QM calculations predict the energetics and infrared spectra of transient glutamine isomers in LOV photoreceptors.

Photosensory receptors containing the flavin-binding light-oxygen-voltage (LOV) domain are modular proteins that fulfil a variety of biological functions ranging from gene expression to phototropism. The LOV photocycle is initiated by blue-light and involves a cascade of intermediate species, including an electronically excited triplet state, that leads to covalent bond formation between the flavin mononucleotide (FMN) chromophore and a nearby cysteine residue. Subsequent conformational changes in the polypeptide chain arise due to the remodelling of the hydrogen bond network in the cofactor binding pocket, whereby a conserved glutamine residue plays a key role in coupling FMN photochemistry with LOV photobiology. Although the dark-to-light transition of LOV photosensors has been previously addressed by spectroscopy and computational approaches, the mechanistic basis of the underlying reactions is still not well understood. Here we present a detailed computational study of three distinct LOV domains: EL222 from Erythrobacter litoralis, AsLOV2 from the second LOV domain of Avena sativa phototropin 1, and RsLOV from Rhodobacter sphaeroides LOV protein. Extended protein-chromophore models containing all known crucial residues involved in the initial steps (femtosecond-to-microsecond) of the photocycle were employed. Energies and rotational barriers were calculated for possible rotamers and tautomers of the critical glutamine side chain, which allowed us to postulate the most energetically favoured glutamine orientation for each LOV domain along the assumed reaction path. In turn, for each evolving species, infrared difference spectra were constructed and compared to experimental EL222 and AsLOV2 transient infrared spectra, the former from original work presented here and the latter from the literature. The good agreement between theory and experiment permitted the assignment of the majority of observed bands, notably the ∼1635 cm-1 transient of the adduct state to the carbonyl of the glutamine side chain after rotation. Moreover, both the energetic and spectroscopic approaches converge in suggesting a facile glutamine flip at the adduct intermediate for EL222 and more so for AsLOV2, while for RsLOV the glutamine keeps its initial configuration. Additionally, the computed infrared shifts of the glutamine and interacting residues could guide experimental research addressing early events of signal transduction in LOV proteins.

Structural and functional analysis of lignostilbene dioxygenases from Sphingobium sp. SYK-6.

Lignostilbene-α,ß-dioxygenases (LSDs) are iron-dependent oxygenases involved in the catabolism of lignin-derived stilbenes. Sphingobium sp. SYK-6 contains eight LSD homologs with undetermined physiological roles. To investigate which homologs are involved in the catabolism of dehydrodiconiferyl alcohol (DCA), derived from ß-5 linked lignin subunits, we heterologously produced the enzymes and screened their activities in lysates. The seven soluble enzymes all cleaved lignostilbene, but only LSD2, LSD3, and LSD4 exhibited high specific activity for 3-(4-hydroxy-3-(4-hydroxy-3-methoxystyryl)-5-methoxyphenyl) acrylate (DCA-S) relative to lignostilbene. LSD4 catalyzed the cleavage of DCA-S to 5-formylferulate and vanillin and cleaved lignostilbene and DCA-S (∼106 M-1 s-1) with tenfold greater specificity than pterostilbene and resveratrol. X-ray crystal structures of native LSD4 and the catalytically inactive cobalt-substituted Co-LSD4 at 1.45 Å resolution revealed the same fold, metal ion coordination, and edge-to-edge dimeric structure as observed in related enzymes. Key catalytic residues, Phe-59, Tyr-101, and Lys-134, were also conserved. Structures of Co-LSD4·vanillin, Co-LSD4·lignostilbene, and Co-LSD4·DCA-S complexes revealed that Ser-283 forms a hydrogen bond with the hydroxyl group of the ferulyl portion of DCA-S. This residue is conserved in LSD2 and LSD4 but is alanine in LSD3. Substitution of Ser-283 with Ala minimally affected the specificity of LSD4 for either lignostilbene or DCA-S. By contrast, substitution with phenylalanine, as occurs in LSD5 and LSD6, reduced the specificity of the enzyme for both substrates by an order of magnitude. This study expands our understanding of an LSD critical to DCA catabolism as well as the physiological roles of other LSDs and their determinants of substrate specificity.

Biochemical characterization and molecular docking analysis of novel esterases from Sphingobium chungbukense DJ77.

