Structure of 3-mercaptopropionic acid dioxygenase with a substrate analog reveals bidentate substrate binding at the iron center.

Thiol dioxygenases are a subset of nonheme iron oxygenases that catalyze the formation of sulfinic acids from sulfhydryl-containing substrates and dioxygen. Among this class, cysteine dioxygenases (CDOs) and 3-mercaptopropionic acid dioxygenases (3MDOs) are the best characterized, and the mode of substrate binding for CDOs is well understood. However, the manner in which 3-mercaptopropionic acid (3MPA) coordinates to the nonheme iron site in 3MDO remains a matter of debate. A model for bidentate 3MPA coordination at the 3MDO Fe-site has been proposed on the basis of computational docking, whereas steady-state kinetics and EPR spectroscopic measurements suggest a thiolate-only coordination of the substrate. To address this gap in knowledge, we determined the structure of Azobacter vinelandii 3MDO (Av3MDO) in complex with the substrate analog and competitive inhibitor, 3-hydroxypropionic acid (3HPA). The structure together with DFT computational modeling demonstrates that 3HPA and 3MPA associate with iron as chelate complexes with the substrate-carboxylate group forming an additional interaction with Arg168 and the thiol bound at the same position as in CDO. A chloride ligand was bound to iron in the coordination site assigned as the O2-binding site. Supporting HYSCORE spectroscopic experiments were performed on the (3MPA/NO)-bound Av3MDO iron nitrosyl (S = 3/2) site. In combination with spectroscopic simulations and optimized DFT models, this work provides an experimentally verified model of the Av3MDO enzyme-substrate complex, effectively resolving a debate in the literature regarding the preferred substrate-binding denticity. These results elegantly explain the observed 3MDO substrate specificity, but leave unanswered questions regarding the mechanism of substrate-gated reactivity with dioxygen.

Initial report of γ-aminobutyric acidergic locomotion regulatory system and its 3-mercaptopropionic acid-sensitivity in metamorphic juvenile of sea urchin, Hemicentrotus pulcherrimus.

The γ-aminobutyric acid (GABA) signal transmission system (GSTS) contributes to larval swimming through the regulation of ciliary beating. However, whether this system also contributes to the primary podia (PP)-generated motility of juveniles remained unclear. The present study aimed to elucidate the involvement of the GSTS in the motility of metamorphic juveniles (juveniles) (1) by immunohistochemically elucidating the location of molecular constituents of the PP, and (2) by inhibiting the activity of GΑΒΑ decarboxylase (GAD) with 3-mercaptopropionic acid (3-MPA). During metamorphosis, the echinus rudiment protrudes its PP out of the body surface in 8-arm plutei. The PP expresses immunopositive signal (-IS) of GAD, GABA, GABAA receptor and tropomyosin, and is constituted with the GABA-IS negative distal tip and the GABA/GAD-IS gaiter region. The latter radiates distal projections to the disc that contains a GAD-IS cellular network. The juvenile body cavity houses a GABA/ßIII-tubulin-IS Penta-radial ring (PrR) that extends branches into each PP and several bridges to the GAD/GABA-IS Penta-radial plate (PrP) on the oral side but does not reach to the gaiter region. 3-MPA reversibly inhibits the juvenile motility and GABA-IS expression in the PrR/PrP complex. This indicates that the complex is the major contributor to the GABAergic motility in juveniles.

Outer-Sphere Tyrosine 159 within the 3-Mercaptopropionic Acid Dioxygenase S-H-Y Motif Gates Substrate-Coordination Denticity at the Non-Heme Iron Active Site.

