Crystal structure of the sugar acid-binding protein CxaP from a TRAP transporter in Advenella mimigardefordensis strain DPN7T.

Recently, CxaP, a sugar acid substrate binding protein (SBP) from Advenella mimigardefordensis strain DPN7T , was identified as part of a novel sugar uptake strategy. In the present study, the protein was successfully crystallized. Although several SBP structures of tripartite ATP-independent periplasmic transporters have already been solved, this is the first structure of an SBP accepting multiple sugar acid ligands. Protein crystals were obtained with bound d-xylonic acid, d-fuconic acid d-galactonic and d-gluconic acid, respectively. The protein shows the typical structure of an SBP of a tripartite ATP-independent periplasmic transporter consisting of two domains linked by a hinge and spanned by a long α-helix. By analysis of the structure, the substrate binding site of the protein was identified. The carboxylic group of the sugar acids interacts with Arg175, whereas the coordination of the hydroxylic groups at positions C2 and C3 is most probably realized by Arg154 and Asn151. Furthermore, it was observed that 2-keto-3-deoxy-d-gluconic acid is bound in protein crystals that were crystallized without the addition of any ligand, indicating that this molecule is prebound to the protein and is displaced by the other ligands if they are available. DATABASE: Structural data of CxaP complexes are available in the worldwide Protein Data Bank (https://www.rcsb.org) under the accession codes 7BBR (2-keto-3-deoxy-d-gluconic acid), 7BCR (d-galactonic acid), 7BCN (d-xylonic acid), 7BCO (d-fuconic acid) and 7BCP (d-gluconic acid).

Algicoccus marinus gen. nov. sp. nov., a marine bacterium isolated from the surface of brown seaweed Laminaria japonica.

A Gram-staining-negative, strictly aerobic, non-motile, ovoid- to rod-shaped bacterium, designated as HZ20T, was isolated from the surface of a brown seaweed (Laminaria japonica) sample collected from the East China Sea. Colonies are 1.0-2.0 mm in diameter, smooth, circular, convex and yellow after grown on MA at 28 °C for 72 h. The strain was found to grow at 4-50 °C (optimum, 37 °C), pH 5.0-9.5 (optimum, pH 7.0-7.5) and with 0-10% (w/v) NaCl (optimum, 1.0-1.5%). Chemotaxonomic analysis showed ubiquinone-8 as the only quinone, C17:0 cyclo, C16:0, summed feature 8 (C18:1ω7c and/or C18:1ω6c) and summed feature 2 (C12:0 aldehyde/unknown 10.9525/C16:1 iso I/C14:0 3OH) as the major fatty acids (> 5%), and diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, one unidentified amino phospholipid, two unidentified phospholipids, five unidentified glycolipid and two unidentified lipids as the polar lipids. The DNA G + C content was 55.5 mol %. 16S rRNA gene sequences of the isolate showed highest similarities to Bordetella flabilis AU10664T (97.1%), Parapusillimonas granuli Ch07T (97.1%), Paracandidimonas soli IMT-305T (97.1%), Kerstersia gyiorum LMG5906T (97.0%) and Bordetella sputigena LMG 28641T (97.0%). The phylogenetic trees using 16S rRNA gene and genome sequences both showed that the strain HZ20T formed a deep branch separated from other related genera, indicating that it represents a novel species of a novel genus. The calculated average nucleotide identity (ANI) and percent of conserved proteins (POCP) values using genome sequences of strain HZ20T and related strains also support this conclusion. Based on the phenotypic properties and phylogenetic distinctiveness, we propose strain HZ20T (= MCCC 1K03465T = KCTC 62330T) to represent a novel species of a novel genus with the name Algicoccus marinus gen. nov. sp. nov.

Protonation state and fine structure of the active site determine the reactivity of dehydratase: hydration and isomerization of ß-myrcene catalyzed by linalool dehydratase/isomerase from Castellaniella defragrans.