We identified three novel microbial esterase (Est1, Est2, and Est3) from Sphingobium chungbukense DJ77. Multiple sequence alignment showed the Est1 and Est3 have distinct motifs, such as tetrapeptide motif HGGG, a pentapeptide sequence motif GXSXG, and catalytic triad residues Ser-Asp-His, indicating that the identified enzymes belong to family IV esterases. Interestingly, Est1 exhibited strong activity toward classical esterase substrates, p-nitrophenyl ester of short-chain fatty acids and long-chain. However, Est3 did not exhibit any activity despite having high sequence similarity and sharing the identical catalytic active residues with Est1. Est3 only showed hydrolytic degradation activity to polycaprolactone (PCL). MOE-docking prediction also provided the parameters consisting of binding energy, molecular docking score, and molecular distance between substrate and catalytic nucleophilic residue, serine. The engineered mutEst3 has hydrolytic activity for a variety of esters ranging from p-nitrophenyl esters to PCL. In the present study, we demonstrated that MOE-docking simulation provides a valuable insight for facilitating biocatalytic performance.

Improved Herbicide Resistance of 4-Hydroxyphenylpyruvate Dioxygenase from Sphingobium sp. TPM-19 through Directed Evolution.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) has attracted extensive interest as a promising target for the genetic engineering of herbicide-resistant crops. However, naturally occurring HPPDs are generally very sensitive to HPPD inhibitors. In this study, random mutagenesis was performed to increase the HPPD inhibitors' resistance of Sphingobium sp. HPPD (SpHPPD). Two mutants, Q258M and Y333F, with improved resistance were obtained. Subsequently, a double-mutant (Q258M/Y333F) was generated through combined mutation. Q258M/Y333F exhibited the highest resistance to four HPPD inhibitors [topramezone, mesotrione, tembotrione, and diketonitrile (DKN)]. The enzyme fitness of Q258M/Y333F to topramezone, mesotrione, tembotrione, and DKN was increased by 4.0-, 4.1-, 4.2-, and 3.2-folds, respectively, in comparison with that of the wild-type. Molecular modeling and docking revealed that Q258M mutation leads to the decrease of enzyme-inhibitor-binding strength by breaking the hydrogen bond between the enzyme and the inhibitor, and Y333F mutation changes the conformational balance of the C-terminal helix H11, which hinders the binding of the inhibitor to the enzyme and thus would contribute to improved herbicide resistance. This study helps to further elucidate the structural basis for herbicide resistance and provides better genetic resources for the genetic engineering of herbicide-resistant crops.

Enhancement of Toxic Efficacy of Alkylated Polycyclic Aromatic Hydrocarbons Transformed by Sphingobium quisquiliarum.

Alkylated polycyclic aromatic hydrocarbons (PAHs) are abundant in crude oils and refined petroleum products and are considered as major contributors to the toxicity of spilled oils. In this study, the microbial degradation of model (alkylated) PAHs (i.e., phenanthrene, 3-methylphenanthrene, 3,6-dimethylphenanthrene (36DMPhe), pyrene, and 1-methylpyrene (1MP)) by the bacterium Sphingobium quisquiliarum EPA505, a known degrader of PAHs, was studied. To evaluate the toxic potential of the metabolic products, reaction mixtures containing metabolites of 36DMPhe and 1MP were fractionated by high-performance liquid chromatography, and their effects on the luminescence inhibition of Aliivibrio fischeri were evaluated. Although the luminescence inhibition of 36DMPhe and 1MP at their solubility levels was not observed, inhibition was observed in their metabolite fractions at the solubility limit of their parent molecule. This indicates that initial biotransformation increases the toxicity of alkylated PAHs because of the increased solubility and/or inherent toxicity of metabolites. Qualitative analysis of the metabolite fractions suggested that mono-oxidation of the methyl group is the main metabolic pathway of 36DMPhe and 1MP.

Investigating the Product Profiles and Structural Relationships of New Levansucrases with Conventional and Non-Conventional Substrates.

