Analysis by the reductive-cleavage method of linkage positions in a polysaccharide containing 4-linked D-glucopyranosyluronic residues

The fate of 4-linked D-glucopyranosyluronic residues under reductive-cleavage conditions was investigated by using the Klebsiella aerogenes type 54 strain A3 capsular polysaccharide. Treatment of the fully methylated polysaccharide with triethylsilane and trimethylsilyl trifluoromethanesulfonate in dichloromethane, followed by in situ acetylation, yielded 1,5-anhydro-2,3,4,6-tetra-O-methyl-D-glucitol, 3,4-di-O-acetyl-1,5-anhydro-2,6-di-O-methyl-D-glucitol, and 3-O-acetyl-1,5-anhydro-2,4-di-O-methyl-L-fucitol, as expected, but the expected product of reductive cleavage of the 4-linked D-glucopyranosyluronic residue, namely, methyl 3-O-acetyl-2,6-anhydro-4,5-di-O-methyl-L-gulonate, was not observed. Instead, methyl 2-O-acetyl-3,6-anhydro-4,5-di-O-methyl-L-gulonate (6) was identified as the sole product of reductive cleavage of the 4-linked D-glucopyranosyluronic residue. That compound 6 arose as a result of rearrangement during reductive cleavage rather than by reductive cleavage of a 5-linked D-glucofuranosyluronic residue, was established by reductive cleavage of the fully methylated polysaccharide following reduction of its ester groups with either lithium aluminum hydride or lithium aluminum deuteride. The products of the latter reductive cleavage were the same as before, except for the absence of 6 and the presence of 4,6-di-O-acetyl-1,5-anhydro-2,3-di-O-methyl-D-glucitol, or its 6,6-dideuterio isomer. Although the reductive-cleavage technique is suitable for the direct analysis of polysaccharides containing 4-linked D-glucopyranosyluronic residues, it does not establish whether the uronic residue is a 4-linked pyranoside or a 5-linked furanoside. The expected product is, however, derived from the 4-linked D-glucopyranosyluronic residue after sequential methylation, reduction of its ester group and reductive cleavage.     

Pyrophosphate as a ligand for delivery of iron to isolated rat-liver mitochondria

Rat liver mitochondria accumulate iron mobilized from transferrin by pyrophosphate. The capacity of the mitochondria to accumulate iron is higher than the capacity of pyrophosphate to mobilize iron from transferrin: with ferric-iron-pyrophosphate as iron donor, iron uptake and heme synthesis are about 10-times that at corresponding concentrations of iron-transferrin plus pyrophosphate. Uptake of iron from ferric-iron-pyrophosphate depends on a functionary respiratory chain and involves reductive cleavage of the ferric-iron-pyrophosphate complex. Apotransferrin inhibits uptake of iron from ferric-iron-pyrophosphate by competing with the mitochondria for iron. The results focus on pyrophosphate as a possible candidate for intracellular iron transport.     

Cofactor dependence of reduction potentials for [4Fe-4S]2+/1+ in lysine 2,3-aminomutase

