Interaction of pyridoxal phosphate with the amino groups at the active site of 5- aminolevulinic acid dehydratase in maize

Pyridoxal 5′-phosphate strongly and reversibly inhibited maize leaf 5-amino levulinic acid dehydratase. The inhibition was linearly competitive with respect to the substrate 5-aminolevulinic acid at pH values between 7 to 9.0. Pyridoxal was also effective as an inhibitor of the enzyme but pyridoxamine phosphate was not inhibitory. The results suggest that pyridoxal 5′-phosphate may be interacting with the enzyme either close to or at the 5-aminolevulinic acid binding site. This conclusion was further corroborated by the detection of a Schiff base between the enzyme and the substrate, 5-aminolevulinic acid and by reduction of pyridoxal phosphate and substrate complexes with sodium borohydride     

<Emphasis Type="Italic">Aspergillus niger</Emphasis> metabolism of citrus furanocoumarin inhibitors of human cytochrome P450 3A4

Fungi metabolize polycyclic aromatic hydrocarbons by a number of detoxification processes, including the formation of sulfated and glycosidated conjugates. A class of aromatic compounds in grapefruit is the furanocoumarins (FCs), and their metabolism in humans is centrally involved in the “grapefruit/drug interactions.” Thus far, the metabolism by fungi of the major FCs in grapefruit, including 6′, 7′-epoxybergamottin (EB), 6′, 7′-dihydroxybergamottin (DHB), and bergamottin (BM), has received little attention. In this study, Aspergillus niger was observed to convert EB into DHB and a novel water-soluble metabolite (WSM). Bergaptol (BT) and BM were also metabolized by A. niger to the WSM, which was identified as BT-5-sulfate using mass spectrometry, UV spectroscopy, chemical hydrolysis, and ¹H and ¹³C nuclear magnetic resonance spectroscopy. Similarly, the fungus had a capability of metabolizing xanthotoxol (XT), a structural isomer of BT, to a sulfated analog of BT-5-sulfate, presumably XT-8-sulfate. A possible enzyme-catalyzed pathway for the grapefruit FC metabolism involving the cleavage of the geranyl group and the addition of a sulfate group is proposed.     

Formation of aerial hyphae and conidia under orthophosphate-limited conditions inNeurospora crassa: Role of repressible cyclic phosphodiesterase

A wild type strain ofNeurospora crassa produced aerial hyphae and luxuriant conidia in standing culture in low phosphate liquid media.nuc-1 andnuc-2, which have no ability to derepress repressible cyclic phosphodiesterase (cPDase) (3′; 5′-cyclic AMP 5′-nucleotidohydrolase, EC 3.1.4.17) and several other repressible enzymes, did not form them. Heterocaryon between them restored the abilities not only to produce aerial hyphae and conidia but also to produce cPDase. Revertants fromnuc-1 and a mutant in alkaline phosphatase,pho-2, produced aerial hyphae and conidia in low phosphate condition, whereas a mutant in cPDase,pho-3, produced only a limited amount of them. In media containing low levels of 2′, 3′-cAMP, the wild type, the revertants fromnuc-1, pho-2 andpho-3 produced aerial hyphae and conidia in abundance, whereas in media containing 3′, 5′-cAMP these strains produced no or only limited amounts of them. In low phosphate medianuc-1, nuc-2 andpho-3 showed higher levels of 3′, 5′-cAMP as compared with those strains which have the ability to derepress cPDase. The cPDase activities in crude mycelial extracts fromnuc-1 andpho-3 grown in low phosphate media were 5.6 and 17.5% of that ofpho-2 when assayed for 3′,5′-cAMP at an intracellular level of 2 μM.     

