Formation of lipid-bound oligosaccharides in yeast

Incubation of a membrane fraction from Saccharomyces cerevisiae with UDP-N-acetyl [¹⁴C] glucosamine catalyzes the tranfer of N-acetylglucosamine to an endeenous lipid fraction as well as a methanol-insoluble polymer. The glycolipid was shown to separate into three compounds by thin-layer chromatography. The biosynthesis of two of them could clearly be stimulated by the addition of dolichol monophosphate to the incubation mixture. Evidence is presented that the substances are dolichol pyrophosphate derivatives: dolichol pyrophosphate N-acetylglucosamine and dolichol pyrophosphate di-N-acetylchitobiose. The formation of the chitobiose-containing lipid was increased by reincubation of the glycolipid with non-radioactive UDP-N-acetylglucosamine.The same particulate preparation transferred mannose from GDPmannose to dolichol pyrophosphate di-N-acetylchitobiose, giving rise to a lipid-bound oligosaccharide. Molecular weight determination of the oligosaccharide moiety gave a value of 780, which is consistent with a tetrasaccharide containing two mannose subunits attached to di-N-acetylchitobiose.The methanol-insoluble radioactive product obtained in the presence of UDP-N-acetyl[¹⁴C]glucosamine was transformed by pronase treatment to a large extent into dialyzable material. It is suggested that the glycolipids described serve as intermediates in the glycosylation of yeast mannoproteins.     

Formation of polyprenol-linked mono- and oligosaccharides in Phaseolus aureus

A crude membrane preparation from Phaseolus aureus hypocotyls catalyzes the incorporation of mannose from GDP-[¹⁴C]mannose into a acid labile glycolipid and a methanol insoluble fraction. Addition of dolichyl monophosphate to the incubation mixture stimulated the formation of both the mannolipid and the methanol insoluble endproduct. Thin-layer chromatography of endogenous lipid and of the stimulated lipid fraction revealed that both compounds run identical. Ficaprenyl monophosphate also stimulates the incorporation of mannose; however, the ficaprenyl monophosphate mannose formed is not identical to the endogenous mannolipid. This suggests that the endogenous acceptor has the properties of an α-saturated polyprenyl monophosphate rather than those of the ficaprenyl phosphate type. The same membrane preparation also incorporates N-acetylglucosamine into an acid labile glyolipid as well as into a polymer fraction. Evidence is presented that the N-acetylglucosamine containing lipid consists of a mixture of dolichyl pyrophosphate N-acetylglucosamine and dolichyl pyrophosphate di-N-acetylchitobiose. It seems likely that the two compounds have a precursor-product relationship. Incubation of dolichyl pyrophosphate di-N-acetylchitobiose together with GDP-mannose gives rise to lipid-bound mannosyl-di-N-acetylchitobiose. Radioactivity from either the [¹⁴C]mannolipid or the N-acetyl[¹⁴C]glucosamine containing lipid is incorporated into a methanol insoluble product to 3.4 and 6.3%, respectively; it seems, at least in part, to be a glycoprotein.     

Biosynthesis of monoterpenes: Preliminary characterization of l-endo-fenchol synthetase from fennel (Foeniculum vulgare) and evidence that no free intermediate is involved in the cyclization of geranyl pyrophosphate to the rearranged product