Lysine 2,3-aminomutase (LAM) catalyzes the interconversion of l-lysine and l-beta-lysine by a free radical mechanism. The 5'-deoxyadenosyl radical derived from the reductive cleavage of S-adenosyl-l-methionine (SAM) initiates substrate-radical formation. The [4Fe-4S](1+) cluster in LAM is the one-electron source in the reductive cleavage of SAM, which is directly ligated to the unique iron site in the cluster. We here report the midpoint reduction potentials of the [4Fe-4S](2+/1+) couple in the presence of SAM, S-adenosyl-l-homocysteine (SAH), or 5'-{N-[(3S)-3-aminocarboxypropyl]-N-methylamino}-5'-deoxyadenosine (azaSAM) as measured by spectroelectrochemistry. The reduction potentials are -430 +/- 2 mV in the presence of SAM, -460 +/- 3 mV in the presence of SAH, and -497 +/- 10 mV in the presence of azaSAM. In the absence of SAM or an analogue and the presence of dithiothreitol, dihydrolipoate, or cysteine as ligands to the unique iron, the midpoint potentials are -479 +/- 5, -516 +/- 5, and -484 +/- 3 mV, respectively. LAM is a member of the radical SAM superfamily of enzymes, in which the CxxxCxxC motif donates three thiolate ligands to iron in the [4Fe-4S] cluster and SAM donates the alpha-amino and alpha-carboxylate groups of the methionyl moiety as ligands to the fourth iron. The results show the reduction potentials in the midrange for ferredoxin-like [4Fe-4S] clusters. They show that SAM elevates the reduction potential by 86 mV relative to that of dihydrolipoate as the cluster ligand. This difference accounts for the SAM-dependent reduction of the [4Fe-4S](2+) cluster by dithionite reported earlier. Analogues of SAM have a weakened capacity to raise the potential. We conclude that the midpoint reduction potential of the cluster ligated to SAM is 1.2 V less negative than the half-wave potential for the one-electron reductive cleavage of simple alkylsulfonium ions in aqueous solution. The energetic barrier in the reductive cleavage of SAM may be overcome through the use of binding energy.     

Site of pyruvate formation and processing of mammalian S-adenosylmethionine decarboxylase proenzyme

Mammalian S-adenosylmethionine decarboxylase was expressed at a high level in an Escherichia coli mutant deficient in this enzyme. The proenzyme form of this enzyme was cleaved and processed to the mature decarboxylase which contains two pairs of nonidentical subunits, the larger of which contains a pyruvate prosthetic group. In order to determine the site of formation of the pyruvate, two approaches were used. First, the mammalian S-adenosylmethionine decarboxylase produced in E. coli was purified to homogeneity and the pyruvate converted to alanine by a reductive amination. The large subunit was then isolated by reversed phase high pressure liquid chromatography and the amino-terminal sequence determined and compared with the sequence of the proenzyme derived from its cDNA. These results indicated that the bond between glutamic acid 67 and serine 68 was the site of cleavage. Second, each of the serine residues in portion of the proenzyme likely to contain the cleavage site were altered by site-directed mutagenesis and the RNA produced from plasmids containing these mutations was translated in a reticulocyte lysate. The translation products were tested for processing and for S-adenosylmethionine decarboxylase activity. Altering the serine residues at positions 50, 66, and 69 to alanines had little effect but changing serine at position 68 to alanine completely prevented both processing and activity. These results indicate that the serine residue at position 68 of the proenzyme which is in the underlined position in the sequence -Leu-Ser-Glu-Ser-Ser-Met- is the residue which is converted to the pyruvate prosthetic group in human S-adenosylmethionine decarboxylase.     

Site-specific DNA cleavage by antisense oligonucleotides covalently linked to phenazine di-N-oxide.

Site-specific degradation of DNA was achieved by the use of DNA oligonucleotides covalently tethered to phenazine 5,10-di-N-oxide. When annealed to a complementary DNA target strand, the antisense oligonucleotide effected alkylation of guanosine residues in proximity to the phenazine di-N-oxide prosthetic group. Admixture of dithiothreitol to the formed duplex resulted in reductive activation of the phenazine di-N-oxide moiety with concomitant generation of diffusible oxygen radicals; the latter effected strand scission of the target DNA oligonucleotide. Several parameters of DNA degradation were studied, including the effect on DNA degradation of chain length in the tether connecting the oligonucleotides and prosthetic group, the relative efficiencies of DNA cleavage when the prosthetic group was in the middle or at the end of the antisense oligonucleotide, and the effect of O2 on DNA degradation. Also studied was the actual chemistry of DNA oligonucleotide degradation and the ability of individual diastereomers of the modified oligonucleotides to mediate degradation of the target DNA.     