The synthesis of complex oligosaccharides is desired for their potential as prebiotics, and their role in the pharmaceutical and food industry. Levansucrase (LS, EC 2.4.1.10), a fructosyl-transferase, can catalyze the synthesis of these compounds. LS acquires a fructosyl residue from a donor molecule and performs a non-Lenoir transfer to an acceptor molecule, via ß-(2→6)-glycosidic linkages. Genome mining was used to uncover new LS enzymes with increased transfructosylating activity and wider acceptor promiscuity, with an initial screening revealing five LS enzymes. The product profiles and activities of these enzymes were examined after their incubation with sucrose. Alternate acceptor molecules were also incubated with the enzymes to study their consumption. LSs from Gluconobacter oxydans and Novosphingobium aromaticivorans synthesized fructooligosaccharides (FOSs) with up to 13 units in length. Alignment of their amino acid sequences and substrate docking with homology models identified structural elements causing differences in their product spectra. Raffinose, over sucrose, was the preferred donor molecule for the LS from Vibrio natriegens, N. aromaticivorans, and Paraburkolderia graminis. The LSs examined were found to have wide acceptor promiscuity, utilizing monosaccharides, disaccharides, and two alcohols to a high degree.

Roles of the Gentisate 1,2-Dioxygenases DsmD and GtdA in the Catabolism of the Herbicide Dicamba in Rhizorhabdus dicambivorans Ndbn-20.

3-Chlorogentisate is a key intermediate in the catabolism of the herbicide dicamba in R. dicambivorans Ndbn-20. In this study, we identified two gentisate 1,2-dioxygenases (GDOs), DsmD and GtdA, from Ndbn-20. The amino acid sequence similarity between DsmD and GtdA is 51%. Both of them are dimers and showed activities to gentisate and 3-chlorogentisate but not 3,6-dichlorogentisate (3,6-DCGA) or 6-chlorogentisate in vitro. The kcat/Km of DsmD for 3-chlorogentisate was 28.7 times higher than that of GtdA, whereas the kcat/Km of DsmD for gentisate was only one-fourth of that of GtdA. Transcription of dsmD was dramatically induced by 3-chlorogentisate but not gentisate, whereas gtdA was not induced. Disruption of dsmD resulted in a significant decline in the degradation rates of 3-chlorogentisate and dicamba but had no effect on the degradation of gentisate, whereas the result of disruption of gtdA was converse; the disruption of both dsmD and gtdA led to the inability to degrade 3-chlorogentisate and gentisate. This study revealed that 3-chlorogentisate but not gentisate or 3,6-DCGA is the ring-cleavage substrate in the dicamba degradation pathway in R. dicambivorans Ndbn-20; DsmD is specifically responsible for cleavage of 3-chlorogentisate, whereas GtdA is a general GDO involved in the catabolism of various natural aromatic compounds.

Description of Erythrobacter mangrovi sp. nov., an aerobic bacterium from rhizosphere soil of mangrove plant (Kandelia candel).

A novel Gram-stain negative, aerobic, non-motile, rod-shaped bacterium, designated as strain EB310T, was isolated from rhizosphere soil of mangrove plant Kandelia candel in Fugong village, Zhangzhou, China. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain EB310T belonged to the genus Erythrobacter, clustering with Erythrobacter pelagi JCM 17468T, Erythrobacter lutimaris KCTC 42109T and Erythrobacter marisflavi KCTC 62896T, and showed the highest 16S rRNA gene sequence similarity of 97.5% to Erythrobacter pelagi JCM 17468T. The genomic average nucleotide identity and in silico DNA-DNA hybridization values between strain EB310T and the reference strains were 71.0-75.5% and 19.8-20.0%, respectively. Growth ranges of the isolate occurred at 10-45 °C (optimum 28-30 °C), pH 5.5-9.5 (optimum pH 7.5) and 0-9.0% NaCl concentrations (optimum 2.0%, w/v). The strain did not produce bacteriochlorophyll a and flexirubin, but produced carotenoids. The strain contained Q-10 as the predominant ubiquinone and summed feature 3 (C16:1 ω7c/C16:1 ω6c) and summed feature 8 (C18:1 ω6c/C18:1 ω7c) as the major fatty acids. The major polar lipids were sphingoglycolipid, phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylcholine. Differential phenotypic characteristics, together with chemotaxonomic, phylogenetic and genomic distinctiveness, indicated that strain EB310T is distinguishable from other members of the genus Erythrobacter. On the basis of the data exhibited, strain EB310T is considered to represent a novel species of the genus Erythrobacter, for which the name Erythrobacter mangrovi sp. nov., is proposed. The type strain is EB310T (= KCTC 72109T = MCCC 1K03690T). The genomic DNA G + C content is 62.9 mol%.