A soluble enzyme preparation from the leaves of fennel (Foeniculum vulgare M.) has been shown to catalyze the cation-dependent cyclization of both geranyl pyrophosphate and neryl pyrophosphate to the bicyclic rearranged monoterpene l-endo-fenchol (R. Croteau, M. Felton, and R. Ronald, 1980 Arch. Biochem. Biophys.200, 524–533). To examine the possible presence of free intermediates between the acyclic precursors and fenchol, and to remove competing cyclase and pyrophosphatase activities, the soluble preparation was partially purified by ammonium sulfate fractionation followed by gel filtration on Sephadex G-150 and ion exchange chromatography on O-diethylaminoethyl-cellulose. Activities for the cyclization of geranyl pyrophosphate and neryl pyrophosphate to fenchol were coincident on Chromatographic fractionation suggesting that the same enzyme was capable of cyclizing both acyclic substrates. No interconversion of the acyclic precursors was detected. Although bornyl pyrophosphate is a free intermediate in the biosynthesis of the related bicyclic monoterpenol borneol, both protein fractionation and isotopic dilution experiments ruled out endo-fenchyl pyrophosphate as a free intermediate in fenchol biosynthesis. Similarly, while construction of the fenchane skeleton was demonstrated to involve the rearrangement of an intermediate pinane skeleton, isotopic dilution experiments ruled out both optical antipodes of α-pinene, β-pinene, cis-2-pinanol, trans-2-pinanol, and the corresponding 2-pinyl pyrophosphates as free intermediates of the enzyme-catalyzed reaction. Furthermore, exhaustive search of the enzymatic reaction products provided no evidence to suggest the involvement of any free intermediate between the acyclic precursor and fenchol. The endo-fenchol synthetase has an apparent molecular weight of 60,000, shows a pH optimum near 7.0, and requires Mn²⁺ (1 mm) for catalytic activity. Co²⁺ can partially substitute for Mn²⁺, but other divalent cations are ineffective. The partially purified synthetase is inhibited by p-hydroxymercuribenzoate and by phenylglyoxal, and it exhibits a preference for geranyl pyrophosphate over neryl pyrophosphate as substrate. An integrated scheme is proposed for the cyclization and rearrangement catalyzed by fenchol synthetase.     

On the metabolic activation of the carcinogen N-hydroxy-N-2-acetylaminofluorene : III. Oxidation with horseradish peroxidase to yield 2-nitrosofluorene and N-acetoxy-N-2-acetylaminofluorene

Previous studies have shown that the carcinogen N-hydroxy-2-acetylaminofluorene is converted by one-electron oxidants to a free nitroxide radical which dismutates to N-acetoxy-2-acetylaminofluorene and 2-nitrosofluorene. The present study shows that the same oxidation can be achieved with horseradish peroxidase and H₂O₂. The free radical intermediate was detected by its ESR signal, and the yields of N-acetoxy-2-acetylaminofluorene and of 2-nitrosofluorene were determined under a number of conditions. Addition of tRNA to the reaction mixture containing N-acetoxy-N-2-acetyl[2′-³H]aminofluorene yielded tRNA-bound radioactivity; addition of guanosine yielded a reaction product which appears to be N-guanosin-8-yl)-2-acetylaminofluorene. The latter compound has previously been identified as a reaction product of N-acetoxy-2-acetylaminofluorene and guanosine. Preliminary attempts to demonstrate the formation of a nitroxide free radical or its dismutation products with rat liver mixed function oxidase systems were not successful.     

Structural insights into the histidine trimethylation activity of EgtD from Mycobacterium smegmatis

EgtD is an S-adenosyl-l-methionine (SAM)-dependent histidine N,N,N-methyltransferase that catalyzes the formation of hercynine from histidine in the ergothioneine biosynthetic process of Mycobacterium smegmatis. Ergothioneine is a secreted antioxidant that protects mycobacterium from oxidative stress. Here, we present three crystal structures of EgtD in the apo form, the histidine-bound form, and the S-adenosyl-l-homocysteine (SAH)/histidine-bound form. The study revealed that EgtD consists of two distinct domains: a typical methyltransferase domain and a unique substrate binding domain. The histidine binding pocket of the substrate binding domain primarily recognizes the imidazole ring and carboxylate group of histidine rather than the amino group, explaining the high selectivity for histidine and/or (mono-, di-) methylated histidine as substrates. In addition, SAM binding to the MTase domain induced a conformational change in EgtD to facilitate the methyl transfer reaction. The structural analysis provides insights into the putative catalytic mechanism of EgtD in a processive trimethylation reaction.     

High-performance liquid chromatographic analysis of neutral glycosphingolipids as their per-O-benzoyl derivatives

Previous reports from this laboratory (1–4) described the perbenzoylation of neutral glycosphingolipids (GSL)¹ with benzoyl chloride in pyridine and analysis of the perbenzoylated derivatives by high performance liquid chromatography (hplc). A disadvantage of this procedure is that N-benzoylation occurs as well as the desired O-benzoylation. This does not permit recovery of the parent GSL after mild alkaline hydrolysis due to formation of a mixture of N-acylated and N-benzoylated GSLs(1). It has also been demonstrated that the benzoylation with benzoic anhydride in pyridine does not lead to the formation of N-benzoylated products. However, the anhydride reaction is sluggish and the benzoyl chloride method has been the preferred procedure.Gupta et al. (5) used N,N-dimethyl-4 amino pyridine (DMAP) as a catalyst in the acylation of phospholipids by the anhydrides of fatty acids. F. B. Jungalwala (private communication) has shown that this catalyst greatly accelerates the reaction of benzoic anhydride with sulfatides.In this communication we report the preparation and hplc analysis of per-O-benzoyl derivatives of GSLs by reaction with benzoic acid anhydride in the presence of DMAP as a catalyst. Reaction with these reagents avoids amide acylation, forms single products with satisfactory chromatographic properties and parent GSLs can be regenerated by mild alkaline hydrolysis.     