Metabolism-based covalent bonding of the heme prosthetic group to its apoprotein during the reductive debromination of BrCCl3 by myoglobin

The reductive metabolism of BrCCl3 by ferrous myoglobin leads to the alteration of the prosthetic heme to form products that can be dissociated from the protein and to those that are irreversibly bound to the protein. The major dissociable or soluble heme metabolites have recently been characterized. In this study, the irreversibly bound heme product was characterized by Edman degradation, amino acid analysis, and electronic absorption and mass spectrometry of peptides derived from the altered protein. It was found that the prosthetic heme was modified by a CCl2 moiety derived from BrCCl3 and was covalently bound to histidine residue 93, the normal proximal ligand to the heme-iron. The data are consistent with a mechanism by which the trichloromethyl radical reacts with the heme to form an intermediate that either can alkylate the proximal histidine residue or form soluble metabolites. The covalent bonding of the heme prosthetic moiety to the apoprotein likely leads to a change in the tertiary structure of the protein that may be responsible for its altered catalytic activity as well as its enhanced susceptibility to proteolysis. Similar processes may account, at least in part, for the covalent alteration of the heme prosthetic group of other hemoproteins caused by xenobiotics and endogenous substrates.     

Iron-reductases in the yeast Saccharomyces cerevisiae

Several NAD(P)H-dependent ferri-reductase activities were detected in sub-cellular extracts of the yeast Saccharomyces cerevisiae. Some were induced in cells grown under iron-deficient conditions. At least two cytosolic iron-reducing enzymes having different substrate specificities could contribute to iron assimilation in vivo. One enzyme was purified to homogeneity: it is a flavoprotein (FAD) of 40 kDa that uses NADPH as electron donor and Fe(III)-EDTA as artificial electron acceptor. Isolated mitochondria reduced a variety of ferric chelates, probably via an 'external' NADH dehydrogenase, but not the siderophore ferrioxamine B. A plasma membrane-bound ferri-reductase system functioning with NADPH as electron donor and FMN as prosthetic group was purified 100-fold from isolated plasma membranes. This system may be involved in the reductive uptake of iron in vivo.     

4-Hydroxyphenylacetate decarboxylases: properties of a novel subclass of glycyl radical enzyme systems

The 4-hydroxyphenylacetate decarboxylases from Clostridium difficile and Clostridium scatologenes, which catalyze the formation of p-cresol, form a distinct group of glycyl radical enzymes (GREs). Cresol formation provides metabolic toxicity, which allows an active suppression of other microbes and may provide growth advantages for the producers in highly competitive environments. The GRE decarboxylases are characterized by a small subunit, which is not similar to any protein of known function in the databases, and provides unique properties that have not been observed in other GREs. Both decarboxylases are functional hetero-octamers (beta(4)gamma(4)), which contain iron-sulfur centers in addition to the glycyl radical prosthetic group. The small subunit is responsible for metal binding and is also involved in the regulation of the enzymes' oligomeric state and activity, which are triggered by reversible serine phosphorylation of the glycyl radical subunits. Biochemical data suggest that the iron-sulfur centers of the decarboxylases could be involved in the radical dissipation of previously activated enzymes in the absence of substrate. The cognate activating enzymes differ from their Pfl and Nrd counterparts in that up to two iron-sulfur centers, in addition to the characteristic SAM cluster, were found. Biochemical data suggested that these [4Fe-4S] centers are involved in the electron transfer to the SAM cluster but do not directly participate in the reductive cleavage of SAM. These data imply a tight regulation of p-cresol formation, which is necessary in order to avoid detrimental effects of the toxic product on the producers.     