Apyrase and 5′-nucleotidase Activities in Synaptosomes from the Cerebral Cortex of Rats Experimentally Demyelinated with Ethidium Bromide and Treated with Interferon-β

Apyrase and 5′-nucleotidase activities were analyzed in an ethidium bromide (EB) demyelinating model associated with interferon-β (IFN-β). The animals were divided in groups: I, control (saline); II, saline and IFN-β; III, EB and IV, EB and IFN-β. After 7, 15 and 30 days the animals (n=5) were sacrificed and the cerebral cortex was removed for synaptosome preparation and enzymatic assays. Apyrase activity using ATP as substrate increased in groups II, III and IV (P<0.001) after 7 days and in groups III and IV (P<0.001) after 15 days. Using ADP as substrate, an activation of this enzyme was observed in group III (P<0.05) after seven and 15 days. The 5′-nucleotidase activity increased in group III (P<0.05) after 7 days and in groups II, III and IV (P<0.001) after 15 days. After 30 days treatment, no significant alteration was observed in enzyme activities. Results showed that apyrase and 5′-nucleotidase activities are altered in demyelination events and that IFN-β was able to regulate the adenine nucleotide hydrolysis.     

Two modes of sulfite oxidation in the extremely thermophilic and acidophilic archaeon Acidianus ambivalens

Sulfite-oxidizing enzyme activities were analyzed in cell-free extracts of aerobically grown cells of Acidianus ambivalens, an extremely thermophilic and chemolithoautotrophic archaeon. In the membrane and cytoplasmic fractions, two distinct enzyme activities were found. In the membrane fraction, a sulfite:acceptor oxidoreductase activity was found [530 mU (mg protein)^–1; apparent K _m for sulfite, 3.6 mM]. In the cytoplasmic fraction the following enzyme activities were found and are indicative of an oxidative adenylylsulfate pathway: adenylylsulfate reductase [138 mU (mg protein)^–1], adenylylsulfate:phosphate adenyltransferase [“ADP sulfurylase”; 86 mU (mg protein)^–1], adenylate kinase [650 mU (mg protein)^–1], and rhodanese [thiosulfate sulfur transferase, 9.2 mU (mg protein)^–1]. In addition, 5′,5′′′-P¹,P⁴-di(adenosine-5′) tetraphosphate (Ap₄A) synthase and Ap₄A pyrophosphohydrolase activities were detected. Received: 17 August 1998 / Accepted: 29 April 1999     

Cis-diammineplatinum(II) forms a macrochelate with 2′-deoxycytidine 5′-monophosphate (dCMP2–)! Reactivity and acid-base properties of cis-Pt(NH3)2(dCMP)

 The synthesis of cis-Pt(NH₃)₂(dCMP) is reported and by various physico-chemical methods it is demonstrated that it is a macrochelate in which Pt(II) is bound simultaneously to the N3 site of cytosine in dCMP^2– and to a phosphate-oxygen atom. According to the NOESY spectra (cross-peaks between cytosine H6 and H2′ and H3′) the cytosine ring adopts an anti orientation. Highly unusual is the significant (1 ppm) downfield shift of the sugar proton H5″ in the ¹H-NMR spectrum and the sensitivity of the cytosine H6 resonance on the protonation state of the phosphate group. Based on these three features a geometry for the macrochelate is proposed. The compound is a major product of the reaction of cis-[Pt(NH₃)₂(H₂O)₂]²⁺ with dCMP^2– at neutral pH, but it even forms at pH 5. By applying pD-dependent NMR spectroscopy (¹H, ³¹P) and potentiometric pH titration, it is demonstrated that the Pt-coordinated phosphate group can be protonated (pK _a/1=3.21±0.10 and 3.31±0.05, respectively), and ¹H- and ³¹P-NMR spectra also indicate deprotonation (pK _a/2=13.35±0.25) of the exocyclic amino group of the cytosine moiety. The metal ion binding affinity of cis-Pt(NH₃)₂(dCMP) is very small, as shown for Cu²⁺ (log K<0.6). The cis-Pt(NH₃)₂(dCMP) complex reacts with nucleosides and nucleotides (L′) by losing its chelate structure and forming mixed ligand complexes, cis-Pt(NH₃)₂(dCMP)(L′); this means that the phosphate group is released from the coordination sphere of Pt(II), indicating that the Pt(II)-O(phosphate) bond is not very strong. Received: 23 October 1997 / Accepted: 17 February 1998     

