Crystallization of a derivative of a new coenzyme,methoxatin

A new compound, derived from a parent compound to which we have given the trivial name, methoxatin, has been isolated from a methanol-oxidizing bacterium, and crystallized. Its chemical structure was determined by X-ray crystallography. Methoxatin is implicated as a coenzyme in the oxidation of substrate alcohols. This report describes the purification and crystallization of the derivative, acetonyl methoxatin.     

Semiquinone radicals of methylamine dehydrogenase, methoxatin, and related o-quinones: a pulse radiolysis study

Methoxatin is a novel o-quinone coenzyme found in a variety of dehydrogenases and oxidases. In this paper we present the visible absorption spectra, apparent pKa, and midpoint potentials of the methoxatin and two analogous phenanthrolinedione semiquinones. We have also obtained absorption spectra for the semiquinone forms of the methoxatin-containing holoenzyme methylamine dehydrogenase and of its resolved methoxatin-containing subunit. The two protein and the methoxatin semiquinone spectra all differ from one another.     

The methoxatin semiquinone: a pulse radiolysis study

Methoxatin is a novel o-quinone found in bacterial dehydrogenases and mammalian plasma amine oxidase. This is the first report of the redox potential and spectrum of the 1-electron reduced methoxatin semiquinone obtained by the method of pulse radiolysis in aqueous solution.     

Reconstitution of glucose dehydrogenases using synthetic methoxatin

Synthetic methoxatin or its reduced derivative, is fully active in the reconstitution of glucose dehydrogenase from Acinetobacter calcoaceticus var. anitratus from apoenzyme, and is also active in a similar reconstitution of enzyme from A. calcoaceticus ATCC 9956. However, we have still not been able to reconstitute holoenzyme of methanol dehydrogenase from Pseudomonas TPI.     

PQQ, the elusive coenzyme

The recently discovered redox coenzyme, PQQ (methoxatin), is widely distributed. Quantitation of protein-bound PQQ has been difficult, but unique redox cycling reactions, which reflect its striking biological properties, reveal trace amounts. PQQ is a potential target for drugs.     

X-ray and NMR study of the fate of the Co(1,10-phenanthroline-5,6-diketone)3 ion in aqueous solution: supramolecular motifs in the packing of 1,10-phenanthroline-5,6-diketone and 1,10-phenanthroline-5,6-diol complexes

X-ray structures are presented of three new cobalt complexes prepared from Co(III) and N,N^′-1,10-phenanthroline-5,6-dione. The cis-aqua-chloro-bis(N,N^′-1,10-phenanthroline-5,6-dione)cobalt(II) nitrate trihydrate (3) and the cis-aqua-bromo-bis(N,N^′-1,10-phenanthroline-5,6-dione)cobalt(II) trifluoro-methanesulfonate tetrahydrate (4), crystalize in the same space group with a similar arrangement of the complex ions. However, on the molecular scale there are important differences. The cobalt complex in 3 has a typical high-spin geometry whereas in 4 the cobalt complex displays a Jahn-Teller distortion characteristic for low-spin compounds. The third structure is di(N,N^′-1,10-phenanthroline-5,6-diol)(N,N^′-1,10-phenanthroline-5,6-dione)cobalt(III) bromide hexahydrate (5). NMR studies of the hydration of the Co(III)(1,10-phenanthroline-5,6-dione)₃ ³⁺ ion in water and DMSO are also presented. The various possible transformations of the N,N^′-1,10-phenanthroline-5,6-dione ligand are discussed.     

Synthesis and characterisation of phenanthroline-oxazine ligands and their Ag(I), Mn(II) and Cu(II) complexes and their evaluation as antibacterial agents

BioMetals - A series of phenanthroline-oxazine ligands were formed by a cyclisation reaction between L-tyrosine amino acid esters and 1,10-phenanthroline-5,6-dione (phendione). The methyl...     