Rationally engineered prolyl endopeptidases from Sphingomonas capsulata with improved hydrolytic activity towards pathogenic peptides of celiac diseases.

Celiac disease affects approximately 1% of the population and is a major public health problem worldwide. It is trigged by gluten-derived peptides, which have unusually high proline-glutamine motif content and are highly resistant to proteolysis by digestive enzymes of the gastrointestinal tract. The only treatment for celiac disease is strict, lifelong adherence to a gluten-free diet, which is effective but costly and difficult to maintain. Therefore, novel non-dietary therapies for celiac disease are urgently needed. Gluten-degrading enzymes are promising non-dietary treatments, and some enzymes have been investigated in preclinical or clinical studies. A combination of prolyl endopeptidase from Sphingomonas capsulata (SC PEP) and a glutamine-specific endoprotease (EP-B2 from barley) known as latiglutenase showed insufficient benefits in phase II clinical trials, likely because of its low enzyme activity in the gastric environment. Therefore, improving enzyme activity is essential for the clinical application of SC PEP. Enzyme activity can be enhanced using computer-aided rational protein design tools. In this study, we combined molecular docking and molecular dynamics simulation to rationally design SC PEP mutants and experimentally evaluated their activities. We identified mutants with up to 90-103% increases in specific activity and up to 80-202% increases in the catalytic rate. We have investigated the mechanism underlying the enhanced activity of these mutants, and found that a conformational transition of the ß-propeller domain and catalytic domain of SC PEP was important for enzyme activity, and this transition was affected by residues in the catalytic domain and at the domain interface; a shorter distance between the substrate Pro and the oxyanion holes was also crucial for improving SC PEP catalytic activity. Our results provide useful information for the rational design of highly active SC PEPs to accelerate the development of enzyme therapeutics candidates for Celiac disease.

A structural framework for unidirectional transport by a bacterial ABC exporter.

The ATP-binding cassette (ABC) transporter of mitochondria (Atm1) mediates iron homeostasis in eukaryotes, while the prokaryotic homolog from Novosphingobium aromaticivorans (NaAtm1) can export glutathione derivatives and confer protection against heavy-metal toxicity. To establish the structural framework underlying the NaAtm1 transport mechanism, we determined eight structures by X-ray crystallography and single-particle cryo-electron microscopy in distinct conformational states, stabilized by individual disulfide crosslinks and nucleotides. As NaAtm1 progresses through the transport cycle, conformational changes in transmembrane helix 6 (TM6) alter the glutathione-binding site and the associated substrate-binding cavity. Significantly, kinking of TM6 in the post-ATP hydrolysis state stabilized by MgADPVO4 eliminates this cavity, precluding uptake of glutathione derivatives. The presence of this cavity during the transition from the inward-facing to outward-facing conformational states, and its absence in the reverse direction, thereby provide an elegant and conceptually simple mechanism for enforcing the export directionality of transport by NaAtm1. One of the disulfide crosslinked NaAtm1 variants characterized in this work retains significant glutathione transport activity, suggesting that ATP hydrolysis and substrate transport by Atm1 may involve a limited set of conformational states with minimal separation of the nucleotide-binding domains in the inward-facing conformation.

Genome-Wide Analysis Reveals Genetic Potential for Aromatic Compounds Biodegradation of Sphingopyxis.

Members of genus Sphingopyxis are frequently found in diverse eco-environments worldwide and have been traditionally considered to play vital roles in the degradation of aromatic compounds. Over recent decades, many aromatic-degrading Sphingopyxis strains have been isolated and recorded, but little is known about their genetic nature related to aromatic compounds biodegradation. In this study, bacterial genomes of 19 Sphingopyxis strains were used for comparative analyses. Phylogeny showed an ambiguous relatedness between bacterial strains and their habitat specificity, while clustering based on Cluster of Orthologous Groups suggested the potential link of functional profile with substrate-specific traits. Pan-genome analysis revealed that 19 individuals were predicted to share 1,066 orthologous genes, indicating a high genetic homogeneity among Sphingopyxis strains. Notably, KEGG Automatic Annotation Server results suggested that most genes pertaining aromatic compounds biodegradation were predicted to be involved in benzoate, phenylalanine, and aminobenzoate metabolism. Among them, ß-ketoadipate biodegradation might be the main pathway in Sphingopyxis strains. Further inspection showed that a number of mobile genetic elements varied in Sphingopyxis genomes, and plasmid-mediated gene transfer coupled with prophage- and transposon-mediated rearrangements might play prominent roles in the evolution of bacterial genomes. Collectively, our findings presented that Sphingopyxis isolates might be the promising candidates for biodegradation of aromatic compounds in pollution sites.