Conversion of [1-3H2,G-14C]geranyl pyrophosphate to cyclic monoterpenes without loss of tritium

Soluble enzyme preparations from Salvia officinalis convert the acyclic precursor [1-³H₂,G-¹⁴C]geranyl pyrophosphate to cyclic monoterpenes of the pinane (α-pinene,β-pinene), isocamphane (camphene), p-menthane (limonene,1,8-cineole), and bornane (bornyl pyrophosphate, determined as borneol) type without loss of tritium, and without significant conversion to other free acyclic intermediates. Similarly, [1-³H₂,G-¹⁴C]geraniol is converted in intact S. officinalis leaves to the cyclic monoterpene olefins and 1,8-cineole, as well as to isothujone and camphor, without loss of tritium from C(1). These results clearly eliminate transcis isomerization of geranyl pyrophosphate to neryl pyrophosphate via aldehyde intermediates prior to cyclization, and they support a scheme whereby the trans precursor is cyclized directly by way of a bound linaloyl intermediate.     

Author index to volume 154

Horseradish peroxidase-catalyzed N-demethylation of aminopyrine and dimethylaniline results in generation of free radical intermediates which can interact with glutathione (GSH) to form a glutathione radical. This can either dimerize to yield glutathione disulfide or react with O₂ to form oxygenated products of glutathione. Ethylmorphine is not a substrate in the peroxidase-mediated reaction, and free radical intermediates which react with GSH, are not formed from aminopyrine and dimethylaniline when the horseradish peroxidase/H₂O₂ system is replaced by liver microsomes and NADPH. Therefore, it appears unlikely that formation of free radical intermediates can be responsible for the depletion of GSH observed during N-demethylation of several drugs in isolated liver cells.     

Affinity labelling of tryptophanyl-transfer RNA synthetase

A double affinity-labelling approach has been developed in order to convert an oligomeric enzyme with multiple active centres into a single-site enzyme.Tryptophanyl-transfer RNA synthetase (EC 6.1.1.2) from beef pancreas is a symmetric dimer, α₂ An ATP analogue, γ-(p-azidoanilide)-ATP does not serve as a substrate for enzymatic aminoacylation of tRNA^Trp but acts as an effective competitive inhibitor in the absence of photochemical reaction, with K₁ = 1 × 10^?3m (K_mfor ATP = 2 × 10^?4m). The covalent photoaddition of azido-ATP³ results in complete loss of enzymatic activity in both the ATP-[³²P]pyrophosphate exchange reaction and tRNA aminoacylation. ATP completely protects the enzyme against inactivation. However, covalent binding of azido-ATP is also observed outside the active centres. The difference between covalent binding of the azido-ATP in the absence and presence of ATP corresponds to 2 moles of the ATP analogue per mole of the enzyme.Two binding sites for tRNA^Trp have been found from complex formation at pH 5.8 in the presence of Mg²⁺. The two tRNA molecules bind, with K_dis = 3.6 × 10^?8m and K_dis = 0.9 × 10^?6m, respectively, pointing to a strong negative co-operativity between the binding sites for tRNA.N-chlorambucilyl-tryptophanyl-tRNA^Trp and TRSase form a complex with K_dis = 5.5 × 10^?8m at pH 5.8 in the presence of 10 mm-Mg²⁺. This value is similar to the value of K_dis for tryptophanyl-tRNA of 4.8 × 10^?8m. Under the same conditions a 1:1 complex (in mol) is formed between the enzyme and Trp-tRNA or N-chlorambucilyl-Trp-tRNA. On incubation, a covalent bond is formed between N-chlorambucilyl-Trp-tRNA and TRSase; 1 mole of affinity reagent alkylates 1 mole of enzyme independently of the concentration of the modifier. The alkylation reaction is completely inhibited by the presence of tRNA^Trp whereas the tRNA devoid of tRNA^Trp does not affect the rate of alkylation. In the presence of either ATP or tryptophan, or a mixture of the two, the alkylation reaction is inhibited even though these ligands have no effect on the complex formation between TRSase and the tRNA analogue. Photoaddition of the azido-ATP completely prevents the reaction of the enzyme with the tRNA analogue, although the non-covalent complex formation is not affected.Exhaustive alkylation of TRSase partially inhibits the reaction of ATP [³²P]pyrophosphate exchange and completely blocks the aminoacylation of tRNA^Trp. Cleavage of the tRNA which is covalently bound to TRSase restores both the ATP-[³²P]pyrophosphate exchange and aminoacylation activity.The TRSase which is covalently-bound to R-Trp-tRNA is able to incorporate only one ATP molecule per dimeric enzyme into the active centre. This doubly modified enzyme is completely enzymatically inactive. Removal of the tRNA residue from the doubly modified enzyme results in the formation of the derivative with one blocked ATP site. Therefore, a “single-site” TRSase may be generated either by alkylation of the enzyme with Cl-R-Trp-tRNA or after the removal of covalently bound tRNA from the doubly labelled protein.Tryptophanyl-tRNA synthetase containing blocked ATP and/or tRNA binding site(s) seems to bo a useful tool for investigation of negative co-operativity and may help in the elucidation of the structure function relationships between the active centres.     