Preference for Internucleotide Linkages as a Function of the Number of Constituents in a Mixture

Phosphoimidazolide-activated ribomononucleotides (*pN; see Scheme I) are useful substrates for the nonenzymatic synthesis of oligonucleotides. In the presence of metal ions dilute neutral aqueous solutions of *pN (0.01 M) typically yield only small amounts of dimers and traces of oligomers; most of *pN hydrolyzes to yield nucleoside 5′-monophosphate (5′NMP). An earlier investigation of *pN reactions in highly concentrated aqueous solutions (up to 1.4 M) showed, as expected, that the percentage yield of the condensation products increases and the yield of the hydrolysis product correspondingly decreases with *pN concentration (Kanavarioti 1997). Here we report product distributions in reactions with one, two, or three reactive components at the same total nucleotide concentration. *pN used as substrates were the nucleoside 5′-phosphate 2-methylimidazolides, 2-MeImpN, with N= cytidine (C), uridine (U), or guanosine (G). Reactions were conducted as self-condensations, i.e., one nucleotide only, with two components in the three binary U,C, U,G, and C,G mixtures, and with three components in the ternary U,C,G mixture. The products are 5′NMP, 5′,5′-pyrophosphate-, 2′,5′-, 3′,5′-linked dimers, cyclic dimers, and a small percentage of longer oligomers. The surprising finding was that, under identical conditions, including the same total monomer concentration, the product distribution differs substantially from one reaction to another, most likely due to changing intermolecular interactions depending on the constituents. Even more unexpected was the observed trend according to which reactions of the U,C,G mixture produce the highest yield of internucleotide-linked dimers, whereas the self-condensations produce the least and the reactions with the binary mixtures produce yields that fall in between. What is remarkable is that the approximately two-fold increase in the percentage yield of internucleotide-linked dimers is not due to a concentration effect or a catalyst, but to the increased complexity of the system from a single to two and three components. These observations, perhaps, provide an example of how increased complexity in relatively simple chemical systems leads to organization of the material and consequently to chemical evolution. A possible link between prebiotic chemistry and the postulated RNA world is discussed. Received: 12 September 1997 / Accepted: 24 November 1997     

A new 5′ terminal murine GAPDH exon identified using 5′RACE LaNe

In this work, a ligation-independent, fully gene-specific, nested polymerase chain reaction (PCR) method for the elucidation of 5′ cDNA sequence is described and demonstrated for the first time. Two manifestations of the method, rapid amplification of cDNA ends (RACE) by lariat-dependent nested PCR 5′ (RACE LaNe), at least as simple to perform as conventional RACE, were successfully applied to the murine housekeeping genes phosphoglycerate kinase 1 (PGK1), β-actin (β-ACT), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the alpha thalassemia mental retardation Y homolog (ATRY) gene of the marsupial, Macropus eugenii. Significantly, a new murine GAPDH 5′ exon, separated by 365 kb of intronic sequence from previously annotated GAPDH sequence, was discovered using 5′RACE LaNe.     

Role of cyclic nucleotides in pigment translocation within the freshwater shrimp, Macrobrachium potiuna, erythrophore