Reactions of site-differentiated [Fe4S4]2+, 1+ clusters with sulfonium cations: reactivity analogues of biotin synthase and other members of the S-adenosylmethionine enzyme family

The first examples of reduced 3:1 site-differentiated Fe(4)S(4) clusters have been synthesized as [Fe(4)S(4)(LS(3))(SR')](3-) (R=Et, Ph) by chemical reduction of previously reported [Fe(4)S(4)(LS(3))(SR')](2-) clusters, and isolated as NBu(4)(+) salts. The reduced clusters were characterized by electrochemistry and EPR, 1H NMR, and M?ssbauer spectroscopies. The reaction of oxidized clusters with the sulfonium ions [PhMeSCH(2)R](+) (R=COPh, p-C(6)H(4)CN) in acetonitrile results in electrophilic attack on coordinated thiolate and production of PhSMe and R'SCH(2)R when the reaction occurs at the unique cluster site. The reactions of reduced clusters with these substrates were examined in relation to the reductive cleavage of the cofactor S-adenosylmethionine, the first step in the catalytic cycle of biotin synthase. Product analysis indicated a approximately 4:1 ratio of reductive cleavage to electrophilic attack. The cleavage products are PhSMe, R'SCH(2)R, and RCH(3) for both clusters, and also PhMeS=CHR and RCH(2)CH(2)R from secondary reactions when the sulfonium cation is [PhMeSCH(2)COPh](+) and [PhMeSCH(2)-p-C(6)H(4)CN](+), respectively. Reaction schemes for reductive cleavage based on product distributions are presented. These results parallel those previously reported for homoleptic [Fe(4)S(4)(SR')(4)](2-,3-) clusters and demonstrate that site-differentiated clusters sustain a high percentage of reductive cleavage, a necessary result in the context of biotin synthase activity preceding an investigation of the mode of binding of sulfonium substrates and inhibitors at the unique iron site. [LS(3)=1,3,5-tris[(4,6-dimethyl-3-mercaptophenyl)thio]-2,4,6-tris(p-tolylthio)benzene(3-)].     

The antiviral protein viperin is a radical SAM enzyme

Viperin, an interferon-inducible antiviral protein, is shown to bind an iron-sulfur cluster, based on iron analysis as well as UV-Vis and electron paramagnetic resonance spectroscopic data. The reduced protein contains a [4Fe-4S]¹⁺ cluster whose g-values are altered upon addition of S-adenosylmethionine (SAM), consistent with SAM coordination to the cluster. Incubation of reduced viperin with SAM results in reductive cleavage of SAM to produce 5′-deoxyadenosine (5′-dAdo), a reaction characteristic of the radical SAM superfamily. The 5′-dAdo cleavage product was identified by a combination of HPLC and mass spectrometry analysis.     

Structural studies of the interaction of S-adenosylmethionine with the [4Fe-4S] clusters in biotin synthase and pyruvate formate-lyase activating enzyme

The diverse reactions catalyzed by the radical-SAM superfamily of enzymes are thought to proceed via a set of common mechanistic steps, key among which is the reductive cleavage of S-adenosyl-L-methionine (SAM) by a reduced [4Fe-4S] cluster to generate an intermediate deoxyadenosyl radical. A number of spectroscopic studies have provided evidence that SAM interacts directly with the [4Fe-4S] clusters in several of the radical-SAM enzymes; however, the molecular mechanism for the reductive cleavage has yet to be elucidated. Selenium X-ray absorption spectroscopy (Se-XAS) was used previously to provide evidence for a close interaction between the Se atom of selenomethionine (a cleavage product of Se-SAM) and an Fe atom of the [4Fe-4S] cluster of lysine-2,3-aminomutase (KAM). Here, we utilize the same approach to investigate the possibility of a similar interaction in pyruvate formate-lyase activating enzyme (PFL-AE) and biotin synthase (BioB), two additional members of the radical-SAM superfamily. The results show that the latter two enzymes do not exhibit the same Fe-Se interaction as was observed in KAM, indicating that the methionine product of reductive cleavage of SAM does not occupy a well-defined site close to the cluster in PFL-AE and BioB. These results are interpreted in terms of the differences among these enzymes in their use of SAM as either a cofactor or a substrate.     