Thiol dioxygenases are non-heme mononuclear iron enzymes that catalyze the O2-dependent oxidation of free thiols (-SH) to produce the corresponding sulfinic acid (-SO2-). Regardless of the phylogenic domain, the active site for this enzyme class is typically comprised of two major features: (1) a mononuclear ferrous iron coordinated by three protein-derived histidines and (2) a conserved sequence of outer Fe-coordination-sphere amino acids (Ser-His-Tyr) spatially adjacent to the iron site (∼3 Å). Here, we utilize a promiscuous 3-mercaptopropionic acid dioxygenase cloned from Azotobacter vinelandii (Av MDO) to explore the function of the conserved S-H-Y motif. This enzyme exhibits activity with 3-mercaptopropionic acid (3mpa), l-cysteine (cys), as well as several other thiol-bearing substrates, thus making it an ideal system to study the influence of residues within the highly conserved S-H-Y motif (H157 and Y159) on substrate specificity and reactivity. The pKa values for these residues were determined by pH-dependent steady-state kinetics, and their assignments verified by comparison to H157N and Y159F variants. Complementary electron paramagnetic resonance and Mössbauer studies demonstrate a network of hydrogen bonds connecting H157-Y159 and Fe-bound ligands within the enzymatic Fe site. Crucially, these experiments suggest that the hydroxyl group of Y159 hydrogen bonds to Fe-bound NO and, by extension, Fe-bound oxygen during native catalysis. This interaction alters both the NO binding affinity and rhombicity of the 3mpa-bound iron-nitrosyl site. In addition, Fe coordination of cys is switched from thiolate only to bidentate (thiolate/amine) for the Y159F variant, indicating that perturbations within the S-H-Y proton relay network also influence cys Fe binding denticity.

Coating Metal Nanoparticle Surfaces with Small Organic Molecules Can Reduce Nonspecific Cell Uptake.

Elucidation of mechanisms of uptake of nanoparticles by cells and methods to prevent this uptake is essential for many applications of nanoparticles. Most recent studies have focused on the role of proteins that coat nanoparticles and have employed PEGylation, particularly dense coatings of PEG, to reduce protein opsonization and cell uptake. Here we show that small molecule coatings on metallic nanoparticles can markedly reduce cell uptake for very sparsely PEGylated nanoparticles. Similar results were obtained in media with and without proteins, suggesting that protein opsonization is not the primary driver of this phenomenon. The reduction in cell uptake is proportional to the degree of surface coverage by the small molecules. Probing cell uptake pathways using inhibitors suggested that the primary role of increased surface coverage is to reduce nanoparticles' interactions with the scavenger receptors. This work highlights an under-investigated mechanism of cell uptake that may have played a role in many other studies and also suggests that a wide variety of molecules can be used alongside PEGylation to stably passivate nanoparticle surfaces for low cell uptake.

Thiol dioxygenase turnover yields benzothiazole products from 2-mercaptoaniline and O2-dependent oxidation of primary alcohols.

Thiol dioxygenases are non-heme mononuclear iron enzymes that catalyze the O2-dependent oxidation of free thiols (-SH) to produce the corresponding sulfinic acid (-SO2-). Previous chemical rescue studies identified a putative FeIII-O2- intermediate that precedes substrate oxidation in Mus musculus cysteine dioxygenase (Mm CDO). Given that a similar reactive intermediate has been identified in the extradiol dioxygenase 2, 3-HCPD, it is conceivable that these enzymes share other mechanistic features with regard to substrate oxidation. To explore this possibility, enzymatic reactions with Mm CDO (as well as the bacterial 3-mercaptopropionic acid dioxygenase, Av MDO) were performed using a substrate analogue (2-mercaptoaniline, 2ma). This aromatic thiol closely approximates the catecholic substrate of homoprotocatechuate of 2, 3-HPCD while maintaining the 2-carbon thiol-amine separation preferred by Mm CDO. Remarkably, both enzymes exhibit 2ma-gated O2-consumption; however, none of the expected products for thiol dioxygenase or intra/extradiol dioxygenase reactions were observed. Instead, benzothiazoles are produced by the condensation of 2ma with aldehydes formed by an off-pathway oxidation of primary alcohols added to aqueous reactions to solubilize the substrate. The observed oxidation of 1º-alcohols in 2ma-reactions is consistent with the formation of a high-valent intermediate similar to what has been reported for cytochrome P450 and mononuclear iron model complexes.

