Secondary metabolites from Diaporthe lithocarpus isolated from Artocarpus heterophyllus.

Fractionation and purification of the ethyl acetate extract of Diaporthe lithocarpus, an endophytic fungus from the leaves of Artocarpus heterophyllus, yielded one new compound, diaporthindoic acid (1), along with seven known compounds (2-8). The new compound was characterized and established by the basis of extensive spectroscopic methods including NMR (1D and 2D) and HRMS. Compound 6 showed the best citotoxicity against murine leukemia P-388 cells with an IC50 value of 0.41 µg/mL. All compounds (1-8) were also tested for their antimicrobial activities. To the best of our knowledge, this is the first chemical evaluation of fungal Diaporthe derived from Artocarpus.

Spectroscopic characterization of the molybdenum cofactor of the sulfane dehydrogenase SoxCD from Paracoccus pantotrophus.

The bacterial sulfane dehydrogenase SoxCD is a distantly related member of the sulfite oxidase (SO) enzyme family that is proposed to oxidize protein-bound sulfide (sulfane) of SoxY as part of a multienzyme mechanism of thiosulfate metabolism. This study characterized the molybdenum cofactor of SoxCD1, comprising the catalytic molybdopterin subunit SoxC and the truncated c-type cytochrome subunit SoxD1. Electron paramagnetic resonance spectroscopy of the Mo(V) intermediate generated by dithionite reduction revealed low- and high-pH species with g and A((95,97)Mo) matrices nearly identical to those of SO, indicating a similar pentacoordinate active site in SoxCD1. However, no sulfite-induced reduction to Mo(V) was detected, nor could a strongly coupled (1)H signal or a phosphate-inhibited species be generated. This indicates that the outer coordination sphere controls substrate binding in SoxCD, permitting access only to protein-bound sulfur via the C-terminal tail of SoxY.

Structural basis for the oxidation of protein-bound sulfur by the sulfur cycle molybdohemo-enzyme sulfane dehydrogenase SoxCD.

The sulfur cycle enzyme sulfane dehydrogenase SoxCD is an essential component of the sulfur oxidation (Sox) enzyme system of Paracoccus pantotrophus. SoxCD catalyzes a six-electron oxidation reaction within the Sox cycle. SoxCD is an α(2)ß(2) heterotetrameric complex of the molybdenum cofactor-containing SoxC protein and the diheme c-type cytochrome SoxD with the heme domains D(1) and D(2). SoxCD(1) misses the heme-2 domain D(2) and is catalytically as active as SoxCD. The crystal structure of SoxCD(1) was solved at 1.33 Å. The substrate of SoxCD is the outer (sulfane) sulfur of Cys-110-persulfide located at the C-terminal peptide swinging arm of SoxY of the SoxYZ carrier complex. The SoxCD(1) substrate funnel toward the molybdopterin is narrow and partially shielded by side-chain residues of SoxD(1). For access of the sulfane-sulfur of SoxY-Cys-110 persulfide we propose that (i) the blockage by SoxD-Arg-98 is opened via interaction with the C terminus of SoxY and (ii) the C-terminal peptide VTIGGCGG of SoxY provides interactions with the entrance path such that the cysteine-bound persulfide is optimally positioned near the molybdenum atom. The subsequent oxidation reactions of the sulfane-sulfur are initiated by the nucleophilic attack of the persulfide anion on the molybdenum atom that is, in turn, reduced. The close proximity of heme-1 to the molybdopterin allows easy acceptance of the electrons. Because SoxYZ, SoxXA, and SoxB are already structurally characterized, with SoxCD(1) the structures of all key enzymes of the Sox cycle are known with atomic resolution.

Interaction between Sox proteins of two physiologically distinct bacteria and a new protein involved in thiosulfate oxidation.

Organisms using the thiosulfate-oxidizing Sox enzyme system fall into two groups: group 1 forms sulfur globules as intermediates (Allochromatium vinosum), group 2 does not (Paracoccus pantotrophus). While several components of their Sox systems are quite similar, i.e. the proteins SoxXA, SoxYZ and SoxB, they differ by Sox(CD)(2) which is absent in sulfur globule-forming organisms. Still, the respective enzymes are partly exchangeable in vitro: P. pantotrophus Sox enzymes work productively with A. vinosum SoxYZ whereas A. vinosum SoxB does not cooperate with the P. pantotrophus enzymes. Furthermore, A. vinosum SoxL, a rhodanese-like protein encoded immediately downstream of soxXAK, appears to play an important role in recycling SoxYZ as it increases thiosulfate depletion velocity in vitro without increasing the electron yield.