The participation of cyclic nucleotide-dependent intracellular signalling pathways in the pigment translocation induced by pigment-dispersing hormone (α -PDH) or pigment-concentrating hormone (PCH) was investigated in the erythrophores of the freshwater shrimp, Macrobrachium potiuna. Cholera toxin, forskolin and dibutyryl cyclic adenosine 3′5′ monophosphate (dbcAMP) were able to induce pigment dispersion with effective agonist concentrations for half maximal response (EC₅₀s) of 2.8 · 10⁻¹¹ mol · l⁻¹, 7.0 · 10⁻⁷ mol · l⁻¹ and 3.3 · 10⁻⁷ mol · l⁻¹, respectively. KT5720 (10⁻⁷ mol · l⁻¹ and 10⁻⁶ mol · l⁻¹) significantly shifted the dose response curve to α -PDH to the right. Dibutyryl cyclic guanosine 3′5′ monophosphate (dbcGMP) was ineffective in inducing either pigment aggregation or dispersion. 2′5′ dideoxyadenosine (DDA) and SQ22,536 essentially elicit a pigment-aggregating response in a dose-dependent manner. These effects were not due to the activation of purinergic receptors, since concentrations up to 10⁻⁴ mol · l⁻¹ of adenosine and adenosine triphosphate (ATP), and up to 10⁻³ mol · l⁻¹ of uracil triphosphate (UTP) did not elicit pigment aggregation. In order to verify if PCH decreased cyclic adenosine 3′5′ monophosphate (cAMP) levels, cumulative dose-response curves to PCH in the absence and presence of pertussis toxin and 8-MOM-IBMX were determined. However, neither drug significantly affected PCH activity. The levels of cAMP in the integument cells of M. potiuna were significantly increased (P < 0.05) by α -PDH (10⁻⁷ mol · l⁻¹) and forskolin (10⁻⁶ mol · l⁻¹), but were not affected by PCH (10⁻⁷ or 10⁻¹⁰ mol · l⁻¹). In conclusion, α -PDH seems to elicit pigment dispersion through the activation of a Gs-protein coupled receptor resulting in cAMP increase and cAMP-dependent protein kinase (PKA) activation. Furthermore, although a decrease in cAMP was assumed to be responsible in turn for the action of PCH, such a decrease could not be directly demonstrated. Accepted: 11 August 1998     

Sequence analysis of the gene encoding a novel l-2,4-diaminobutyrate decarboxylase of Acinetobacter baumannii: similarity to the group II amino acid decarboxylases

The gene (ddc) encoding a novel enzyme, l-2,4-diaminobutyrate decarboxylase (DABA-DC; EC 4.1.1.-) in Acinetobacter baumannii was sequenced, and an open reading frame of 1,530 nucleotides was detected. The sequence of 20 N-terminal amino acids of purified DABA-DC and of its proteolytic peptide fragments coincided with those deduced from the nucleotide sequence determined. Comparison of the predicted amino acid sequence of the A. baumannii enzyme with those of other pyridoxal 5′-phosphate-dependent decarboxylases revealed significant similarity to the group II amino acid decarboxylases and conservation of the putative pyridoxal 5′-phosphate-binding domain. Received:20 February 1996 / Accepted 15 April 1996     

Specificity of recognition sequence forEscherichia coli primase

Summary We have surveyed the frequency of each of 64 trinucleotide permutations at every nucleotide frame located from 1 to 15 nucleotides upstream of primer RNA-DNA transition sites mapped within a 1.5 kb region of the bacteriophage lambda genome and a 1.4 kb region of theEscherichia coli genome. We have demonstrated that in both systems initiation of DNA synthesis strongly correlates with a CAG sequence located 11 nucleotides upstream of the DNA start sites. Based on the examination of various reports of the priming reaction catalyzed byE. coli primase in vivo and in vitro, we propose that (i)E. coli primase itself recognizes a 3′GTC 5′ sequence on the template strand, (ii) DnaB helicase releases the specificity ofE. coli primase and, (iii) the consensus recognition sequence forE. coli primase associated with DnaB helicase is 3′PuPyPy 5′.     