Characterization of a Linuron-Specific Amidohydrolase from the Newly Isolated Bacterium Sphingobium sp. Strain SMB.

The phenylurea herbicide linuron is globally used and has caused considerable concern because it leads to environmental pollution. In this study, a highly efficient linuron-transforming strain Sphingobium sp. SMB was isolated, and a gene (lahB) responsible for the hydrolysis of linuron to 3,4-dichloroaniline and N,O-dimethylhydroxylamine was cloned from the genome of strain SMB. The lahB gene encodes an amidohydrolase, which shares 20-53% identity with other biochemically characterized amidohydrolases, except for the newly reported linuron hydrolase Phh (75%). The optimal conditions for the hydrolysis of linuron by LahB were determined to be pH 7.0 and 30 °C, and the Km value of LahB for linuron was 37.3 ± 1.2 µM. Although LahB and Phh shared relatively high identity, LahB exhibited a narrow substrate spectrum (specific for linuron) compared to Phh (active for linuron, diuron, chlortoluron, etc.). Sequence analysis and site-directed mutagenesis revealed that Ala261 of Phh was the key amino acid residue affecting the substrate specificity. Our study provides a new amidohydrolase for the specific hydrolysis of linuron.

Production and Characterization of Bioplastic by Polyhydroxybutyrate Accumulating Erythrobacter aquimaris Isolated from Mangrove Rhizosphere.

The synthesis of bioplastic from marine microbes has a great attendance in the realm of biotechnological applications for sustainable eco-management. This study aims to isolate novel strains of poly-ß-hydroxybutyrate (PHB)-producing bacteria from the mangrove rhizosphere, Red Sea, Saudi Arabia, and to characterize the extracted polymer. The efficient marine bacterial isolates were identified by the phylogenetic analysis of the 16S rRNA genes as Tamlana crocina, Bacillus aquimaris, Erythrobacter aquimaris, and Halomonas halophila. The optimization of PHB accumulation by E. aquimaris was achieved at 120 h, pH 8.0, 35 °C, and 2% NaCl, using glucose and peptone as the best carbon and nitrogen sources at a C:N ratio of 9.2:1. The characterization of the extracted biopolymer by Fourier-transform infrared spectroscopy (FTIR), Nuclear magnetic resonance (NMR), and Gas chromatography-mass spectrometry (GC-MS) proves the presence of hydroxyl, methyl, methylene, methine, and ester carbonyl groups, as well as derivative products of butanoic acid, that confirmed the structure of the polymer as PHB. This is the first report on E. aquimaris as a PHB producer, which promoted the hypothesis that marine rhizospheric bacteria were a new area of research for the production of biopolymers of commercial value.

Isolation and Characterization of a Topramezone-Resistant 4-Hydroxyphenylpyruvate Dioxygenase from Sphingobium sp. TPM-19.

Topramezone is a 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor. Due to its broad-spectrum, high efficiency, and low toxicity, topramezone is a candidate herbicide for the construction of genetically modified (GM) herbicide-resistant crops. In the present study, we screened a topramezone-resistant isolate Sphingobium sp. TPM-19 and cloned a topramezone-resistant HPPD gene (SphppD) from this isolate. SpHPPD shared the highest similarity (53%) with an HPPD from Vibrio vulnificus CMCP6. SpHPPD was synthesized in Escherichia coli BL21(DE3) and purified to homogeneity using Co2+-affinity chromatography. SpHPPD was found to be a monomer. The Km and kcat of SpHPPD for 4-hydroxyphenylpyruvate (4-HPP) were 82.8 µM and 15.0 s-1, respectively. SpHPPD showed high resistance to topramezone with half maximal inhibitory concentration (IC50) and Ki values of 5.2 and 2.5 µM, respectively. Additionally, SpHPPD also showed high resistance to isoxaflutole (DKN) (IC50: 8.7 µM; Ki: 6.0 µM) and mesotrione (IC50: 4.2 µM; Ki: 1.3 µM) and moderate resistance to tembotrione (IC50: 2.5 µM; Ki: 1.0 µM). The introduction of the SphppD gene into Arabidopsis thaliana enhanced obvious resistance against topramezone. In conclusion, this study provides a novel topramezone-resistant HPPD gene for the genetic engineering of GM herbicide-resistant crops.