Identification of two inactive forms of the central sulfur cycle protein SoxYZ of Paracoccus pantotrophus.

The central protein of the sulfur-oxidizing enzyme system of Paracoccus pantotrophus, SoxYZ, reacts with three different Sox proteins. Its active site Cys110(Y) is on the carboxy-terminus of the SoxY subunit. SoxYZ "as isolated" consisted mainly of the catalytically inactive SoxY-Y(Z)(2) heterotetramer linked by a Cys110(Y)-Cys110(Y) interprotein disulfide. Sulfide activated SoxYZ "as isolated" 456-fold, reduced the disulfide, and yielded an active SoxYZ heterodimer. The reductant tris(2-carboxyethyl)phosphine (TCEP) inactivated SoxYZ. This form was not re-activated by sulfide, which identified it as a different inactive form. In analytical gel filtration, the elution of "TCEP-treated" SoxYZ was retarded compared to active SoxYZ, indicating a conformational change. The possible enzymes involved in the re-activation of each inactive form of SoxYZ are discussed.

Activation of the heterodimeric central complex SoxYZ of chemotrophic sulfur oxidation is linked to a conformational change and SoxY-Y interprotein disulfide formation.

The central protein of the four component sulfur oxidizing (Sox) enzyme system of Paracoccus pantotrophus, SoxYZ, carries at the SoxY subunit the covalently bound sulfur substrate which the other three proteins bind, oxidize, and release as sulfate. SoxYZ of different preparations resulted in different specific thiosulfate-oxidizing activities of the reconstituted Sox enzyme system. From these preparations SoxYZ was activated up to 24-fold by different reductants with disodium sulfide being the most effective and yielded a uniform specific activity of the Sox system. The activation comprised the activities with hydrogen sulfide, thiosulfate, and sulfite. Sulfide-activation decreased the predominant beta-sheet character of SoxYZ by 4%, which caused a change in its conformation as determined by infrared spectroscopy. Activation of SoxYZ by sulfide exposed the thiol of the C-terminal Cys-138 of SoxY as evident from alkylation by 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid. Also, SoxYZ activation enhanced the formation of the Sox(YZ)2 heterotetramer as evident from density gradient gel electrophoresis. The tetramer was formed due to an interprotein disulfide between SoxY to yield a SoxY-Y dimer as determined by combined high pressure liquid chromatography and mass spectrometry. The significance of the conformational change of SoxYZ and the interprotein disulfide between SoxY-Y is discussed.

The unusal redox centers of SoxXA, a novel c-type heme-enzyme essential for chemotrophic sulfur-oxidation of Paracoccus pantotrophus.

The heterodimeric hemoprotein SoxXA, essential for lithotrophic sulfur oxidation of the aerobic bacterium Paracoccus pantotrophus, was examined by a combination of spectroelectrochemistry and EPR spectroscopy. The EPR spectra for SoxXA showed contributions from three paramagnetic heme iron centers. One highly anisotropic low-spin (HALS) species (gmax = 3.45) and two "standard" cytochrome-like low-spin heme species with closely spaced g-tensor values were identified, LS1 (gz = 2.54, gy = 2.30, and gx = 1.87) and LS2 (gz = 2.43, gy = 2.26, and gx = 1.90). The crystal structure of SoxXA from P. pantotrophus confirmed the presence of three heme groups, one of which (heme 3) has a His/Met axial coordination and is located on the SoxX subunit [Dambe et al. (2005) J. Struct. Biol. 152, 229-234]. This heme was assigned to the HALS species in the EPR spectra of the isolated SoxX subunit. The LS1 and LS2 species were associated with heme 1 and heme 2 located on the SoxA subunit, both of which have EPR parameters characteristic for an axial His/thiolate coordination. Using thin-layer spectroelectrochemistry the midpoint potentials of heme 3 and heme 2 were determined: Em3 = +189 +/- 15 mV and Em2 = -432 +/- 15 mV (vs NHE, pH 7.0). Heme 1 was not reducible even with 20 mM titanium(III) citrate. The Em2 midpoint potential turned out to be pH dependent. It is proposed that heme 2 participates in the catalysis and that the cysteine persulfide ligation leads to the unusually low redox potential (-436 mV). The pH dependence of its redox potential may be due to (de)protonation of the Arg247 residue located in the active site.