Cis-2′, 3′-Dihydrodiol production on flavone B-Ring by Biphenyl Dioxygenase from Pseudomonas pseudoalcaligenes KF707 expressed in Escherichia coli

Escherichia coli JM109 strains expressing either toluene dioxygenase from Pseudomonas putida F1 or biphenyl dioxygenase from Pseudomonas pseudoalcaligenes KF707 were examined for their ability to catalyze flavones. Biphenyl dioxygenase produced metabolites from flavone and 5,7-dihydroxyflavone which were not found in the control experiments. The absorption maxima of UV-visible spectra for the metabolites from flavone and 5,7-dihydroxyflavone were found at 337 and 348 nm respectively by using a photodiode array detector in the HPLC. Liquid chromatography/mass spectroscopy (LC/MS) showed molecular weights 256 and 288 for the metabolites, respectively. The metabolite of flavone, which was isolated and purified from the bacterial culture, was further subjected to analysis by ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy. Based on the LC/MS and NMR results, biphenyl dioxygenase inserted oxygen at C2′ and C3′ on the B-ring of flavone, resulting in the formation of flavone cis-2′, 3′-dihydrodiol (2-[3,4-dihydroxy-1.5-cyclohexadienyl]-4H-chromen-4-one). Since this product is not found in Chemical Abstracts, this compound is considered a novel one. In addition, biotransformation of flavones by biphenyl dioxygenase suggested a potential role of bacterial dioxygenase to synthesize novel compounds from plant secondary metabolites. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of dephosphorylated 2′,5′-trioligoadenylate on the entry of sodium ions into human neuroblastoma cells

Using a radioisotope technique, we studied the effect of dephosphorylated 2′,5′-trioligoadenylate (2′,5′ ApApA) on the entry of sodium ions into cultured human neuroblastoma cells (IMR 32 strain). Short-term (nearly 1 h) action of 2′,5′ ApApA did not influence the entry of sodium ions through voltage-operated sodium channels in the absence of neurotoxins modulating the characteristics of these channels (toxin of a scorpion, Leiurus quinquestriatus, + veratrine). At the same time, 2′,5′ ApApA weakened in a dose-dependent manner the entry of sodium ions through sodium channels opened upon the action of the above neurotoxins. In cells cultured for 22 h in a medium containing 5 · 10⁻⁶ M 2′,5′ ApApA, the entry of sodium ions in the absence of neurotoxins was 25% greater, while in the presence of neurotoxins it was 24% smaller than that in the control cells. Tetrodotoxin (TTX, 4 · 10⁻⁷ M) blocked completely sodium entry through sodium channels in cells cultured in the absence of 2′,5′ ApApA, while in cells cultured in the presence of this adenylate TTX decreased the entry by 64%. It is hypothesized that long-lasting action of 2′,5′ ApApA results in the appearance of voltage-operated TTX-insensitive sodium channels in the plasma membrane of IMR 32 cells. Our data show that 2′,5′ ApApA is capable of modulating to a considerable extent the functioning of mechanisms controlling transport of sodium ions in cells of human neuroblastoma cells of the IMR 32 strain. Neirofiziologiya/Neurophysiology, Vol. 40, No. 1, pp. 3–8, January–February, 2008.     

Complementation of an <Emphasis Type="Italic">E. coli</Emphasis> cysteine auxotrophic mutant for the structural modification study of 3′(2′),5′-bisphosphate nucleotidase

The Arabidopsis AHL gene encodes a 3′(2′),5′-bisphosphate nucleotidase (BPNTase) involved in the reductive sulfate activation pathway. A bacterial expression vector containing AHL cDNA was randomly mutagenized with hydroxylamine and transformed into the E. coli cysteine auxotrophic mutant cysQ. Bacterial colonies that did not show evidence of complementation, i.e. those that exhibited slower growth on cysteine-free medium, were selected for further study. Sequencing of the AHL cDNA in one such clone revealed the conversion of cytosine 635 (C₆₃₅) to thymine, resulting in an Alanine (A₂₁₂) to Valine substitution. This microbial complementation procedure is useful in BPNTase structure-activity studies for biotechnological applications.     