Theoretical insight into the hydroxylamine oxidoreductase mechanism

The multiheme enzyme hydroxylamine oxidoreductase from the autotrophic bacteria Nitrosomonas europaea catalyzes the conversion of hydroxylamine to nitrite, with a complicate arrangement of heme groups in three subunits. As a distinctive feature, the protein has a covalent linkage between a tyrosyl residue of one subunit and a meso carbon atom of the heme active site of another. We studied the influence of this bond in the catalysis from a theoretical perspective through electronic structure calculations at the density functional theory level, starting from the crystal structure of the protein. Geometry optimizations of proposed reaction intermediates were used to calculate the dissociation energy of different nitrogen containing ligands, considering the presence and absence of the meso tyrosyl residue. The results indicate that the tyrosine residue enhances the binding of hydroxylamine, and increases the stability of a Fe^IIINO intermediate, while behaving indifferently in the Fe^IINO form. The calculations performed on model systems including neighboring aminoacids revealed the probable formation of a bidentate hydrogen bond between the Fe^IIIH₂O complex and Asp 257, in a high-spin aquo complex as the resting state. Characterization of non-planar heme distortions showed that the meso-substituent induces significant ruffling in the evaluated intermediates.     

Specific Interactions of Clausin, a New Lantibiotic, with Lipid Precursors of the Bacterial Cell Wall

We investigated the specificity of interaction of a new type A lantibiotic, clausin, isolated from Bacillus clausii, with lipid intermediates of bacterial envelope biosynthesis pathways. Isothermal calorimetry and steady-state fluorescence anisotropy (with dansylated derivatives) identified peptidoglycan lipids I and II, embedded in dodecylphosphocholine micelles, as potential targets. Complex formation with dissociation constants of ∼0.3 μM and stoichiometry of ∼2:1 peptides/lipid intermediate was observed. The interaction is enthalpy-driven. For the first time, to our knowledge, we evidenced the interaction between a lantibiotic and C₅₅-PP-GlcNAc, a lipid intermediate in the biosynthesis of other bacterial cell wall polymers, including teichoic acids. The pyrophosphate moiety of these lipid intermediates was crucial for the interaction because a strong binding with undecaprenyl pyrophosphate, accounting for 80% of the free energy of binding, was observed. No binding occurred with the undecaprenyl phosphate derivative. The pentapeptide and the N-acetylated sugar moieties strengthened the interaction, but their contributions were weaker than that of the pyrophosphate group. The lantibiotic decreased the mobility of the pentapeptide. Clausin did not interact with the water-soluble UDP-MurNAc- and pyrophosphoryl-MurNAc-pentapeptides, pointing out the importance of the hydrocarbon chain of the lipid target.     