Roles of the D-ribose and 5,6-dimethylbenzimidazole moieties of the nucleotide loop of adenosylcobalamin in manifestation of coenzymic function in the diol dehydrase reaction

Coenzyme analogs in which the D-ribose moiety of the nucleotide loop was replaced by an oligomethylene group and a trimethylene analog containing imidazole instead of 5,6-dimethylbenzimidazole were synthesized. Coordination of the 5,6-dimethylbenzimidazole to the cobalt atom in these analogs was much weaker than that in cobalamins. The replacement of this base with imidazole did not significantly alter the strength of the coordination to the cobalt atom. 5,6-Dimethylbenzimidazolyl trimethylene and tetramethylene and imidazolyl trimethylene analogs were partially active as coenzymes in the diol dehydrase reaction in this order as judged by kcat, but the others were not active as coenzymes and were weak competitive inhibitors. This indicates that neither the alpha-D-ribofuranose ring nor the functional groups of the ribose moiety are essential for coenzymic function. There was an optimum loop size of the analogs for catalysis and for tight binding to the apoenzyme, which corresponds to the loop size of cobalamins. Therefore, the D-ribose moiety seems important as a spacer to keep the base in the proper position. The reaction with the imidazolyl trimethylene analog as coenzyme was accompanied with concomitant rapid inactivation during catalysis. The inactivation occurred only in the presence of substrate. Upon inactivation with this analog, 5'-deoxyadenosine and a B12r-like species were formed from the adenosyl group and the rest of the analog molecule, respectively, without modification of the apoenzyme. Therefore, it can be concluded that this is a kind of suicide inactivation which occurred from one of the intermediates in the normal catalytic process. The dimethylbenzo moiety of the regular coenzyme thus seems to play an important role in preventing the intermediate complexes from inactivation during catalysis.     

Palladium(II) complexes with N,N'-dialkyl-1,10-phenanthroline-2,9-dimathanamine: synthesis, characterization and cytotoxic activity

Five new tetradentate ligands and their corresponding palladium complexes, [Pd(L)]Cl2 (L = N,N'-dimethyl-1,10-phenanthroline-2,9-dimathanamine, N,N'-diethyl-1,10-phenanthroline-2,9-dimathanamine, N,N'-dipropyl-1,10-phenanthroline-2,9-dimathanamine, N,N'-ditert-butyl-1,10-phenanthroline-2,9-dimathanamine, N,N'-dicyclohexyl-1,10-phenanthroline-2,9-dimathanamine) have been synthesized. The ligands and their complexes have been characterized by elemental analysis, IR, and 1H NMR. The complexes have been assayed for antitumor activity in vitro against the mouse leukemia L1210 and the mouse liver carcinoma Bel7402 cell lines. The results showed that the activities of these complexes are significantly dependent on the nature of the alkyl groups on the coordinated amine moieties, and three of these palladium complexes showed lower ID50 values against the two cell lines than cisplatin.     

Influence of steric and electronic factors in the stabilization of five-coordinate ethylene complexes of platinum(II): X-ray crystal structure of [PtCl2(2,9-dimethyl-1,10-phenanthroline-5,6-dione)]

Reactions of Zeise’s salt (K[Pt(η²-C₂H₄)Cl₃]) with oxidized phenanthroline ligands (1,10-phenanthroline-5,6-dione, phedon, and 2,9-dimethyl-1,10-phenanthroline-5,6-dione, Me₂phedon) are reported. Comparison with analogous reactions involving unoxidized phen (1,10-phenanthroline) and Me₂phen (2,9-dimethyl-1,10-phenanthroline) ligands indicates that these latter ligands are less capable to stabilize the five-coordinate species [PtCl₂(η²-C₂H₄)(phenanthroline)] in which the phenanthroline and the olefin share the trigonal plane and two chlorines are in the axial positions. The X-ray structure of the four-coordinate species [PtCl₂(Me₂phedon)] indicates that the major difference between oxidized and unoxidized phenanthrolines is the loss of aromaticity of the central ring of phenanthroline. As a consequence, the oxidized phenanthroline becomes more flexible and can undergo a bow-like distortion so to reduce steric interaction between ortho substituents of phenanthroline and cis chlorine ligands. The increase in stability of the four-coordinate species with Me₂phedon is concomitant with an increase in stability of the five-coordinate precursor complex with ethylene. In the latter case the stabilization is not of sterical origin but stems from reduced electron-donor properties of oxidized phenanthrolines. The balance of the two effects is such that the equilibrium between five- and four-coordinate species is more shifted in favour of the former species in the case of Me₂phedon than in the case of Me₂phen.     