Occurrence and biosynthesis of 3-mercaptopropionic acid in Methanocaldococcus jannaschii.

In a non-targeted analysis of thiol-containing compounds in the hyperthermophilic methanogen Methanocaldococcus jannaschii, we discovered three unexpected metabolites: 3-mercaptopropionic acid (MPA), 2-hydroxy-4-mercaptobutyric acid (HMBA) and 4-mercapto-2-oxobutyric acid (MOB). HMBA and MOB have never been reported as natural products, while MPA is highly prevalent in aquatic environments as a result of biotic and abiotic processing of sulfur-containing compounds. This report provides evidence that HMBA and MOB are part of a biosynthetic pathway to generate MPA in M. jannaschii We show that HMBA can be biosynthesized from malate semialdehyde and hydrogen sulfide, likely using a mechanism similar to that proposed for coenzyme M, coenzyme B and homocysteine biosynthesis in methanogens, where an aldehyde is converted to a thiol. The L-sulfolactate dehydrogenase, derived from the MJ1425 gene, is shown to catalyze the NAD-dependent oxidation of HMBA to MOB. Finally, we demonstrate that HMBA can be used as a biosynthetic precursor to MPA in M. jannaschii cell extracts. This proposed pathway may contribute to the wide occurrence of MPA in marine environments and indicates that MPA must serve some important function in M. jannaschii.

Substrate and Cofactor Range Differences of Two Cysteine Dioxygenases from Ralstonia eutropha H16.

Cysteine dioxygenases (Cdos), which catalyze the sulfoxidation of cysteine to cysteine sulfinic acid (CSA), have been extensively studied in eukaryotes because of their roles in several diseases. In contrast, only a few prokaryotic enzymes of this type have been investigated. In Ralstonia eutropha H16, two Cdo homologues (CdoA and CdoB) have been identified previously. In vivo studies showed that Escherichia coli cells expressing CdoA could convert 3-mercaptopropionate (3MP) to 3-sulfinopropionate (3SP), whereas no 3SP could be detected in cells expressing CdoB. The objective of this study was to confirm these findings and to study both enzymes in detail by performing an in vitro characterization. The proteins were heterologously expressed and purified to apparent homogeneity by immobilized metal chelate affinity chromatography (IMAC). Subsequent analysis of the enzyme activities revealed striking differences with regard to their substrate ranges and their specificities for the transition metal cofactor, e.g., CdoA catalyzed the sulfoxidation of 3MP to a 3-fold-greater extent than the sulfoxidation of cysteine, whereas CdoB converted only cysteine. Moreover, the dependency of the activities of the Cdos from R. eutropha H16 on the metal cofactor in the active center could be demonstrated. The importance of CdoA for the metabolism of the sulfur compounds 3,3'-thiodipropionic acid (TDP) and 3,3'-dithiodipropionic acid (DTDP) by further converting their degradation product, 3MP, was confirmed. Since 3MP can also function as a precursor for polythioester (PTE) synthesis in R. eutropha H16, deletion of cdoA might enable increased synthesis of PTEs.

Release of ecologically relevant metabolites by the cyanobacterium Synechococcus elongates CCMP 1631.

Photoautotrophic plankton in the surface ocean release organic compounds that fuel secondary production by heterotrophic bacteria. Here we show that an abundant marine cyanobacterium, Synechococcus elongatus, contributes a variety of nitrogen-rich and sulfur-containing compounds to dissolved organic matter. A combination of targeted and untargeted metabolomics and genomic tools was used to characterize the intracellular and extracellular metabolites of S. elongatus. Aromatic compounds, such as 4-hydroxybenzoic acid and phenylalanine, as well as nucleosides (e.g. thymidine, 5'-methylthioadenosine, xanthosine), the organosulfur compound 3-mercaptopropionate, and the plant auxin indole 3-acetic acid, were released by S. elongatus at multiple time points during its growth. Further, the amino acid kynurenine was found to accumulate in the media even though it was not present in the predicted metabolome of S. elongatus. This indicates that some metabolites, including those not predicted by an organism's genome, are likely excreted into the environment as waste; however, these molecules may have broader ecological relevance if they are labile to nearby microbes. The compounds described herein provide excellent targets for quantitative analysis in field settings to assess the source and lability of dissolved organic matter in situ.