The crystal structures of ornithine carbamoyltransferase from Mycobacterium tuberculosis and its ternary complex with carbamoyl phosphate and L-norvaline reveal the enzyme's catalytic mechanism

Mycobacterium tuberculosis ornithine carbamoyltransferase (Mtb OTC) catalyzes the sixth step in arginine biosynthesis; it produces citrulline from carbamoyl phosphate (CP) and ornithine (ORN). Here, we report the crystal structures of Mtb OTC in orthorhombic (form I) and hexagonal (form II) space groups. The molecules in form II are complexed with CP and l-norvaline (NVA); the latter is a competitive inhibitor of OTC. The asymmetric unit in form I contains a pseudo hexamer with 32 point group symmetry. The CP and NVA in form II induce a remarkable conformational change in the 80s and the 240s loops with the displacement of these loops towards the active site. The displacement of these loops is strikingly different from that seen in other OTC structures. In addition, the ligands induce a domain closure of 4.4° in form II. Sequence comparison of active-site residues of Mtb OTC with several other OTCs of known structure reveals that they are virtually identical. The interactions involving the active-site residues of Mtb OTC with CP and NVA and a modeling study of ORN in the form II structure strongly rule out an earlier proposed mechanistic role of Cys264 in catalysis and suggest a possible mechanism for OTC. Our results strongly support the view that ORN with an already deprotonated N^ε atom is the species that binds to the enzyme and that one of the phosphate oxygen atoms of CP is likely to be involved in accepting a proton from the doubly protonated N^ε atom of ORN. We have interpreted this deprotonation as part of the collapse of the transition state of the reaction.     

cDNA cloning and characterization of human and mouse Ca-independent phosphatidylethanolamine N-acyltransferases

The formation of N-acylphosphatidylethanolamine by N-acylation of phosphatidylethanolamine (PE) is the initial step in the biosynthetic pathway of bioactive N-acylethanolamines, including the endocannabinoid anandamide and the anti-inflammatory substance N-palmitoylethanolamine. We recently cloned a rat enzyme capable of catalyzing this reaction, and referred to the enzyme as Ca²⁺-independent N-acyltransferase (iNAT). Here we report cDNA cloning and characterization of human and mouse iNATs. We cloned iNAT-homologous cDNAs from human and mouse testes, and overexpressed them in COS-7 cells. The purified recombinant proteins abstracted an acyl group from both sn-1 and sn-2 positions of phosphatidylcholine, and catalyzed N-acylation of PE as well as phospholipase A₁/A₂-like hydrolysis. The iNAT activity was mainly detected in soluble rather than particulate fractions, and was only slightly increased by Ca²⁺. These results demonstrated that the human and mouse homologues function as iNAT. As for the organ distribution of iNAT, human testis and pancreas and mouse testis exhibited by far the highest expression level, suggesting its physiological importance in the specific organs. Moreover, mutagenesis studies showed crucial roles of His-154 and Cys-241 of rat iNAT in the catalysis and a possible role of the N-terminal domain in membrane association or protein–protein interaction.     

A dolichol-linked trisaccharide from central nervous tissue: structure and biosynthesis.