Assessing interactions, predicting function, and increasing degradation potential of a PAH-degrading bacterial consortium by effect of an inoculant strain.

A natural phenanthrene-degrading consortium CON was inoculated with an exogenous strain Sphingobium sp. (ex Sp. paucimobilis) 20006FA yielding the consortium called I-CON, in order to study ecological interactions into the bacterial community. DGGE and proteomic profiles and analyses by HTS (High-Throughput Sequencing) technologies demonstrated inoculant establishment and changes on CON composition. Inoculation increased degradation efficiency in I-CON and prevented intermediate HNA accumulation. This could be explained not only by the inoculation, but also by enrichment in Achromobacter genus at expense of a decrease in Klebsiella genus. After inoculation, cooperation between Sphingobium and Achromobacter genera were improved, thereby, some competition could have been generated, and as a consequence, species in minor proportion (cheaters), as Inquilinus sp. and Luteibacter sp., were not detected. Sequences of Sphingobium (corresponding to the inoculated strain) did not vary. PICRUSt predicted a network with bacterial phylotypes connected with enzymes, showing functional redundancy in the phenanthrene pathway, with exception of the first enzymes biphenyl-2,3-diol 1,2-dioxygenase and protocatechuate 4,5-dioxygenase that were only encoded in Sphingobium sp. This is the first report where a natural consortium that has been characterized by HTS technologies is inoculated with an exogenous strain in order to study competitiveness and interactions.

Sulfur-Containing Carotenoids from A Marine Coral Symbiont Erythrobacter flavus Strain KJ5.

Erythrobacter flavus strain KJ5 (formerly called Erythrobacter sp. strain KJ5) is a yellowish marine bacterium that was isolated from a hard coral Acropora nasuta in the Karimunjawa Islands, Indonesia. The complete genome sequence of the bacterium has been reported recently. In this study, we examined the carotenoid composition of this bacterium using high-performance liquid chromatography coupled with ESI-MS/MS. We found that the bacterium produced sulfur-containing carotenoids, i.e., caloxanthin sulfate and nostoxanthin sulfate, as the most abundant carotenoids. A new carotenoid zeaxanthin sulfate was detected based on its ESI-MS/MS spectrum. The unique presence of sulfated carotenoids found among the currently known species of the Erythrobacter genus were discussed.

Enantioselective Catabolism of Napropamide Chiral Enantiomers in Sphingobium sp. A1 and B2.

Napropamide [ N, N-diethyl-2-(1-naphthalenyloxy)propenamide, NAP] is a highly efficient and broad-spectrum amide herbicide. Little is known about the bacterial catabolism of its different enantiomers. Here, we report the isolation of two NAP-degrading strains of Sphingobium sp., A1 and B2, and the different catabolic pathways of different enantiomers in these two strains. Strain A1 dioxygenated NAP at different positions of the naphthalene ring of different enantiomers, leading to the complete degradation of R-NAP while producing a dead-end product from S-NAP. Strain B2 cleaved the amido bonds of both enantiomers, but only the product from S-NAP could be further transformed to form α-naphthol and mineralize in strain B2. The degradation rates of R-NAP and S-NAP in the combination degradation by strains A1 and B2 were 24.8 and 7.5 times that in the single-strain degradation by strain B2 or A1, respectively, showing enhanced synergistic catabolism between strains A1 and B2. This study provides new insights into the enantioselective catabolic network of the chiral herbicide NAP in microorganisms.

Sphingobium aromaticivastans sp. nov., a novel aniline- and benzene-degrading, and antimicrobial compound producing bacterium.