The periplasmic thioredoxin SoxS plays a key role in activation in vivo of chemotrophic sulfur oxidation of Paracoccus pantotrophus.

The significance of the soxS gene product on chemotrophic sulfur oxidation of Paracoccus pantotrophus was investigated. The thioredoxin SoxS was purified, and the N-terminal amino acid sequence identified SoxS as the soxS gene product. The wild-type formed thiosulfate-oxidizing activity and Sox proteins during mixotrophic growth with succinate plus thiosulfate, while there was no activity, and only traces of Sox proteins, under heterotrophic conditions. The homogenote mutant strain GBOmegaS is unable to express the soxSR genes, of which soxR encodes a transcriptional regulator. Strain GBOmegaS cultivated mixotrophically showed about 22 % of the specific thiosulfate-dependent O(2) uptake rate of the wild-type, and when cultivated heterotrophically it produced 35 % activity. However, under both mixotrophic and heterotrophic conditions, strain GBOmegaS formed Sox proteins essential for sulfur oxidation in vitro at the same high level as the wild-type produced them during mixotrophic growth. Genetic complementation of strain GBOmegaS with soxS restored the activity upon mixotrophic and heterotrophic growth. Chemical complementation by reductants such as L-cysteine, DTT and tris(2-carboxyethyl)phosphine also restored the activity of strain GBOmegaS in the presence of chloramphenicol, which is an inhibitor of de novo protein synthesis. The data demonstrate that SoxS plays a key role in activation of the Sox enzyme system, and this suggests that SoxS is part of a novel type of redox control in P. pantotrophus.

The flavoprotein SoxF functions in chemotrophic thiosulfate oxidation of Paracoccus pantotrophus in vivo and in vitro.

Paracoccus pantotrophus strain GBsoxFDelta carries a deletion in the soxF gene that inactivates flavoprotein SoxF-sulfide dehydrogenase. This strain grew with thiosulfate slower than the wild type. GBsoxFDelta cells oxidized thiosulfate at a rate of 40% and hydrogen sulfide at a rate of 45% of the wild type. Complementation of GBsoxFDelta with plasmid pRIsoxF carrying the soxF gene increased these rates to 83% and 70%, respectively. However, GBsoxFDelta and GBsoxFDelta (pRIsoxF) oxidized thiosulfate and hydrogen sulfide to sulfate as evident from the yield of electrons. The thiosulfate oxidation rate of cell-free extracts of strain GBsoxFDelta was increased when supplemented with SoxF isolated from the wild type. However, SoxF did not affect the thiosulfate-oxidizing activity of the Sox enzyme system as reconstituted from the 'as-isolated' four Sox proteins. These data demonstrated that SoxF enhanced chemotrophic thiosulfate oxidation in vivo and acted on some component or condition present in whole cells and cell-free extracts but not present in the reconstituted system.

Structure of the cytochrome complex SoxXA of Paracoccus pantotrophus, a heme enzyme initiating chemotrophic sulfur oxidation.

The sulfur-oxidizing enzyme system (Sox) of the chemotroph Paracoccus pantotrophus is composed of several proteins, which together oxidize hydrogen sulfide, sulfur, thiosulfate or sulfite and transfers the gained electrons to the respiratory chain. The hetero-dimeric cytochrome c complex SoxXA functions as heme enzyme and links covalently the sulfur substrate to the thiol of the cysteine-138 residue of the SoxY protein of the SoxYZ complex. Here, we report the crystal structure of the c-type cytochrome complex SoxXA. The structure could be solved by molecular replacement and refined to a resolution of 1.9A identifying the axial heme-iron coordination involving an unusual Cys-251 thiolate of heme2. Distance measurements between the three heme groups provide deeper insight into the electron transport inside SoxXA and merge in a better understanding of the initial step of the aerobic sulfur oxidation process in chemotrophic bacteria.

Multifrequency EPR analysis of the dimanganese cluster of the putative sulfate thiohydrolase SoxB of Paracoccus pantotrophus.