Calf brain membranes have previously been shown to enzymatically transfer N-acetyl[¹⁴C]glucosamine from UDP-N-acetyl[¹⁴C]glucosamine into N-acetyl[¹⁴C]glucosami-nylpyrophosphoryldolichol, N,N′-diacetyl[¹⁴C]chitobiosylpyrophosphoryldolichol and a minor labeled product with the chemical and chromatographic properties of a [¹⁴C]trisaccharide lipid (Waechter, C. J., and Harford, J. B. (1977) Arch. Biochem. Biophys.181, 185–198). This paper demonstrates that incubating calf brain membranes containing endogenous, prelabeled N-acetyl[¹⁴C]glucosaminyl lipids with unlabeled GDP-mannose enhances the formation of the [¹⁴C]trisaccharide lipid. The intact [¹⁴C]trisaccharide lipid behaves like a dolichol-bound trisaccharide, in which the glycosyl group is linked via a pyrophosphate bridge, when chromatographed on SG-81 paper or DEAE-cellulose. Mild acid treatment releases a water-soluble product that comigrates with authentic β-Man-(1→4)-β-GlcNAc(1→4)-GlcNAc. The free [¹⁴C]trisaccharide is converted to N,N′-diacetyl[¹⁴C]chitobiose by incubation with a highly purified β-mannosidase. These findings indicate that the trisaccharide lipid formed by calf brain membranes is β-mannosyl-N,N′-diacetylchito-biosylpyrophosphoryldolichol. The two glycosyltransferases responsible for the enzymatic conversion of the N-acetylglucosaminyl lipid to the trisaccharide lipid have been studied using exogenous, purified [¹⁴C]glycolipid substrates. Calf brain membranes enzymatically transfer N-acetylglucosamine from UDP-N-acetylglucosamine to exogenous N-acetyl[¹⁴C] glucosaminylpyrophosphoryldolichol to form [¹⁴C]disaccharide lipid. The biosynthesis of [¹⁴C]disaccharide lipid is stimulated by unlabeled UDP-N-acetylglucosamine under conditions that inhibit N-acetylglucosaminylpyrophosphoryldolichol synthesis. Unlike the formation of N-acetylglucosaminylpyrophosphoryldolichol the enzymatic addition of the second N-acetylglucosamine residue is not inhibited by tunicamycin. Exogenous purified [¹⁴C] disaccharide lipid is enzymatically mannosylated by calf brain membranes to form the [¹⁴C] trisaccharide lipid. The formation of the [¹⁴C]trisaccharide lipid from exogenous [¹⁴C] disaccharide lipid is stimulated by unlabeled GDP-mannose and Mg²⁺, and inhibited by EDTA. Exogenous dolichyl monophosphate is also inhibitory. These results strongly suggest that the calf brain mannosyltransferase involved in the synthesis of the trisaccharide lipid requires a divalent cation and utilizes GDP-mannose, not mannosylphosphoryldolichol, as the direct mannosyl donor.     

Reactive derivative of adenosine-5′-triphosphate formed by irradiation of ATP γ-p-azidoanilide

UV and ¹H NMR spectra changes demonstrate that p-azidoanilides of ATP, ITP, pyrophosphate after irradiation at 313 nm, transform to similar reactive intermediates. The latter react readily with morpholine, iso-propylamine, tert-butylamine, imidazole, mercaptoethanol the reactivity being qualitatively correlated with nucleophilicity. By analogy with the transformation of p-azidoanilide, it is concluded that the reactive intermediate is the respective derivative of quinone diimine. Photoaffinity labelling of proteins with p-azidoanilide derivatives may proceed as the reaction of the nucleophilic centers of proteins with this quinone diimine derivative, rather than as a direct attack by a nitrene biradical.     

Synthesis, characterization and X-ray crystal structures of dinuclear nickel(II) complexes of a novel potentially hexadentate but functionally tetradentate binucleating ligand

A binucleating potentially hexadentate chelating agent containing oxygen, nitrogen and sulfur as potential donor atoms (H₂ONNO) has been synthesized by condensing α,α-xylenebis(N-methyldithiocarbazate) with 2,4-pentanedione. An X-ray crystallographic structure determination shows that the Schiff base remains in its ketoimine tautomeric form with the protons attached to the imine nitrogen atoms. The reaction of the Schiff base with nickel(II) acetate in a 1:1 stoichiometry leads to the formation of a dinuclear nickel(II) complex [Ni(ONNO)]₂ (ONNO²⁻ = dianionic form of the Schiff base) containing N,O-chelated tetradentate ligands, the sulfur donors remaining uncoordinated. A single crystal X-ray structure determination of this dimer reveals that each ligand binds two low spin nickel(II) ions, bridged by a xylyl group. The nickel(II) atoms adopt a distorted square-planar geometry in a trans-N₂O₂ donor environment. Reaction of the Schiff base with nickel(II) acetate in the presence of excess pyridine leads to the formation of a similar dinuclear complex, [Ni(ONNO)(py)]₂, but in this case comprises five coordinate high-spin Ni(II) ions with pyridine ligands occupying the axial coordination sites as revealed by X-ray crystallographic analysis.     

Glucosamine metabolism in Drosophila virilis salivary glands: Ontogenetic changes of enzyme activities and metabolite synthesis

In Drosophila virilis salivary glands the in vitro activities of enzymes involved in the glucosamine pathway were examined during the third larval instar and in the prepupa. While glutamine-fructose-6-phosphate aminotransferase (EC 5.3.1.19) becomes inactive at the time of puparium formation, glucosamine-6-phosphate isomerase (EC 5.3.1.10) and glucosamine-6-phosphate N-acetyltransferase (EC 2.3.1.3) show maximal activities in the prepupal gland. The activity of UDP-N-acetylglucosamine pyrophosphorylase (EC 2.7.7.23) may also decrease prior to puparium formation. Incubation of larval and prepupal glands in medium containing [³H]glucose + [¹⁴C]-uridine or [¹⁴C]glucosamine and subsequent separation of intermediates of the glucosamine pathway by chromatographic procedures reveal that the capacity of the glands to incorporate the isotopes into these intermediates decreases significantly at the time of puparium formation. The results suggest that in D. virilis salivary glands the formation of aminosugars is mainly controlled by the activities of the two enzymes glutamine-fructose-6-phosphate aminotransferase and UDP-N-acetylglucosamine pyrophosphorylase.     