Structures of Arg- and Gln-type bacterial cysteine dioxygenase homologs.

In some bacteria, cysteine is converted to cysteine sulfinic acid by cysteine dioxygenases (CDO) that are only ∼15-30% identical in sequence to mammalian CDOs. Among bacterial proteins having this range of sequence similarity to mammalian CDO are some that conserve an active site Arg residue ("Arg-type" enzymes) and some having a Gln substituted for this Arg ("Gln-type" enzymes). Here, we describe a structure from each of these enzyme types by analyzing structures originally solved by structural genomics groups but not published: a Bacillus subtilis "Arg-type" enzyme that has cysteine dioxygenase activity (BsCDO), and a Ralstonia eutropha "Gln-type" CDO homolog of uncharacterized activity (ReCDOhom). The BsCDO active site is well conserved with mammalian CDO, and a cysteine complex captured in the active site confirms that the cysteine binding mode is also similar. The ReCDOhom structure reveals a new active site Arg residue that is hydrogen bonding to an iron-bound diatomic molecule we have interpreted as dioxygen. Notably, the Arg position is not compatible with the mode of Cys binding seen in both rat CDO and BsCDO. As sequence alignments show that this newly discovered active site Arg is well conserved among "Gln-type" CDO enzymes, we conclude that the "Gln-type" CDO homologs are not authentic CDOs but will have substrate specificity more similar to 3-mercaptopropionate dioxygenases.

Formation by yeast of 2-furanmethanethiol and ethyl 2-mercaptopropionate aroma compounds in Japanese soy sauce.

Two aroma compounds of volatile thiols, 2-furanmethanethiol (2FM) and ethyl 2-mercaptopropionate (ET2MP), were formed in five types of Japanese soy sauce during fermentation by yeast. The concentrations of 2FM and ET2MP in the soy sauce samples increased during alcoholic fermentation. The concentrations of 2FM and ET2MP were higher in the soy sauce fermented by Zygosaccharomyces rouxii than in that fermented by Candida versatilis. The enantiomers of ET2MP were separated by gas chromatography in a capillary column. The average enantiomeric ratio of ET2MP in the soy sauce was approximately 1:1. 2FM was formed by yeast in a medium prepared from cysteine and furfural, and cysteine is considered the key precursor of 2FM by yeast in soy sauce.

Polythioester synthesis in Ralstonia eutropha H16: novel insights into 3,3'-thiodipropionic acid and 3,3'-dithiodipropionic acid catabolism.

Ralstonia eutropha H16 is capable of utilizing 3,3'-thiodipropionic acid (TDP) and 3,3'-dithiodipropionic acid (DTDP) as precursor substrates for biosynthesis of a polythioester (PTE) heteropolymer consisting of 3-hydroxybutyric acid (3HB) and 3-mercaptopropionic acid (3MP). To elucidate the hitherto unknown catabolic pathways of TDP and DTDP in R. eutropha H16, 19 defined deletion mutants were generated based on extensive functional genome analyses. Deletions of two ABC-type transporter clusters (H16_A0357-0359, H16_A3658-3660) resulted in an alteration of poly(3HB-co-3MP) composition with TDP as precursor to only 10.2±1.9mol% 3MP in comparison to 15.1±5.5mol% in the wild type. A mutant strain of H16 lacking Bordetella uptake gene-like substrate binding proteins (H16_A2779, H16_A0337) incorporated only 7.4±3.8mol% 3MP into PTE heteropolymers with DTDP as precursor in comparison to 24.5±14.5mol% in the wild type. Therefore, both gene products are probably involved in transport processes of this compound into the cells. However, the most significant reduction in 3MP contents of the heteropolymers with DTDP as precursor occurred upon the deletion of a gene encoding the putative thiol-disulfide interchange protein DsbD (H16_A3455, 3.9±2.6mol% 3MP). DsbD is proposed to be involved in the reduction of DTDP into two molecules of 3MP, the common cleavage product of TDP and DTDP.