Differences in coenzyme specificity of the N5-methyltetrahydrofolate-homocysteine methyltransferases of various species: implications for corrin binding loci

Investigations of the coenzyme specificity of N5-methyltetrahydrofolate-homocysteine methyltransferases of diverse biological origin revealed previously unrecognized differences between Escherichia coli methyltransferase and the corresponding enzymes of other species. Cyanocobalamin (CNCbl) actively supports methyltransferase in extracts of animal tissues and E. coli. Cobinamide is more active than CNCbl with rat liver methyltransferase; however, it is non-competitively inhibitory with E. coli enzyme. E. coli methyltransferase, but not rat liver enzyme, is competitively inhibited by alpha-ribazole 3'-phosphate and 5,6-dimethyl-benzimidazole, two moieties of the nucleotide loop. This suggests that animal enzyme binds its corrinoid coenzyme at a site on the corrin macro-ring, while E. coli enzyme binds to the nucleotide loop as well as the macro-ring.     

Overexpression,homology modeling and coenzyme docking studies of the cytochrome P450nor2 from <Emphasis Type="Italic">Cylindrocarpon tonkinense</Emphasis>

Cytochrome P450nor catalyzes an unusual reaction that transfers electrons from NADP/NADPH to bound heme directly. To improve the expression level of P450nor2 from Cylindrocarpon tonkinense (C.P450nor2), Escherichia coli system was utilized to substitute the yeast system we constructed for expression of the P450nor2 gene, and the protein was purified in soluble form using Ni⁺-NTA affinity chromatography. In contrast to P450nor from Fusarium oxysporum (F.P450nor) and P450nor1 from Cylindrocarpon tonkinense (C.P450nor1), C.P450nor2 shows a dual specificity for using NADH or NADPH as electron donors. The present study developed a computational approach in order to illustrate the coenzyme specificity of C.P450nor2 for NADH and NADPH. This study involved homology modeling of C.P450nor2 and docking analyses of NADH and NADPH into the crystal structure of F.P450nor and the predictive model of C.P450nor2, respectively. The results suggested that C.P450nor2 and F.P450nor have different coenzyme specificity for NADH and NADPH; whilst the space around the B'-helix of the C.P450nor2, especially the Ser79 and Gly81, play a crucial role for the specificity of C.P450nor2. In the absence of the experimental structure of C.P450nor2, we hope that our model will be useful to provide rational explanation on coenzyme specificity of C.P450nor2.     

Direct labeling microRNA with an electrocatalytic moiety and its application in ultrasensitive microRNA assays

An ultrasensitive procedure for the detection of microRNA (miRNA) in total RNA is described in this work. The miRNA is directly labeled with a redox active and electrocatalytic moiety, Ru(PD)(2)Cl(2) (PD=1,10-phenanthroline-5,6-dione), through coordinative bonds with purine bases in the miRNA molecule. The excellent electrocatalytic activity of the Ru(PD)(2)Cl(2) towards the oxidation of hydrazine makes it possible to conduct ultrasensitive miRNA detection. Under optimized experimental conditions, the assay allows the detection of miRNAs in the range of 0.50-400 pM with a detection limit of 0.20 pM in 2.5 microl (0.50 amole). MicroRNA quantitation is therefore performed in as little as 10 ng of total RNA, providing a much-needed platform for miRNA expression analysis.     

D-Serine inhibits serine palmitoyltransferase, the enzyme catalyzing the initial step of sphingolipid biosynthesis

Serine palmitoyltransferase (SPT), responsible for the initial step of sphingolipid biosynthesis, catalyzes condensation of palmitoyl coenzyme A and L-serine to produce 3-ketodihydrosphingosine (KDS). For determination of the stereochemical specificity of the amino acid substrate, a competition analysis of the production of [(3)H]KDS from L-[(3)H]serine was performed using purified SPT. D-Serine inhibited [(3)H]KDS production as effectively as non-radioactive L-serine, whereas neither D-alanine nor D-threonine showed any significant effect. Incubation of purified SPT with [palmitoyl 1-(14)C]palmitoyl coenzyme A and D-serine did not produce [(14)C]KDS, while the control incubation with L-serine did. These results suggest that D-serine competes with L-serine for the amino acid recognition site of SPT, but that D-serine is not utilized by this enzyme to produce KDS.     