A strictly aerobic, orange-pigmented strain was isolated and designated as UCM-25T. This strain is capable of degrading aniline and benzene, while is also producing antimicrobial compounds which inhibit the growth of some common pathogenic microbes. A near full-length 16S rRNA gene sequence revealed similarity to Sphingobium chlorophenolicum NBRC 16172T (98.6%). The level of DNA-DNA hybridization between the new isolate and the related species suggests UCM-25T to be a new species belonging to the genus Sphingobium. The bacterial cells contained phosphatidylglycerol, phosphatidylethanolamine, sphingoglycolipid, phosphatidylcholine, phosphatidylmonomethylethanolamine, phosphatidyldimethylethanolamine, three unidentified polar lipids, and an unidentified aminophospholipid. Ubiquinone Q-10 was the major quinone and spermidine was the major polyamine. The G+C content in the DNA of strain UCM-25T was 62.9 mol%. Cells contained summed feature 8 (C18:1ω7c and/or C18:1ω6c), summed feature 3 (C16:1ω7c and/or C16:1ω6c), C16:0, and C14:0 2-OH as major fatty acids. Based on the comparison of phenotypic, genotypic, and chemotaxonomic characteristics, strain UCM-25T represents a new member of the genus Sphingobium, for which the name S. aromaticivastans sp. nov. is proposed. The type strain is UCM-25T (=KACC 19288T =DSM 105181T).

Novosphingobium aromaticivorans uses a Nu-class glutathione S-transferase as a glutathione lyase in breaking the ß-aryl ether bond of lignin.

As a major component of plant cell walls, lignin is a potential renewable source of valuable chemicals. Several sphingomonad bacteria have been identified that can break the ß-aryl ether bond connecting most phenylpropanoid units of the lignin heteropolymer. Here, we tested three sphingomonads predicted to be capable of breaking the ß-aryl ether bond of the dimeric aromatic compound guaiacylglycerol-ß-guaiacyl ether (GGE) and found that Novosphingobium aromaticivorans metabolizes GGE at one of the fastest rates thus far reported. After the ether bond of racemic GGE is broken by replacement with a thioether bond involving glutathione, the glutathione moiety must be removed from the resulting two stereoisomers of the phenylpropanoid conjugate ß-glutathionyl-γ-hydroxypropiovanillone (GS-HPV). We found that the Nu-class glutathione S-transferase NaGSTNu is the only enzyme needed to remove glutathione from both (R)- and (S)-GS-HPV in N. aromaticivorans We solved the crystal structure of NaGSTNu and used molecular modeling to propose a mechanism for the glutathione lyase (deglutathionylation) reaction in which an enzyme-stabilized glutathione thiolate attacks the thioether bond of GS-HPV, and the reaction proceeds through an enzyme-stabilized enolate intermediate. Three residues implicated in the proposed mechanism (Thr51, Tyr166, and Tyr224) were found to be critical for the lyase reaction. We also found that Nu-class GSTs from Sphingobium sp. SYK-6 (which can also break the ß-aryl ether bond) and Escherichia coli (which cannot break the ß-aryl ether bond) can also cleave (R)- and (S)-GS-HPV, suggesting that glutathione lyase activity may be common throughout this widespread but largely uncharacterized class of glutathione S-transferases.

Topological analysis of the lipoprotein organophosphate hydrolase from Sphingopyxis wildii reveals a periplasmic localisation.

Organophosphate hydrolase (OPH) is a membrane-associated lipoprotein. It translocates across the inner membrane via the twin-arginine transport pathway and remains anchored to the periplasmic face of the inner membrane through a diacylglycerol moiety linked to the invariant cysteine residue found at the junction of a SpaseII cleavage site. Due to the existence of a transmembrane helix at the C-terminus of the mature OPH, an inner-membrane topology was predicted suggesting the C-terminus of OPH is cytoplasmic. The predicted topology was validated by generating OPH variants either fused in-frame with ß-lactamase or with unique cysteine residues. Sphingopyxis wildii cells expressing OPH variants with Bla fused at the N-terminal, C-terminal or central regions all grew in the presence of ampicillin. Supporting the ß-lactamase reporter assay, the OPH variants having unique cysteine residues at different strategic locations were accessible to the otherwise membrane-impermeant PEG-Mal (methoxypolyethylene glycol maleimide) revealing that, with the exception of the lipoprotein anchor, the entire OPH is in the periplasmic space.