Mechanistic implications of persulfenate and persulfide binding in the active site of cysteine dioxygenase.

Describing the organization of substrates and substrate analogues in the active site of cysteine dioxygenase identifies potential intermediates in this critical yet poorly understood reaction, the oxidation of cysteine to cysteine sulfinic acid. The fortuitous formation of persulfides under crystallization conditions has allowed their binding in the active site of cysteine dioxygenase to be studied. The crystal structures of cysteine persulfide and 3-mercaptopropionic acid persulfide bound to iron(II) in the active site show that binding of the persulfide occurs via the distal sulfide and, in the case of the cysteine persulfide, the amine also binds. Persulfide was detected by mass spectrometry in both the crystal and the drop, suggesting its origin is chemical rather than enzymatic. A mechanism involving the formation of the relevant disulfide from sulfide produced by hydrolysis of dithionite is proposed. In comparison, persulfenate {observed bound to cysteine dioxygenase [Simmons, C. R., et al. (2008) Biochemistry 47, 11390]} is shown through mass spectrometry to occur only in the crystal and not in the surrounding drop, suggesting that in the crystalline state the persulfenate does not lie on the reaction pathway. Stabilization of both the persulfenate and the persulfides does, however, suggest the position in which dioxygen binds during catalysis.

Employing a recombinant strain of Advenella mimigardefordensis for biotechnical production of Homopolythioesters from 3,3'-dithiodipropionic acid.

Advenella mimigardefordensis strain DPN7(T) was genetically modified to produce poly(3-mercaptopropionic acid) (PMP) homopolymer by exploiting the recently unraveled process of 3,3'-dithiodipropionic acid (DTDP) catabolism. Production was achieved by systematically engineering the metabolism of this strain as follows: (i) deletion of its inherent 3MP dioxygenase-encoding gene (mdo), (ii) introduction of the buk-ptb operon (genes encoding the butyrate kinase, Buk, and the phosphotransbutyrylase, Ptb, from Clostridium acetobutylicum), and (iii) overexpression of its own polyhydroxyalkanoate synthase (phaC(Am)). These measures yielded the potent PMP production strain A. mimigardefordensis strain SHX22. The deletion of mdo was required for adequate synthesis of PMP due to the resulting accumulation of 3MP during utilization of DTDP. Overexpression of the plasmid-borne buk-ptb operon caused a severe growth repression. This effect was overcome by inserting this operon into the genome. Polyhydroxyalkanoate (PHA) synthases from different origins were compared. The native PHA synthase of A. mimigardefordensis (phaC(Am)) was obviously the best choice to establish homopolythioester production in this strain. In addition, the cultivation conditions, including an appropriate provision of the carbon source, were further optimized to enhance PMP production. The engineered strain accumulated PMP up to approximately 25% (wt/wt) of the cell dry weight when cultivated in mineral salts medium containing glycerol as the carbon source in addition to DTDP as the sulfur-providing precursor. According to our knowledge, this is the first report of PMP homopolymer production by a metabolically engineered bacterium using DTDP, which is nontoxic, as the precursor substrate.

Investigations on the microbial catabolism of the organic sulfur compounds TDP and DTDP in Ralstonia eutropha H16 employing DNA microarrays.