Analysis of a pyruvic acid acetal-containing polysaccharide by the reductive-cleavage method.

The applicability of the reductive-cleavage method to the analysis of polysaccharides bearing pyruvic acid acetals has been demonstrated. Direct reductive cleavage of fully methylated gum xanthan yielded the expected products, including 1,5-anhydro-4,6-O-[(S)-1-methoxycarbonylethylidene]-2,3-di-O-methy l-D- mannitol. The latter product was not observed when reductive cleavage was performed subsequent to reduction of ester groups in the fully methylated polysaccharide and mild hydrolysis to remove pyruvic acid acetal substituents. Instead, the latter experiment yielded 1,5-anhydro-2,3-di-O-methyl-D-mannitol, establishing the presence in the polysaccharide of terminal (nonreducing) D-mannopyranosyl groups bearing 4,6-O-(1-carboxyethylidene) substituents. The products of reductive cleavage were characterized, where appropriate, by comparison of the gas chromatographic retention times and chemical ionization- and electron ionization-mass spectra of their acetates to those of authentic standards. Alternatively, the products of reductive cleavage could be characterized without resort to comparison with authentic standards by analysis of the 1H-n.m.r. spectra of their benzoates, which were obtained in pure form by high-performance liquid chromatography. By either method of product characterization, this two-step procedure of analysis reveals the presence of pyruvic-acetal residues in polysaccharides and establishes both the identity of the sugar residue to which they are attached and their positions of attachment.     

Biosynthesis of triphosphoribosyl-dephospho-coenzyme A, the precursor of the prosthetic group of malonate decarboxylase

Malonate decarboxylase from Klebsiella pneumoniae consists of four subunits MdcA, D, E, and C and catalyzes the cleavage of malonate to acetate and CO(2). The smallest subunit MdcC is an acyl carrier protein to which acetyl and malonyl thioester residues are bound via a 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA prosthetic group and turn over during the catalytic mechanism. We report here on the biosynthesis of holo acyl carrier protein from the unmodified apoprotein. The prosthetic group biosynthesis starts with the MdcB-catalyzed condensation of dephospho-CoA with ATP to 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA. In this reaction, a new alpha (1' ' --> 2') glycosidic bond between the two ribosyl moieties is formed, and thereby, the adenine moiety of ATP is displaced. MdcB therefore is an ATP:dephospho-CoA 5'-triphosphoribosyl transferase. The second protein involved in holo ACP synthesis is MdcG. This enzyme forms a strong complex with the 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA prosthetic group precursor. This complex, called MdcG(i), is readily separated from free MdcG by native polyacrylamide gel electrophoresis. Upon incubation of MdcG(i) with apo acyl carrier protein, holo acyl carrier protein is synthesized by forming the phosphodiester bond between the 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA prosthetic group and serine 25 of the protein. MdcG corresponds to a 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA:apo ACP 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA transferase. In absence of the prosthetic group precursor, MdcG catalyzes at a low rate the adenylylation of apo acyl carrier protein using ATP as substrate. The adenylyl ACP thus formed is an unphysiological side product and is not involved in the biosynthesis of holo ACP. The 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA precursor of the prosthetic group has been purified and its identity confirmed by mass spectrometry and enzymatic analysis.     

Enzymatic redox chemistry: a proposed reaction pathway for the six-electron reduction of SO3(2-) to S2- by the assimilatory-type sulfite reductase from Desulfovibrio vulgaris (Hildenborough)

A detailed reaction pathway for the six-electron reduction of SO3(2-) to S2- by the assimilatory-type sulfite reductase (SiR) from Desulfovibrio vulgaris (Hildenborough) has been deduced from experiments with 35S-labeled enzyme and the relative reaction rates of nitrogenous substrates. The ligand bridging the prosthetic [Fe4S4]-siroheme center is apparently exchanged by 35S2- in both oxidized and reduced enzyme. This 35S2- label was retained in the course of SO3(2-) reduction, implicating substrate binding to the nonbridging axial site of the siroheme. A reaction mechanism is proposed in which SO3(2-) binds to Fe2+ through the sulfur atom, followed by a series of two-electron reductive cleavages of S-O bonds. Protonation of oxygen facilitates bond cleavage, giving hydroxide as leaving group. The bridge remains intact throughout the course of the reaction, providing an efficient coupling pathway for electron transfer between the cluster and siroheme.     