A detailed analysis of the EPR signatures at X-band and Q-band of an enzyme (SoxB) involved in sulfur oxidation from Paracoccus pantotrophus is presented. EPR spectra are attributed to an exchange-coupled dimanganese Mn(2)(II,II) complex. An antiferromagnetic exchange interaction of J=-7.0 (+/-1) cm(-1) (H=-2JS ( 1 ) S ( 2 )) is evidenced by a careful examination of the temperature dependence of the EPR spectra. The spin Hamiltonian parameters for a total spin of S ( T ) =1, 2 and 3 are obtained and an inter-manganese distance of 3.4 (+/-0.1) A is estimated. The comparison with exchange coupling and inter-manganese distance data of other dimanganese proteins and model compounds leads to a tentative assignment of the Mn bridging ligands to bis(mu-hydroxo) (mu-carboxylato).

Prokaryotic sulfur oxidation.

Recent biochemical and genomic data differentiate the sulfur oxidation pathway of Archaea from those of Bacteria. From these data it is evident that members of the Alphaproteobacteria harbor the complete sulfur-oxidizing Sox enzyme system, whereas members of the beta and gamma subclass and the Chlorobiaceae contain sox gene clusters that lack the genes encoding sulfur dehydrogenase. This indicates a different pathway for oxidation of sulfur to sulfate. Acidophilic bacteria oxidize sulfur by a system different from the Sox enzyme system, as do chemotrophic endosymbiotic bacteria.

Sulfur dehydrogenase of Paracoccus pantotrophus: the heme-2 domain of the molybdoprotein cytochrome c complex is dispensable for catalytic activity.

Sulfur dehydrogenase, Sox(CD)(2), is an essential part of the sulfur-oxidizing enzyme system of the chemotrophic bacterium Paracoccus pantotrophus. Sox(CD)(2) is a alpha(2)beta(2) complex composed of the molybdoprotein SoxC (43 442 Da) and the hybrid diheme c-type cytochrome SoxD (37 637 Da). Sox(CD)(2) catalyzes the oxidation of protein-bound sulfur to sulfate with a unique six-electron transfer. Amino acid sequence analysis identified the heme-1 domain of SoxD proteins to be specific for sulfur dehydrogenases and to contain a novel ProCysMetXaaAspCys motif, while the heme-2 domain is related to various cytochromes c(2). Purification of sulfur dehydrogenase without protease inhibitor yielded a dimeric SoxCD(1) complex consisting of SoxC and SoxD(1) of 30 kDa, which contained only the heme-1 domain. The heme-2 domain was isolated as a new cytochrome SoxD(2) of about 13 kDa. Both hemes of SoxD in Sox(CD)(2) are redox-active with midpoint potentials at E(m)1 = 218 +/- 10 mV and E(m)2 = 268 +/- 10 mV, while SoxCD(1) and SoxD(2) both exhibit a midpoint potential of E(m) = 278 +/- 10 mV. Electrochemically induced FTIR difference spectra of Sox(CD)(2), SoxCD(1), and SoxD(2) were distinct. A carboxy group is protonated upon reduction of the SoxD(1) heme but not for SoxD(2). The specific activity of SoxCD(1) and Sox(CD)(2) was identical as was the yield of electrons with thiosulfate in the reconstituted Sox enzyme system. To examine the physiological significance of the heme-2 domain, a mutant was constructed that was deleted for the heme-2 domain, which produced SoxCD(1) and transferred electrons from thiosulfate to oxygen. These data demonstrated the crucial role of the heme-1 domain of SoxD for catalytic activity, electron yield, and transfer of the electrons to the cytoplasmic membrane, while the heme-2 domain mediated the alpha(2)beta(2) tetrameric structure of sulfur dehydrogenase.

Sulfide dehydrogenase activity of the monomeric flavoprotein SoxF of Paracoccus pantotrophus.

