphate reacts with 1 mol.prop. of ammonia to yield 1 mol.prop. each of adenosine 5′-phosphoramidate and sulphate; no AMP is found. The highly purified enzyme does not catalyse any of the known reactions of adenosine 5′-phosphosulphate, including those catalysed by ATP sulphurylase, adenosine 5′-phosphosulphate kinase, adenosine 5′-phosphosulphate sulphotransferase or ADP sulphurylase. Adenosine 5′-phosphoramidate is found in old samples of the ammonium salt of adenosine 5′-phosphosulphate and can be formed non-enzymically if adenosine 5′-phosphosulphate and ammonia are boiled. In the non-enzymic reaction both adenosine 5′-phosphoramidate and AMP are formed. Thus the enzyme forms adenosine 5′-phosphoramidate by selectively speeding up an already favoured reaction.     

Partial purification and properties of an inducible uridine 5'-diphosphate-glucose-salicylic Acid glucosyltransferase from oat roots

A salicylic acid (SA)-inducible uridine 5′-diphosphate (UDP)-glucose:SA 3-O-glucosyltransferase was extracted from oat (Avena sativa L. cv Dal) roots. Reverse phase high-performance liquid chromatography or anion exchange chromatography was used to separate SA from the product, β-O-d-glucosylsalicylic acid. The soluble enzyme was purified 176-fold with 5% recovery using a combination of pH fractionation, anion exchange, gel filtration, and chromatofocusing chromatography. The partially purified protein had a native molecular weight of about 50,000, an apparent isoelectric point at pH 5.0, and maximum activity at pH 5.5. The enzyme had a K_m of 0.28 mm for UDP-glucose and was highly specific for this sugar donor. More than 20 hydroxybenzoic and hydroxycinnamic acid derivatives were assayed as potential glucose acceptors. UDP-glucose:SA 3-O-glucosyltransferase activity was highly specific toward SA (K_m = 0.16 mm). The enzyme was inhibited by UDP and uridine 5′-triphosphate but not by up to 7.5 mm uridine 5′-monophosphate.     

Enzymic Properties of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Purified from Rice Leaves

The enzymic properties of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase purified from rice (Oryza sativa L.) leaves were studied. Rice RuBPcarboxylase, activated by preincubation with CO₂ and Mg²⁺ like other higher plant carboxylases, had an activation equilibrium constant (K_cK_Mg) of 1.90 × 10⁵ to 2.41 × 10⁵ micromolar² (pH 8.2 and 25°C). Kinetic parameters of carboxylation and oxygenation catalyzed by the completely activated enzyme were examined at 25°C and the respective optimal pHs. The K_m(CO₂), K_m(RuBP), and V_max values for carboxylation were 8 micromolar, 31 micromolar, and 1.79 units milligram⁻¹, respectively. The K_m(O₂), K_m(RuBP), and V_max values for oxygenation were 370 micromolar, 29 micromolar, and 0.60 units milligram⁻¹, respectively.

Comparison of rice leaf RuBP carboxylase with other C₃ plant carboxylases showed that it had a relatively high affinity for CO₂ but the lowest catalytic turnover number (V_max) among the species examined.

     

Cytokinin oxidase from wheat: partial purification and general properties

As part of the study of the possible role(s) of CBF-1, a cytokinin-binding protein abundant in wheat embryo, a cytokinin oxidase was found in wheat (Triticum aestivum L.) germ and partially purified by conventional purification techniques and high performance chromatofocusing. This preparation catalyzes conversion of N⁶-(Δ²-isopentenyl)adenosine to adenosine at a V_max of 0.4 nanomol per milligram protein per minute at 30°C and pH 7.5, the K_m being 0.3 micromolar. This high affinity and the apparent molecular weight of 40,000 estimated by high performance gel permeation on a Spherogel TSK-3000 SW column indicate that this enzyme is different from other cytokinin oxidases previously reported. Oxygen is required for the reaction, as for other cytokinin oxidases already described. N⁶-(Δ²-isopentenyl)adenine and zeatin riboside are also degraded, but N⁶-(Δ²-isopentenyl)adenosine-5′-monophosphate is apparently not a substrate. Benzyladenine is degraded, but to a small extent, and it inhibits slightly the degradation of N⁶-(Δ²-isopentenyl)adenosine. The degradation of N⁶-(Δ²-isopentenyl)adenosine is strongly inhibited by diphenylurea and its highly active derivative N-(2-chloro-4-pyridyl)-N′-phenylurea.     