Mode of action of oxathiin systemic fungicides. V. Effect on electron transport system of Ustilago maydis and Saccharomyces cerevisiae

The systemic fungicide carboxin (5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide) at 100 mum inhibited succinate cytochrome c reductase in mitochondria from Ustilago maydis and Saccharomyces cerevisiae. It did not have any effect on reduced nicotinamide adenine dinucleotide (NADH) cytochrome c reductase. Succinate coenzyme Q reductase was also inhibited, but NADH coenzyme Q reductase was not. When dichlorophenolindophenol (DCIP) was used as the terminal acceptor of electrons from the oxidation of succinate, carboxin was very effective in inhibiting succinate-DCIP reductase. Carboxin was inhibitory to succinic dehydrogenase assayed with phenazine methosulfate plus DCIP when intact mitochondria were used as the enzyme source but not when solubilized enzyme was used. The main site of action of carboxin, therefore, appears to lie between succinate and coenzyme Q. The dioxide analogue of carboxin was also effective in inhibiting succinate-cytochrome c reductase, succinate-coenzyme Q reductase, or succinate-DCIP reductase, whereas the monoxide analogue was less effective in inhibiting these enzymes.     

Synthesis, structure, spectroscopic, magnetic and electrochemical studies of Ni phen-dione complex

The first crystal structure of Ni^II phen-dione complex is reported. This compound is [Ni(bpy)₂(phen-dione)](PF₆)₂ (bpy = 2,2′-bipyridine and phen-dione = 1,10-phenanthroline-5,6-dione). The complex has been characterized by elemental analysis, IR and electronic absorption spectroscopy and cyclic voltammetry. The electrochemical behavior and electronic spectrum of [Ni(bpy)₂(phen-dione)](OAc)₂ has also been studied in buffered solutions at pH between 1 and 8. ORTEP drawing of [Ni(bpy)₂(phen-dione)](PF₆)₂ · 2CH₃CN shows that the coordination geometry around the Ni^II is a distorted octahedron, with bite angles of 78.1-78.8° for all three bidentate ligands and the two pyridyl rings of the bpy ligands are nearly co-planar, as are the two pyridyl rings of phen-dione.     

Design, synthesis, and antiviral evaluation of 2-deoxy-D-ribosides of substituted benzimidazoles as potential agents for human cytomegalovirus infections

Stereoselective glycosylation of 2,5,6-trichlorobenzimidazole (1b), 2-bromo-5,6-dichlorobenzimidazole (1c), 5,6-dichlorobenzimidazole (1d), 5,6-dichlorobenzimidazole-2-thione (1e), 5,6-dichloro-2-(methylthio)benzimidazole (1f), 2-(benzylthio)-5,6-dichlorobenzimidazole (1g), and 2-chloro-5,6-dimethylbenzimidazole (1h) with 2-deoxy-3,5-di-O-p-toluoyl-alpha-D-erythro-pentofuranosyl chloride was achieved to give the desired beta nucleosides 2b-h. Subsequent deprotection afforded the corresponding free beta-D-2-deoxyribosides 3b-h. The 2-methoxy derivative 3i was synthesized by the treatment of 2b with methanolic sodium methoxide. Displacement of the 2-chloro group of 2b with lithium azide followed by a removal of the protective groups gave the 2-azido-5,6-dichlorobenzimidazole derivative (5). The 2-amino derivative (6) was obtained by hydrogenolysis of 5 over Raney nickel. 5,6-Dichloro-2-isopropylamino-1-(2-deoxy-beta-D-erythro- pentofuranosyl)benzimidazole (10) was prepared using 2'-deoxyuridine (7), N-deoxyribofuranosyl transferase and 1d followed by functionalization of the C2 position. Antiviral evaluation of target compounds established that compounds 3b and 3c were active against human cytomegalovirus (HCMV) at non-cytotoxic concentrations. The activity of these 2-deoxy ribosides, however, was less than the activity of the parent riboside, 2,5,6-trichloro-1-beta-D-ribofuranosylbenzimidazole (TCRB). Compared to TCRB, 3b and 3c were somewhat more cytotoxic and active against herpes simplex virus type 1. Compounds 3d-i with other substituents in the 2-position were inactive against both viruses and non-cytotoxic. In contrast, compounds with amine substituents in the 2-position (5, 6, 10) were active against HCMV albeit less so than TCRB. These results establish that 2-deoxy-D-ribosyl benzimidazoles are less active against the DNA virus HCMV than are the corresponding D-ribosides.     