Linalool dehydratase/isomerase (LinD) from Castellaniella defragrans is a bifunctional enzyme that catalyzes the hydration of ß-myrcene to (S)-linalool and the isomerization of (S)-linalool to geraniol. In this paper, on the basis of recently obtained crystal structures, the catalytic mechanism of LinD has been explored by a combined quantum mechanics and molecular mechanics (QM/MM) approach. Two computational models have been constructed. Model I (LinD-linalool) was derived from the crystal structure of the selenomethionine derivative of LinD (Semet-LinD) in complex with the natural substrate geraniol, whereas model II (LinD-ß-myrcene) was constructed from the crystal structure of LinD in complex with ß-myrcene. In addition to a minor conformational difference of the active sites, the two models also differ in the protonation state of key residues. In model I, the pocket residue Asp39' was set to be deprotonated and His129 was protonated on ND1, whereas in model II, Asp38' was set to be deprotonated and His128 was protonated on NE2. Our calculations reveal model II as the active one, which implies that hydration and dehydration are sensitive to the protonation state and fine structure of the active site. On the basis of model II, the conversion details from ß-myrcene to geraniol can be obtained. Firstly, ß-myrcene is hydrated by a crystal water (W14) and is converted into the stable intermediate (S)-linalool, then linalool is isomerized to geraniol with an overall energy barrier of 24.6 kcal mol-1. Besides, linalool can also reversibly convert into the reactant with an energy barrier of 24.1 kcal mol-1. It is also found that the intermediate IM1 can directly transform to geraniol without first converting to (S)-linalool. His128 and Tyr65 form hydrogen bonds to stabilize the structure of the active site, but they do not act as general acid/base catalysts during the catalytic reactions.

Limonene dehydrogenase hydroxylates the allylic methyl group of cyclic monoterpenes in the anaerobic terpene degradation by Castellaniella defragrans.

The enzymatic functionalization of hydrocarbons is a central step in the global carbon cycle initiating the mineralization of methane, isoprenes, and monoterpenes, the most abundant biologically produced hydrocarbons. Also, terpene-modifying enzymes have found many applications in the energy-economic biotechnological production of fine chemicals. Here, we describe a limonene dehydrogenase that was purified from the facultatively anaerobic betaproteobacterium Castellaniella defragrans 65Phen grown on monoterpenes under denitrifying conditions in the absence of molecular oxygen. The purified limonene:ferrocenium oxidoreductase activity hydroxylated the methyl group of limonene (1-methyl-4-(1-methylethenyl)-cyclohex-1-ene) yielding perillyl alcohol ([4-(prop-1-en-2-yl)cyclohex-1-en-1-yl]methanol). The enzyme had a DTT:perillyl alcohol oxidoreductase activity yielding limonene. Mass spectrometry and molecular size determinations revealed a heterodimeric enzyme comprising CtmA and CtmB. Recently, the two proteins had been identified by transposon mutagenesis and proteomics as part of the cyclic terpene metabolism (ctm) in C. defragrans and are annotated as FAD-dependent oxidoreductases of the protein domain family phytoene dehydrogenases and related proteins (COG1233). CtmAB is the first heterodimeric enzyme in this protein superfamily. Flavins in the purified CtmAB are oxidized by ferrocenium and are reduced by limonene. Heterologous expression of CtmA, CtmB, and CtmAB in Escherichia coli demonstrated that limonene dehydrogenase activity required both subunits, each carrying a flavin cofactor. Native CtmAB oxidized a wide range of monocyclic monoterpenes containing the allylic methyl group motif (1-methyl-cyclohex-1-ene). In conclusion, we have identified CtmAB as a hydroxylating limonene dehydrogenase and the first heteromer in a family of FAD-dependent dehydrogenases acting on allylic methylene or methyl CH-bonds. We suggest placing in Enzyme Nomenclature as new entry EC 1.17.99.8.

Azohydromonas riparia sp. nov. and Azohydromonas ureilytica sp. nov. isolated from a riverside soil in South Korea.