In this study, we have investigated the transcriptome of Ralstonia eutropha H16 during cultivation with gluconate in presence of 3,3'-thiodipropionic acid (TDP) or 3,3'-dithiodipropionic acid (DTDP) during biosynthesis of poly(3-hydroxybutyrate-co-3-mercaptopropionate). Genome-wide transcriptome analyses revealed several genes which were upregulated during cultivation in presence of the above-mentioned compounds. Obtained data strongly suggest that two ABC-type transport system and three probable extracytoplasmic solute receptors mediate the uptake of TDP and DTDP, respectively. In addition, genes encoding the hydrolase S-adenosylhomocysteinase AhcY and the thiol-disulfide interchange proteins DsbA, DsbD, and FrnE were upregulated during cultivation on DTDP and, in case of AhcY and FrnE, on TDP as well. It is assumed that the corresponding enzymes are involved in the cleavage of TDP and DTDP. Several genes of the fatty acid metabolism exhibited increased expression levels: genes encoding two acetyltransferases, a predicted acyltransferase, the acetoacetyl-CoA reductase phaB3, an enoyl-CoA hydratase as well as an acyl dehydratase, an acetyl-CoA synthetase, two acyl-CoA dehydrogenases, the methylmalonyl-CoA mutase encoded by sbm1 and sbm2 and phaY1 were detected. Furthermore, ORF H16_A0217 encoding a hypothetical protein and exhibiting 54% amino acids identical to an acyl-CoA thioesterase from Saccharomonospora viridis was found to be highly upregulated. As the 2-methylcitrate synthase PrpC exhibited a three- to fourfold increased activity in cells grown in presence of TDP or DTDP as compared to gluconate, metabolization of the cleavage products 3MP and 3-hydroxypropionate to propionyl-CoA is proposed.

Dihydrolipoamide dehydrogenases of Advenella mimigardefordensis and Ralstonia eutropha catalyze cleavage of 3,3'-dithiodipropionic acid into 3-mercaptopropionic acid.

The catabolism of the disulfide 3,3'-dithiodipropionic acid (DTDP) is initiated by the reduction of its disulfide bond. Three independent Tn5::mob-induced mutants of Advenella mimigardefordensis strain DPN7(T) were isolated that had lost the ability to utilize DTDP as the sole source of carbon and energy and that harbored the transposon insertions in three different sites of the same dihydrolipoamide dehydrogenase gene encoding the E3 subunit of the pyruvate dehydrogenase multi-enzyme complex of this bacterium (LpdA(Am)). LpdA(Am) was analyzed in silico and compared to homologous proteins, thereby revealing high similarities to the orthologue in Ralstonia eutropha H16 (PdhL(Re)). Both bacteria are able to cleave DTDP into two molecules of 3-mercaptopropionic acid (3MP). A. mimigardefordensis DPN7(T) converted 3MP to 3-sulfinopropionic acid, whereas R. eutropha H16 showed no growth with DTDP as the sole carbon source but was instead capable of synthesizing heteropolythioesters using the resulting cleavage product 3MP. Subsequently, the genes lpdA(Am) and pdhL(Re) were cloned, heterologously expressed in Escherichia coli applying the pET23a expression system, purified, and assayed by monitoring the oxidation of NADH. The physiological substrate lipoamide was reduced to dihydrolipoamide with specific activities of 1,833 mkat/kg of protein (LpdA(Am)) or 1,667 mkat/kg of protein (PdhL(Re)). Reduction of DTDP was also unequivocally detected with the purified enzymes, although the specific enzyme activities were much lower: 0.7 and 0.5 mkat/kg protein, respectively.

Biological assays and noncovalent interactions of pyridine-2-carbaldehyde thiosemicarbazonecopper(II) drugs with [poly(dA-dT)](2), [poly(dG-dC)] (2), and calf thymus DNA.