Effect of gem 2,2′-disubstitution and base in the formation of spiro- and ansa-1,3-propandioxy derivatives of cyclotriphosphazenes

The gem-dialkyl effect has been investigated in the reactions of cyclotriphosphazene, N₃P₃Cl₆1, with various 2,2′-derivatives of 1,3-propandiol, CXY(CH₂OH)₂, in either THF or DCM to form spiro (6-membered) and ansa (8-membered ring) derivatives. The reactions were made with a number of symmetrically-substituted (X = Y, methyl, ethyl, n-butyl and a malonate ester) and unsymmetrically-substituted (X ≠ Y, methyl/H, phenyl/H, methyl/n-propyl, ethyl/n-butyl and Br/NO₂) 1,3-propandiols. The products were analysed by ¹H and ³¹P NMR spectroscopy and some of the spiro and ansa derivatives were also characterized by X-ray crystallography. Reactions of 1 with unsymmetrically-substituted 1,3-propandiols results in the formation of two structural isomers of ansa-substituted compounds, both isomers (endo and exo) have been structurally-characterized by X-ray crystallography for the ethyl/n-butyl derivative. It is found that the regioselectivity of the reaction is changed when the base is changed. The relative proportions of spiro and ansa compounds formed under different reaction conditions were quantified by ³¹P NMR measurements of the reaction mixtures. The results were rationalised mainly in terms of the electronic effect of the substituents, whereas the steric effect has a secondary role in the formation of both spiro and ansa compounds.     

Aeribactin synthesis in a cell-free system of Aerobacter Aerogenes 62-1

The supernatant fraction of the cell-free system of Aerobacter aerogenes 62-1 was found to contain enzyme(s) capable of synthesising aerobactin from citrate and N⁶-acetyl-N⁶-hydroxylysine. Studies with partially purified enzyme systems revealed that aerobactin production was dependent on Mg²⁺ and ATP, the latter being used for the activation of both the precursors, citrate and N⁶-acethyl-N⁶-hydroxylysine. The generation of ADP in the activation process was consistent with the formation of phosphorylated precursor intermediates in the reactions leading to the peptide bond formation.     

Two novel dATP analogs for DNA photoaffinity labeling

Two new photoreactive dATP analogs, N⁶-[4-azidobenzoyl–(2-aminoethyl)]-2′-deoxyadenosine-5′-triphosphate (AB-dATP) and N⁶-[4-[3-(trifluoromethyl)-diazirin-3-yl]benzoyl-(2-aminoethyl)]-2′-deoxyadenosine-5′-triphosphate (DB-dATP), were synthesized from 2′-deoxyadenosine-5′-monophosphate in a six step procedure. Synthesis starts with aminoethylation of dAMP and continues with rearrangement of N¹-(2-aminoethyl)-2′-deoxyadenosine-5′-monophosphate to N⁶-(2-aminoethyl)-2′-deoxyadenosine-5′-monophosphate (N⁶-dAMP). Next, N⁶-dAMP is converted into the triphosphate form by first protecting the N-6 primary amino group before coupling the pyrophosphate. After pyrophosphorylation, the material is deprotected to yield N⁶-(2-aminoethyl)-2′-deoxyadenosine-5′-triphosphate (N⁶-dATP). The N-6 amino group is subsequently used to attach either a phenylazide or phenyldiazirine and the photoreactive nucleotide is then enzymatically incorporated into DNA. N⁶-dATP and its photoreactive analogs AB-dATP and DB-dATP were successfully incorporated into DNA using the exonuclease-free Klenow fragment of DNA polymerase I in a primer extension reaction. UV irradiation of the primer extension reaction with AB-dATP or DB-dATP showed specific photocrosslinking of DNA polymerase I to DNA.