Purification and characterization of UDP-glucose: anthocyanin 3′,5′-<Emphasis Type="Italic">O</Emphasis>-glucosyltransferase from <Emphasis Type="Italic">Clitoria ternatea</Emphasis>

A UDP-glucose: anthocyanin 3′,5′-O-glucosyltransferase (UA3′5′GT) (EC 2.4.1.-) was purified from the petals of Clitoria ternatea L. (Phaseoleae), which accumulate polyacylated anthocyanins named ternatins. In the biosynthesis of ternatins, delphinidin 3-O-(6″-O-malonyl)-β-glucoside (1) is first converted to delphinidin 3-O-(6″-O-malonyl)-β-glucoside-3′-O-β-glucoside (2). Then 2 is converted to ternatin C5 (3), which is delphinidin 3-O-(6″-O-malonyl)-β-glucoside-3′,5′-di-O-β-glucoside. UA3′5′GT is responsible for these two steps by transferring two glucosyl groups in a stepwise manner. Its substrate specificity revealed the regioselectivity to the anthocyanin′s 3′- or 5′-OH groups. Its kinetic properties showed comparable k _cat values for 1 and 2, suggesting the subequality of these anthocyanins as substrates. However, the apparent K _m value for 1 (3.89 × 10⁻⁵ M), which is lower than that for 2 (1.38 × 10⁻⁴ M), renders the k _cat/K _m value for 1 smaller, making 1 catalytically more efficient than 2. Although the apparent K _m value for UDP-glucose (6.18 × 10⁻³ M) with saturated 2 is larger than that for UDP-glucose (1.49 × 10⁻³ M) with saturated 1, the k _cat values are almost the same, suggesting the UDP-glucose binding inhibition by 2 as a product. UA3′5′GT turns the product 2 into a substrate possibly by reversing the B-ring of 2 along the C2-C1′ single bond axis so that the 5′-OH group of 2 can point toward the catalytic center. K. Kogawa, N. Kato, K. Kazuma, and N. Noda contributed equally to this work.     

Alginate promotes production of various enzymes by Catharanthus roseus cells

When 1.0 g/l of alginate was added to a Catharanthus roseus L. cell culture, many proteins were released from the cells as detected by SDS polyacrylamide gel electrophoresis. In particular, production and/or release of 5′-phosphodiesterase (5′-PDase), catalase and chitinase by C. roseus L. cells were promoted by the addition of alginate. The promotive effects of alginate on 5′-PDase production were observed for various C. roseus cell lines and similar results were obtained when different alginates with various mannuronate/guluronate ratios and viscosities were used. In contrast, agar, agarose, and chitosan did not promote 5′-PDase production. The promotion of 5′-PDase production was not due to cell mutation, the alginate acted rather as a kind of elicitor. During 82 subcultures (577 days) in Murashige and Skoog medium containing 1.0 g/l of alginate, production and release of 5′-PDase by C. roseus L. cells were promoted without inhibition of cell growth. Received: 27 February 1997 / Revision received: 10 July 1997 / Accepted: 20 July 1997     

Divalent cations modify adsorption of 5′-AMP onto precipitated calcium phosphate: A model for cation modulation of adsorptive processes in primitive aqueous environments

The adsorption of 5′-AMP onto precipitated calcium phosphate (CaPi) requires the presence of soluble calcium and this dependence exhibits a Michaelian-like behavior. This result suggests that the formation of a complex between 5′-AMP and free Ca²⁺ (CaAMP) is a prelude to the adsorption of the nucleotide in the solid matrix. At concentrations one order of magnitude higher, Mn²⁺ and Mg²⁺ can substitute for soluble Ca²⁺ in the adsorption of 5′-AMP onto solid CaPi. However, when added simultaneously with 5′-AMP to a heterogeneous mixture that contains CaPi and soluble Ca²⁺, Mn²⁺ and Mg²⁺ inhibit the adsorption of 5′-AMP in a concentration-dependent manner. This suggests the formation of complexes that are much less effective for 5′-AMP adsorption than the CaAMP complex. On the other hand, Mn²⁺ and Mg²⁺ cannot promote desorption of the nucleotide attached to the precipitate in the presence of soluble Ca²⁺ if they are added after adsorption has attained equilibrium. Although desorption of 5′-AMP can be obtained by a sequential dilution of the soluble phase with buffer and no nucleotide in a process that obeys a Langmuir equation, the lack of effect of Mn²⁺ or Mg²⁺ when adsorption has attained its maximal value suggests strong interactions between the CaAMP complex and the solid matrix when adsorption equilibrium is reached. The divalent cations present in the matrix also participate with different selectivity in the attachment of the CaAMP complex, indicating that a cation-exchange mechanism could have acted in the modulation of adsorptive/desorptive processes involving biomonomers and phosphate surfaces in primitive aqueous environments. Received: 11 December 1995 / Accepted: 5 April 1996     