Bile pigment--protein interactions. Coupled oxidation of cytochrome c

A new methodology is described for the chemical modification of the heme prosthetic group of horse heart cytochrome c. The selective modification of the heme moiety of cytochrome c is facilitated by utilizing coupling oxidation conditions. Comparison of the absorption spectra of this chemically modified cytochrome c species in two different solvents (aqueous pyridine and carbon monoxide saturated 6 M guanidinium chloride) with those of two model compounds [bis(pyridine)(2,3,7,8,12,13,17,18-octaethyl-5-oxaporphyrinato)iron(II) tetrafluoroborate salt and (pyridine)carbonyl-(2,3,7,8,12,13,17,18-octaethyl-5-oxaporphyrinato)iron(II) tetrafluoroborate salt] shows that coupled oxidation of cytochrome c affords a new protein with a covalently bound iron(II) oxaporphyrin prosthetic group. Amino acid analysis of this protein-bound iron(II) oxaporphyrin species reveals that only limited modification of the primary structure of the apoprotein occurs during coupled oxidation of cytochrome c. This protein-bound iron(II) oxaporphyrin species is also interconvertible to a protein-bound bilatriene species under hydrolytic conditions. The synthetic utility of the coupled oxidation of cytochrome c for the preparation of chromoproteins which possess covalently bound iron(II) oxaporphyrin and bilatriene prosthetic groups is considered.     

Mycobacterium tuberculosis can utilize heme as an iron source

Most iron in mammals is found within the heme prosthetic group. Consequently, many bacterial pathogens possess heme acquisition systems to utilize iron from the host. Here, we demonstrate that Mycobacterium tuberculosis can utilize heme as an iron source, suggesting that M. tuberculosis possesses a yet-unknown heme acquisition system.     

Microbial Degradation of Diphenylamine Under Anoxic Conditions

Diphenylamine (DPA) was cometabolically degraded in anoxic sediment-water batch enrichments and in cultures of newly isolated sulfate-reducing bacteria. In gas chromatography–mass spectrometry (GC-MS) measurements, aniline was identified as a major breakdown product of the diphenylamine structure. After its identification, aniline was quantified by reversed phase high pressure liquid chromatography (HPLC). The fate of the other carbon ring system remained unclear, because benzene (as a product of reductive cleavage), phenol (as a product of hydrolytic cleavage), and/or other ring cleavage products of diphenylamine were not observed in our experiments with the methods employed. Received: 20 May 1997 / Accepted: 25 June 1997     

Reductive and nucleophilic activation products of dynemicin A with methyl thioglycolate. A rational mechanism for DNA cleavage of the thiol-activated dynemicin A

The reaction products of methyl thioglycolate with dynemicin A, dynemicin H and dynemicin S, were isolated by HPLC purification and identified spectroscopically. The major product, dynemicin H (C30H23NO9), was determined to be a C-8 hydrogen analogue of dynemicins L and N in which the enediyne core is aromatized. The minor product, dynemicin S (C33H27No11S), is an adduct of methyl thioglycolate at the C-8 position. By using NADPH instead of methyl thioglycolate, the reaction with dynemicin A also gives the same major product (dynemicin H). The nucleotide-specific cleavage of dynemicin A induced by addition of methyl thioglycolate is remarkably similar to that induced by addition of NADPH, whereas dynemicins H and S show no DNA cleavage activities. The formation of dynemicins H and S provides a rationale for the reductive and nucleophilic activations of dynemicin A.