Flavocytochrome c-sulfide dehydrogenases (FCSDs) are complexes of a flavoprotein with a c-type cytochrome performing hydrogen sulfide-dependent cytochrome c reduction in vitro. The amino acid sequence analysis revealed that the phylogenetic relationship of different flavoproteins reflected the relationship of sulfur-oxidizing bacteria. The flavoprotein SoxF of Paracoccus pantotrophus is 29-67% identical to the flavoprotein subunit of FCSD of phototrophic sulfur-oxidizing bacteria. Purification of SoxF yielded a homogeneous emerald-green monomeric protein of 42 797 Da. SoxF catalyzed sulfide-dependent horse heart cytochrome c reduction at the optimum pH of 6.0 with a k(cat) of 3.9 s(-1), a K(m) of 2.3 microM for sulfide, and a K(m) of 116 microM for cytochrome c, as determined by nonlinear regression analysis. The yield of 1.9 mol of cytochrome c reduced per mole of sulfide suggests sulfur or polysulfide as the product. Sulfide dehydrogenase activity of SoxF was inhibited by sulfur (K(i) = 1.3 microM) and inactivated by sulfite. Cyanide (1 mM) inhibited SoxF activity at pH 6.0 by 25% and at pH 8.0 by 92%. Redox titrations in the infrared spectral range from 1800 to 1200 cm(-1) and in the visible spectral range from 400 to 700 nm both yielded a midpoint potential for SoxF of -555 +/- 10 mV versus Ag/AgCl at pH 7.5 and -440 +/- 20 mV versus Ag/AgCl at pH 6.0 (-232 mV versus SHE') and a transfer of 1.9 electrons. Electrochemically induced FTIR difference spectra of SoxF as compared to those of free flavin in solution suggested a strong cofactor interaction with the apoprotein. Furthermore, an activation/variation of SoxF during the redox cycles is observed. This is the first report of a monomeric flavoprotein with sulfide dehydrogenase activity.

Sulfur oxidation in Paracoccus pantotrophus: interaction of the sulfur-binding protein SoxYZ with the dimanganese SoxB protein.

The central protein of the sulfur-oxidizing enzyme system of Paracoccus pantotrophus, SoxYZ, formed complexes with subunits associated and covalently bound. In denaturing SDS-polyacrylamide gel electrophoresis (PAGE) SoxY migrated at 12 and SoxZ at 16kDa. SDS-PAGE of homogeneous SoxYZ without reductant separated dimeric complexes of 25, 29, and 32kDa identified by the N-terminal amino acid sequences as SoxY-Y, SoxY-Z, and SoxZ-Z, and subunit cleavage by reduction suggested their linkage via protein disulfide bonds. SoxYZ was reversibly redox active between -0.25 and 0.2V, as monitored by a combined electrochemical and FTIR spectroscopic approach. The dimanganese SoxB protein (58.611Da) converted the covalently linked heterodimer SoxY-Z to SoxYZ with associated subunits which in turn aggregated to the heterotetramer Sox(YZ)(2). This reaction depended on time and the SoxB concentration, and demonstrated the interaction of these two Sox proteins.

Inducible aluminum resistance of Acidiphilium cryptum and aluminum tolerance of other acidophilic bacteria.

Aluminum ions are highly soluble in acidic environments. Toxicity of aluminum ions for heterotrophic, facultatively and obligately chemolithoautotrophic acidophilic bacteria was examined. Acidiphilium cryptum grew in glucose-mineral medium, pH 3, containing 300 mM aluminum sulfate [Al(2)(SO(4))(3)] after a lag phase of about 120 h with a doubling time of 7.6 h, as compared to 5.2 h of growth without aluminum. Precultivation with 1 mM Al(2)(SO(4))(3) and transfer to a medium with 300 mM Al(2)(SO(4))(3) reduced the lag phase from 120 to 60 h, and immediate growth was observed when A. cryptum was precultivated with 50 mM Al(2)(SO(4))(3), suggesting an aluminum-induced resistance. Aluminum resistance was not induced by Fe(3+) ions and divalent cations. Upon exposure of A. cryptum to 300 mM Al(2)(SO(4))(3), the protein profile changed significantly as determined by SDS-PAGE. When other acidophiles were cultivated with 50-200 mM aluminum sulfate, no lag phase was observed while the growth rates and the cellular yields were significantly reduced. This growth response was observed with Acidobacterium capsulatum, Acidiphilium acidophilum, Acidithiobacillus ferrooxidans, and Acidithiobacillus thiooxidans. Precultivation of these strains with aluminum ions did not alter the growth response caused by aluminum. The content of A. cryptum cultivated with 300 mM Al(2)(SO(4))(3)was 0.44 microg Al/mg cell dry weight, while that of the other strains cultivated with 50 mM Al(2)(SO(4))(3) ranged from 0.30 to 3.47 microg Al/mg cell dry weight.