FAD requirement for the reduction of coenzyme F420 by formate dehydrogenase from Methanobacterium formicicum.

The partial purification of the formate dehydrogenase from cell-free extracts of Methanobacterium formicicum decreased the rate of coenzyme F420 reduction 175-fold relative to the rate of methyl viologen reduction. FAD, isolated from this organism, reactivated the coenzyme F420-dependent activity of purified formate dehydrogenase and restored the activity ratio (coenzyme F420/methyl viologen) to near that in cell-free extracts. Neither flavin mononucleotide nor FADH2 replaced FAD. The reduced form of FAD inhibited the reactivation of coenzyme F420-dependent formate dehydrogenase activity by the oxidized form. The results suggest that native formate dehydrogenase from Methanobacterium formicicum contains noncovalently bound FAD that is required for coenzyme F420-dependent activity.     

Homoadenosylcobalamins as probes for exploring the active sites of coenzyme B12-dependent diol dehydratase and ethanolamine ammonia-lyase

[Omega-(Adenosyl)alkyl]cobalamins (homoadenosylcobalamins) are useful analogues of adenosylcobalamin to get information about the distance between Co and C5', which is critical for Co-C bond activation. In order to use them as probes for exploring the active sites of enzymes, the coenzymic properties of homoadenosylcobalamins for diol dehydratase and ethanolamine ammonia-lyase were investigated. The kcat and kcat/Km values for adenosylmethylcobalamin were about 0.27% and 0.15% that for the regular coenzyme with diol dehydratase, respectively. The kcat/kinact value showed that the holoenzyme with this analogue becomes inactivated on average after about 3000 catalytic turnovers, indicating that the probability of inactivation during catalysis is almost 500 times higher than that for the regular holoenzyme. The kcat value for adenosylmethylcobalamin was about 0.13% that of the regular coenzyme for ethanolamine ammonia-lyase, as judged from the initial velocity, but the holoenzyme with this analogue underwent inactivation after on average about 50 catalytic turnovers. This probability of inactivation is 3800 times higher than that for the regular holoenzyme. When estimated from the spectra of reacting holoenzymes, the steady state concentration of cob(II)alamin intermediate from adenosylmethylcobalamin was very low with either diol dehydratase or ethanolamine ammonia-lyase, which is consistent with its extremely low coenzymic activity. In contrast, neither adenosylethylcobalamin nor adeninylpentylcobalamin served as active coenzyme for either enzyme and did not undergo Co-C bond cleavage upon binding to apoenzymes.     

Metal complexes of 1,10-phenanthroline-5,6-dione alter the susceptibility of the yeast Candida albicans to Amphotericin B and Miconazole

Growth of the pathogenic yeast Candida albicans in sub-MIC (minimum inhibitory concentration) levels of Cu(ClO4)2 6H2O and [Cu(phendio)3](ClO4)2 4H2O (phendio = 1,10-phenanthroline-5,6-dione) increased the concentration of miconazole and amphotericin B required to achieve the MIC90 whereas pre-growth in AgClO4 and [Ag(phendio)2]ClO4 resulted in a small decrease in the relevant MIC90 values. The copper complexes reduce the oxygen consumption of C. albicans while the silver complexes increase oxygen consumption. In addition, pre-growth of cells in the copper complexes resulted in a lower ergosterol content while the silver complexes induced an elevation in ergosterol synthesis. The ability of copper and silver complexes to alter the susceptibility of C. albicans to miconazole and amphotericin B may be influenced by their action on respiration, since reduced respiration rates correlate with reduced cellular ergosterol which is the target for amphotericin B. Lower levels of ergosterol have previously been associated with elevated tolerance to this drug. In the case of reduced sensitivity to miconazole, tolerance may be mediated by lower ergosterol synthesis giving rise to fewer toxic side products once biosynthesis is inhibited by miconazole.