Flower colour and cytochromes P450

Flavonoids are major constituents of flower colour. Plants accumulate specific flavonoids and thus every species often exhibits a limited flower colour range. Three cytochromes P450 play critical roles in the flavonoid biosynthetic pathway. Flavonoid 3′-hydroxylase (F3′H, CYP75B) and flavonoid 3′,5′-hydroxylase (F3′5′H, CYP75A) catalyze the hydroxylation of the B-ring of flavonoids and are necessary to biosynthesize cyanidin-(red to magenta) and delphinidin-(violet to blue) based anthocyanins, respectively. Pelargonidin-based anthocyanins (orange to red) are synthesized in their absence. Some species such as roses, carnations and chrysanthemums do not have violet/blue flower colour due to deficiency of F3′5′H. Successful expression of heterologous F3′5′H genes in roses and carnations results in delphinidin production, causing a novel blue/violet flower colour. Down-regulation of F3′H and F3′5′H genes has yielded orange petunia and pink torenia colour that accumulate pelargonidin-based anthocyanins. Flavone synthase II (CYP93B) catalyzes the synthesis of flavones that contribute to the bluing of flower colour, and modulation of FNSII gene expression in petunia and tobacco changes their flower colour. Extensive engineering of the anthocyanin pathway is therefore now possible, and can be expected to enhance the range of flower colours.     

Preparation and characterization of several new immobilized derivatives of pyridoxal 5′-phosphate

Two types of new Sepharose-bound pyridoxal 5′-phosphate, N-immobilized and 3-0-immobilized pyridoxal 5′-phosphate analogues, were prepared by reacting pyridoxal 5′-phosphate with a bromoacetyl derivative of Sepharose 4B in dimethylformamide (50% v/v) and in potassium phosphate buffer (pH 6.0) for approx. 70 h at room temperature in the dark, respectively. The properties of these immobilized pyridoxal 5′-phosphate derivatives including their catalytic activities in the non-enzymatic cleavage reaction of tryptophan were studied in comparison with those of the 6-immobilized pyridoxal 5′-phosphate analogue reported previously by the present authors. The usefulness of these pyridoxal 5′-phosphate analogues in the preparation of immobilized tryptophanase was demonstrated.     

One-pot Microbial Synthesis of 2′-deoxyribonucleoside from Glucose, Acetaldehyde, and a Nucleobase

A one-pot enzymatic synthesis of 2′-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase was established. Glycolysis by baker’s yeast (Saccharomyces cerevisiae) generated ATP which was used to produce d-glyceraldehyde 3-phosphate production from glucose via fructose 1,6-diphosphate. The d-glyceraldehyde 3-phosphate produced was transformed to 2′-deoxyribonucleoside via 2-deoxyribose 5-phosphate and then 2-deoxyribose 1-phosphate in the presence of acetaldehyde and a nucleobase by deoxyriboaldolase, phosphopentomutase expressed in Escherichia coli, and a commercial nucleoside phosphorylase. About 33 mM 2′-deoxyinosine was produced from 600 mM glucose, 333 mM acetaldehyde and 100 mM adenine in 24 h. 2′-Deoxyinosine was produced from adenine due to the adenosine deaminase activity of E. coli transformants.