The interaction of the Cu(II) drugs CuL(NO(3)) and CuL'(NO(3)) (HL is pyridine-2-carbaldehyde thiosemicarbazone and HL' is pyridine-2-carbaldehyde 4N-methylthiosemicarbazone, in water named [CuL](+) and [CuL'](+)) with [poly(dA-dT)](2), [poly(dG-dC)](2), and calf thymus (CT) DNA has been probed in aqueous solution at pH 6.0, I = 0.1 M, and T = 25 degrees C by absorbance, fluorescence, circular dichroism, and viscosity measurements. The results reveal that these drugs act as groove binders with [poly(dA-dT)](2), with a site size n = 6-7, whereas they act as external binders with [poly(dG-dC)](2) and/or CT-DNA, thus establishing overall electrostatic interaction with n = 1. The binding constants with [CuL'](+) were slightly larger than with [CuL](+). The title compounds display some cleavage activity in the presence of thiols, bringing about the rupture of the DNA strands by the reactive oxygen species formed by reoxidation of Cu(I) to Cu(II); this feature was not observed in the absence of thiols. Mutagenic assays performed both in the presence and in the absence of S9 mix, probed by the Ames test on TA 98, TA 100, and TA 102, were negative. Weak genotoxic activity was detected for [CuL](+) and [CuL'](+), with a significative dose-response effect for [CuL'](+), which was shown to be more cytotoxic in the Ames test and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell proliferation assays. Methylation of the terminal NH(2) group enhances the antiproliferative activity of the pyridine-2-carbaldehyde thiosemicarbazones.

3-mercaptopropionate dioxygenase, a cysteine dioxygenase homologue, catalyzes the initial step of 3-mercaptopropionate catabolism in the 3,3-thiodipropionic acid-degrading bacterium variovorax paradoxus.

The thioether 3,3-thiodipropionic acid can be used as precursor substrate for biotechnological synthesis of 3-mercaptopropionic acid-containing polythioesters. Therefore, the hitherto unknown catabolism of this compound was elucidated to engineer novel and improved polythioester biosynthesis pathways in the future. Bacteria capable of using 3,3-thiodipropionic acid as the sole source of carbon and energy for growth were enriched from the environment. From eleven isolates, TBEA3, TBEA6, and SFWT were morphologically and physiologically characterized. Their 16 S rDNAs and other features affiliated these isolates to the beta-subgroup of the proteobacteria. Tn5::mob mutagenesis of isolate Variovorax paradoxus TBEA6 yielded ten mutants fully or partially impaired in growth on 3,3-thiodipropionic acid. Genotypic characterization of two 3,3-thiodipropionic acid-negative mutants demonstrated the involvement of a bacterial cysteine dioxygenase (EC 1.13.11.22) homologue in the further catabolism of the 3,3-thiodipropionic acid cleavage product 3-mercaptopropionic acid. Detection of 3-sulfinopropionate in the supernatant of one of these mutants during cultivation on 3,3-thiodipropionic acid as well as in vivo and in vitro enzyme assays using purified protein demonstrated oxygenation of 3-mercaptopropionic acid to 3-sulfinopropionate by this enzyme; cysteine and cysteamine were not used as substrate. Beside cysteine dioxygenase and cysteamine dioxygenase, this 3-mercaptopropionic acid dioxygenase is the third example for a thiol dioxygenase and the first report about the microbial catabolism of 3-mercaptopropionic acid. Insertion of Tn5::mob in a gene putatively coding for a family III acyl-CoA-transferase resulted in the accumulation of 3-sulfinopropionate during cultivation on 3,3-thiodipropionic acid, indicating that this compound is further metabolized to 3-sulfinopropionyl-CoA and subsequently to propionyl-CoA.

Novel pathway for catabolism of the organic sulfur compound 3,3'-dithiodipropionic acid via 3-mercaptopropionic acid and 3-Sulfinopropionic acid to propionyl-coenzyme A by the aerobic bacterium Tetrathiobacter mimigardefordensis strain DPN7.