Transport and metabolism of 1'-fluorosucrose, a sucrose analog not subject to invertase hydrolysis

The novel sucrose derivative 1′-fluorosucrose (α-d-glucopyranosyl-β- d-1-deoxy-1-fluorofructofuranoside) was synthesized in order to help define mechanisms of sucrose entry into plant cells. Replacement of the 1′-hydroxyl by fluorine very greatly reduces invertase hydrolysis of the derivative (hydrolysis at 10 millimolar 1′-fluorosucrose is less than 2% that of sucrose) but does not reduce recognition, binding, or transport of 1′-fluorosucrose by a sucrose carrier. Transport characteristics of 1′-fluorosucrose were studied in three different tissues. The derivative is transported by the sucrose carrier in the plasmalemma of developing soybean cotyledon protoplasts with a higher affinity than sucrose (K_m 1′-fluorosucrose 0.9 millimolar, K_m sucrose 2.0 millimolar). 1′-Fluorosucrose is a competitive inhibitor of sucrose uptake with an apparent K_i also of 0.9 millimolar, while the K_i of sucrose competition of 1′-fluorosucrose uptake was 2.0 millimolar. Thus, both sugars are recognized at the same binding site in the plasmalemma. Both sucrose and 1′-fluorosucrose show very similar patterns of phloem translocation from an abraded leaf surface through the petiole indicating that recognition of 1′-fluorosucrose by sucrose carriers involved in phloem loading is likely as well.     

Identification of a 5′-Deoxyadenosine Deaminase in Methanocaldococcus jannaschii and Its Possible Role in Recycling the Radical S-Adenosylmethionine Enzyme Reaction Product 5′-Deoxyadenosine

We characterize here the MJ1541 gene product from Methanocaldococcus jannaschii, an enzyme that was annotated as a 5′-methylthioadenosine/S-adenosylhomocysteine deaminase (EC 3.5.4.31/3.5.4.28). The MJ1541 gene product catalyzes the conversion of 5′-deoxyadenosine to 5′-deoxyinosine as its major product but will also deaminate 5′-methylthioadenosine, S-adenosylhomocysteine, and adenosine to a small extent. On the basis of these findings, we are naming this new enzyme 5′-deoxyadenosine deaminase (DadD). The K_m for 5′-deoxyadenosine was found to be 14.0 ± 1.2 μM with a k_cat/K_m of 9.1 × 10⁹ M⁻¹ s⁻¹. Radical S-adenosylmethionine (SAM) enzymes account for nearly 2% of the M. jannaschii genome, where the major SAM derived products is 5′-deoxyadenosine. Since 5′-dA has been demonstrated to be an inhibitor of radical SAM enzymes; a pathway for removing this product must be present. We propose here that DadD is involved in the recycling of 5′-deoxyadenosine, whereupon the 5′-deoxyribose moiety of 5′-deoxyinosine is further metabolized to deoxyhexoses used for the biosynthesis of aromatic amino acids in methanogens.     

Partial Purification and Characterization of Indol-3-Ylacetylglucosemyo-Inositol Indol-3-Ylacetyltransferase (Indoleacetic Acid-Inositol Synthase)

A procedure is described for the purification of the enzyme indol-3-ylacetylglucose:myo-inositol indol-3-ylacetyltransferase (IAA-myo-inositol synthase). This enzyme catalyzes the transfer of indol-3-ylacetate from 1-0-indol-3-ylacetyl-β-d-glucose to myo-inositol to form indol-3-ylacetyl-myo-inositol and glucose. A hexokinase or glucose oxidase based assay system is described. The enzyme has been purified approximately 16,000-fold, has an isoelectric point of pH 6.1 and yields three catalytically inactive bands upon acrylamide gel electrophoresis of the native protein. The enzyme shows maximum transferase activity with myo-inositol but shows some transferase activity with scyllo-inositol and myo-inosose-2. No transfer of IAA occurs with myo-inositol-d-galactopyranose, cyclohexanol, mannitol, or glycerol as acyl acceptor. The affinity of the enzyme for 1-0-indol-3-ylacetyl-β-d-glucose is, K_m = 30 micromolar, and for myo-inositol is, K_m = 4 millimolar. The enzyme does not catalyze the exchange incorporation of glucose into IAA-glucose indicating the reaction mechanism involves binding of IAA glucose to the enzyme with subsequent hydrolytic cleavage of the acyl moiety by the hydroxyl of myo-inositol to form IAA myo-inositol ester.     

Thiolase from Clostridium acetobutylicum ATCC 824 and Its Role in the Synthesis of Acids and Solvents

Thiolase (acetyl-coenzyme A [CoA] acetyltransferase, E.C. 2.3.1.19) from Clostridium acetobutylicum ATCC 824 has been purified 70-fold to homogeneity. Unlike the thiolase in Clostridium pasteurianum, this thiolase has high relative activity throughout the physiological range of internal pH of 5.5 to 7.0, indicating that change in internal pH during acid production is not an important factor in the regulation of this thiolase. In the condensation direction, the thiolase is inhibited by micromolar levels of CoA, and this may be an important factor in modulating the net condensation of acetyl-CoA to acetoacetyl-CoA. Other cofactors and metabolites that were tested and shown to be inhibitors are ATP and butyryl-CoA. The native enzyme consists of four 44,000-molecular-weight subunits. The kinetic binding mechanism is ping-pong. The K_m value for acetyl-CoA is 0.27 mM at 30°C and pH 7.4. The K_m values for sulfhydryl-CoA and acetoacetyl-CoA are, respectively, 0.0048 and 0.032 mM at 30°C and pH 8.0. The active site apparently contains a sulfhydryl group, but unlike other thiolases, this thiolase is relatively stable in the presence of 5,5′-dithiobis(2-nitrobenzoic acid). Studies of thiolase specific activity under various types of continuous fermentations show that regulation of this enzyme at both the genetic and enzyme levels is important.