White and pale yellow coloured bacteria were isolated from the riverside soil, Daejeon, South Korea, and were designated UCM-11T, UCM-F25, and UCM-80T. We found that all strains were able to reduce nitrate, and the cells were aerobic and motile. The DNA G+C contents of UCM-11T, UCM-F25, and UCM-80T were between 68.9 to 71.2 mol% and the main ubiquinone was observed as Q-8. Based on16S rRNA gene sequences, strains UCM-11T and UCM-F25 were found to closely match with Azohydromonas australica IAM 12664T (98.48-98.55%), and the strain UCM-80T was the closest match with Azohydromonas lata IAM 12599T (98.34%). The presence of summed feature 3 (C16:1 ω7c and/or C16:1 ω6c), C16:0, summed feature 8 (C18:1 ω7c and/or C18:1 ω6c) as well as twokinds of hydroxyfatty acids consisting of C10:0 3-OH and C12:0 2-OH, and branched fatty acids containing C16:0 iso and C17:0 cyclo were detected in all the strains. Phosphatidylethanolamine was a major polar lipid. DNA-DNA relatedness confirmed UCM-11T, UCM-F25 and UCM-80T as novel members of the genus Azohydromonas. Based on the morphological, physiological, biochemical and genotypic characteristics, we suggest that strains UCM-11T, UCM-F25, and UCM-80T represent novel species within the genus Azohydromonas. The names Azohydromonas riparia sp. nov., and Azohydromonas ureilytica sp. nov. are proposed for the type strains UCM-11T (=KACC 18570T =NBRC 111646T) and UCM-80T (=KACC 18576T =NBRC 111658T), respectively.

Intracellular delivery of peptide cargos using polyhydroxybutyrate based biodegradable nanoparticles: Studies on antitumor efficacy of BCL-2 converting peptide, NuBCP-9.

Faster biodegradation, immunogenicity and lack of cell penetrative capabilities are hurdles in development of peptidyl drugs for cancer therapy. Polymeric carriers can be used to overcome these problems. The present study is focused on the use of polyhydroxybutyrate as a potential nanovehicle for the delivery of anticancer peptides. PHB (72kDa) was produced by thermal treatment of high molecular weight PHB (300kDa) under melt conditions and then conjugated with PEG (4kDa) by Steglich esterification reaction. Anticancer peptide NuBCP-9 (FSRSLHSLL) encapsulated PHB(72K)-PEG(4K) NPs were prepared by double emulsion solvent evaporation method. PHB(72K)-PEG(4K) NPs showed encapsulation efficiency of 61% and exhibited sustained release of peptide over a period of 26days at physiological pH. NuBCP-9 loaded PHB(72K)-PEG(4K) NPs showed an IC50 value of 2.2µM & 1.6µM in MCF-7 cells in 48h and 72h respectively. Confocal laser microscopy confirmed efficient cellular uptake and induction of apoptosis by peptide loaded NPs in a time dependent manner. In vivo intraperitonial administration of 20mg/kg NuBCP-9/NPs twice a week for three weeks triggered 90% tumor regression in Ehrlich syngeneic mouse model. Our results illustrated the potential of PHB(72K)-PEG(4K) based nanoformulation as a tool for targeting intracellular proteins.

Unravelling the complete genome sequence of Advenella mimigardefordensis strain DPN7T and novel insights in the catabolism of the xenobiotic polythioester precursor 3,3'-dithiodipropionate.

Advenella mimigardefordensis strain DPN7(T) is a remarkable betaproteobacterium because of its extraordinary ability to use the synthetic disulfide 3,3'-dithiodipropionic acid (DTDP) as the sole carbon source and electron donor for aerobic growth. One application of DTDP is as a precursor substrate for biotechnically synthesized polythioesters (PTEs), which are interesting non-degradable biopolymers applicable for plastics materials. Metabolic engineering for optimization of PTE production requires an understanding of DTDP conversion. The genome of A. mimigardefordensis strain DPN7(T) was sequenced and annotated. The circular chromosome was found to be composed of 4,740,516 bp and 4112 predicted ORFs, whereas the circular plasmid consisted of 23,610 bp and 24 predicted ORFs. The genes participating in DTDP catabolism had been characterized in detail previously, but knowing the complete genome sequence and with support of Tn5: :mob-induced mutants, putatively involved transporter proteins and a transcriptional regulator were also identified. Most probably, DTDP is transported into the cell by a specific tripartite tricarboxylate transport system and is then cleaved by the disulfide reductase LpdA, sulfoxygenated by the 3-mercaptopropionate dioxygenase Mdo, activated by the CoA ligase SucCD and desulfinated by the acyl-CoA dehydrogenase-like desulfinase AcdA. Regulation of this pathway is presumably performed by a transcriptional regulator of the xenobiotic response element family. The excessive sulfate that is inevitably produced is secreted by the cells by a unique sulfate exporter of the CPA (cation : proton antiporter) superfamily.