The hitherto unstudied microbial degradation of the organic disulfide 3,3'-dithiodipropionic acid (DTDP) was investigated with the recently described bacterium Tetrathiobacter mimigardefordensis strain DPN7(T) (DSM 17166(T); LMG 22922(T)), which is able to use DTDP as the sole carbon source for growth. 3-Mercaptopropionic acid (3MP) and 3-sulfinopropionic acid (3SP) were detected in the growth medium and occurred as intermediates during DTDP degradation. To identify genes coding for enzymes of DTDP catabolism, Tn5::mob-induced mutants of T. mimigardefordensis were generated. Screening of transposon mutant libraries yielded many mutants fully or partially impaired in utilizing DTDP as a carbon source. Mapping of the insertion loci in some mutants identified four disrupted open reading frames (ORFs) with putative metabolic functions. The ORFs were assigned function on the basis of homologies with lpdA (EC 1.8.1.4), cdo (EC 1.13.11.20), sucCD (EC 6.2.1.5), and acnB (EC 4.2.1.3). Tn5::mob insertions occurred additionally in the vicinity of heat shock protein-encoding genes. The predicted function of the LpdA homologue in T. mimigardefordensis is cleavage of the disulfide bond of DTDP to form two molecules of 3MP. Cdo catalyzes the conversion of the sulfhydryl group of 3MP, yielding the corresponding sulfinic acid, 3SP. SucCD exhibits thiokinase activity, ligating coenzyme A (CoA) with 3SP to form 3SP-CoA. Afterwards, an elimination of sulfite via a putative desulfinase is expected. acnB encodes a putative 2-methylisocitrate dehydratase. Therefore, a new pathway is proposed for the catabolism of DTDP via 3MP, 3SP, and 3SP-CoA toward propionyl-CoA, which is then further catabolized via the 2-methylcitric acid cycle in T. mimigardefordensis.

Mercaptopropionaldehyde from homocysteine: implications for Alzheimer's disease.

There has been evidence for a causal relationship between homocysteine and Alzheimer's disease for several years but the mechanism is unclear. In vivo, some homocysteine is converted to the thiolactone. This report describes a novel reaction between homocysteine thiolactone and dehydroascorbic acid in which the homocysteine thiolactone is converted to 3-mercaptopropionaldehyde. This product is shown to react with proteins causing their precipitation (probably by cross-linking). The two reactions are extremely facile and appear to be physiologically compatible suggesting a mechanism by which homocysteine may promote the deposition of proteins in nerve cells as amyloid plaques and fibrillary tangles.

Improving simvastatin bioconversion in Escherichia coli by deletion of bioH.

Simvastatin is an important cholesterol lowering compound and is currently synthesized from the natural product lovastatin via multistep chemical synthesis. We have previously reported the use of an Escherichia coli strain BL21(DE3)/pAW31 as the host for whole-cell biocatalytic conversion of monacolin J acid to simvastatin acid. During fermentation and bioconversion, unknown E. coli enzyme(s) hydrolyzed the membrane permeable thioester substrate dimethylbutyryl-S-methyl mercaptopropionate (DMB-S-MMP) to the free acid, significantly decreased the efficiencies of the whole-cell bioconversion and the downstream purification steps. Using the Keio K-12 Singe-Gene Knockout collection, we identified BioH as the sole enzyme responsible for the observed substrate hydrolysis. Purification and reconstitution of E. coli BioH activity in vitro confirmed its function. BioH catalyzed the rapid hydrolysis of DMB-S-MMP with kcat and Km values of 260+/-45 s(-1) and 229+/-26 microM, respectively. This is in agreement with previous reports that BioH can function as a carboxylesterase towards fatty acid esters. YT2, which is a delta bioH mutant of BL21(DE3), did not hydrolyze DMB-S-MMP during prolonged fermentation and was used as an alternative host for whole-cell biocatalysis. The rate of simvastatin acid synthesis in YT2 was significantly faster than in BL21(DE3) and 99% conversion of 15 mM simvastatin acid in less than 12 h was achieved. Furthermore, the engineered host required significantly less DMB-S-MMP to be added to accomplish complete conversion. Finally, simvastatin acid synthesized using YT2 can be readily purified from fermentation broth and no additional steps to remove the hydrolyzed dimethylbutyryl-S-mercaptopropionic acid is required. Together, the proteomic and metabolic engineering approaches render the whole-cell biocatalytic process more robust and economically attractive.