Characterization of a novel Rieske-type alkane monooxygenase system in Pusillimonas sp. strain T7-7.

The cold-tolerant bacterium Pusillimonas sp. strain T7-7 is able to utilize diesel oils (C5 to C30 alkanes) as a sole carbon and energy source. In the present study, bioinformatics, proteomics, and real-time reverse transcriptase PCR approaches were used to identify the alkane hydroxylation system present in this bacterium. This system is composed of a Rieske-type monooxygenase, a ferredoxin, and an NADH-dependent reductase. The function of the monooxygenase, which consists of one large (46.711 kDa) and one small (15.355 kDa) subunit, was further studied using in vitro biochemical analysis and in vivo heterologous functional complementation tests. The purified large subunit of the monooxygenase was able to oxidize alkanes ranging from pentane (C5) to tetracosane (C24) using NADH as a cofactor, with greatest activity on the C15 substrate. The large subunit also showed activity on several alkane derivatives, including nitromethane and methane sulfonic acid, but it did not act on any aromatic hydrocarbons. The optimal reaction condition of the large subunit is pH 7.5 at 30°C. Fe(2+) can enhance the activity of the enzyme evidently. This is the first time that an alkane monooxygenase system belonging to the Rieske non-heme iron oxygenase family has been identified in a bacterium.

Estimation of fundamental kinetic parameters of polyhydroxybutyrate fermentation process of Azohydromonas australica using statistical approach of media optimization.

Polyhydroxybutyrate or PHB is a biodegradable and biocompatible thermoplastic with many interesting applications in medicine, food packaging, and tissue engineering materials. The present study deals with the enhanced production of PHB by Azohydromonas australica using sucrose and the estimation of fundamental kinetic parameters of PHB fermentation process. The preliminary culture growth inhibition studies were followed by statistical optimization of medium recipe using response surface methodology to increase the PHB production. Later on batch cultivation in a 7-L bioreactor was attempted using optimum concentration of medium components (process variables) obtained from statistical design to identify the batch growth and product kinetics parameters of PHB fermentation. A. australica exhibited a maximum biomass and PHB concentration of 8.71 and 6.24 g/L, respectively in bioreactor with an overall PHB production rate of 0.75 g/h. Bioreactor cultivation studies demonstrated that the specific biomass and PHB yield on sucrose was 0.37 and 0.29 g/g, respectively. The kinetic parameters obtained in the present investigation would be used in the development of a batch kinetic mathematical model for PHB production which will serve as launching pad for further process optimization studies, e.g., design of several bioreactor cultivation strategies to further enhance the biopolymer production.

Advenella incenata gen. nov., sp. nov., a novel member of the Alcaligenaceae, isolated from various clinical samples.

A polyphasic taxonomic study of 14 isolates recovered from various human and veterinary clinical samples was performed. Phenotypically these isolates shared several characteristics with members of the Alcaligenaceae and related genera. Random amplified polymorphic DNA fingerprinting and whole-cell protein analysis suggested the presence of multiple genomic groups, which was confirmed by DNA-DNA hybridization experiments. 16S rRNA gene sequence analysis indicated that these isolates were related to the genera Pelistega, Taylorella, Oligella, Pigmentiphaga, Alcaligenes, Kerstersia, Achromobacter and Bordetella and belonged to the family Alcaligenaceae. Based on the results of the present study the organisms were classified in a novel genus, Advenella gen. nov. This genus comprises one named species, Advenella incenata sp. nov. (type strain LMG 22250T=CCUG 45225T) and five currently unnamed genomic species. The DNA G+C content of members of the novel genus Advenella is between 54.0 and 57.7 mol%.