Roseovarius pelagicus sp. nov., a facultatively anaerobic bacterium with potential for degrading polypropylene, isolated from Arctic seawater.

A Gram-stain-negative, facultatively anaerobic, non-motile, rod-shaped bacterial strain, designated HL-MP18T, was isolated from Arctic seawater after a prolonged incubation employing polypropylene as the sole carbon source. Phylogenetic analyses of the 16S rRNA gene sequence revealed that strain HL-MP18T was affiliated to the genus Roseovarius with close relatives Roseovarius carneus LXJ103T (96.8 %) and Roseovarius litorisediminis KCTC 32327T (96.5 %). The complete genome sequence of strain HL-MP18T comprised a circular chromosome of 3.86 Mbp and two circular plasmids of 0.17 and 0.24 Mbp. Genomic comparisons based on average nucleotide identity and digital DNA-DNA hybridization showed that strain HL-MP18T was consistently discriminated from its closely related taxa in the genus Roseovarius. Strain HL-MP18T showed optimal growth at 25 °C, pH 7.0 and 2.5 % (w/v) sea salts. The major cellular fatty acids were C18 : 1 ω6c and/or C18 : 1 ω7c (49.6 %), C19 : 0 cyclo ω8c (13.5 %), and C16 : 0 (12.8 %). The major respiratory quinone was ubiquinone-10. The polar lipids consisted of phosphatidylcholine, phosphatidylglycerol, an unidentified aminolipid and three unidentified lipids. The genomic DNA G+C content of the strain was 59.2 mol%. The phylogenetic, genomic, phenotypic and chemotaxonomic results indicate that strain HL-MP18T is distinguishable from the recognized species of the genus Roseovarius. Therefore, we propose that strain HL-MP18T represents a novel species belonging to the genus Roseovarius, for which the name Roseovarius pelagicus sp. nov. is proposed. The type strain is HL-MP18T (=KCCM 90405T=JCM 35639T).

Efficient NADPH-dependent dehalogenation afforded by a self-sufficient reductive dehalogenase.

Reductive dehalogenases are corrinoid and iron-sulfur cluster-containing enzymes that catalyze the reductive removal of a halogen atom. The oxygen-sensitive and membrane-associated nature of the respiratory reductive dehalogenases has hindered their detailed kinetic study. In contrast, the evolutionarily related catabolic reductive dehalogenases are oxygen tolerant, with those that are naturally fused to a reductase domain with similarity to phthalate dioxygenase presenting attractive targets for further study. We present efficient heterologous expression of a self-sufficient catabolic reductive dehalogenase from Jhaorihella thermophila in Escherichia coli. Combining the use of maltose-binding protein as a solubility-enhancing tag with the btuCEDFB cobalamin uptake system affords up to 40% cobalamin occupancy and a full complement of iron-sulfur clusters. The enzyme is able to efficiently perform NADPH-dependent dehalogenation of brominated and iodinated phenolic compounds, including the flame retardant tetrabromobisphenol, under both anaerobic and aerobic conditions. NADPH consumption is tightly coupled to product formation. Surprisingly, corresponding chlorinated compounds only act as competitive inhibitors. Electron paramagnetic resonance spectroscopy reveals loss of the Co(II) signal observed in the resting state of the enzyme under steady-state conditions, suggesting accumulation of Co(I)/(III) species prior to the rate-limiting step. In vivo reductive debromination activity is readily observed, and when the enzyme is expressed in E. coli strain W, supports growth on 3-bromo-4-hydroxyphenylacetic as a sole carbon source. This demonstrates the potential for catabolic reductive dehalogenases for future application in bioremediation.

Substrate Specificity of the 3-Methylmercaptopropionyl Coenzyme A Dehydrogenase (DmdC1) from Ruegeria pomeroyi DSS-3.

The acyl-coenzyme A (CoA) dehydrogenase family enzyme DmdC catalyzes the third step in the dimethylsulfoniopropionate (DMSP) demethylation pathway, the oxidation of 3-methylmercaptopropionyl-CoA (MMPA-CoA) to 3-methylthioacryloyl-CoA (MTA-CoA). To study its substrate specificity, the recombinant DmdC1 from Ruegeria pomeroyi was characterized. In addition to MMPA-CoA, the enzyme was highly active with short-chain acyl-CoAs, with Km values for MMPA-CoA, butyryl-CoA, valeryl-CoA, caproyl-CoA, heptanoyl-CoA, caprylyl-CoA, and isobutyryl-CoA of 36, 19, 7, 11, 14, 10, and 149 µM, respectively, and kcat values of 1.48, 0.40, 0.48, 0.73, 0.46, 0.23, and 0.01 s-1, respectively. Among these compounds, MMPA-CoA was the best substrate. The high affinity of DmdC1 for its substrate supports the model for kinetic regulation of the demethylation pathway. In contrast to DmdB, which catalyzes the formation of MMPA-CoA from MMPA, CoA, and ATP, DmdC1 was not affected by physiological concentrations of potential effectors, such as DMSP, MMPA, ATP, and ADP. Thus, compared to the other enzymes of the DMSP demethylation pathway, DmdC1 has only minimal adaptations for DMSP metabolism compared to other enzymes in the same family with similar substrates, supporting the hypothesis that it evolved relatively recently from a short-chain acyl-CoA dehydrogenase involved in fatty acid oxidation. IMPORTANCE We report the kinetic properties of DmdC1 from the model organism R. pomeroyi and close an important gap in the literature. While the crystal structure of this enzyme was recently solved and its mechanism of action described (X. Shao, H. Y. Cao, F. Zhao, M. Peng, et al., Mol Microbiol 111:1057-1073, 2019, https://doi.org/10.1111/mmi.14211), its substrate specificity and sensitivity to potential effectors was never examined. We show that DmdC1 has a high affinity for other short-chain acyl-CoAs in addition to MMPA-CoA, which is the natural substrate in DMSP metabolism and is not affected by the potential effectors tested. This evidence supports the hypothesis that DmdC1 possesses few adaptations to DMSP metabolism and likely evolved relatively recently from a short-chain acyl-CoA dehydrogenase involved in fatty acid oxidation. This work is important because it expands our understanding of the adaptation of marine bacteria to the increased availability of DMSP about 250 million years ago.

Structure and ion-release mechanism of PIB-4-type ATPases.

Transition metals, such as zinc, are essential micronutrients in all organisms, but also highly toxic in excessive amounts. Heavy-metal transporting P-type (PIB) ATPases are crucial for homeostasis, conferring cellular detoxification and redistribution through transport of these ions across cellular membranes. No structural information is available for the PIB-4-ATPases, the subclass with the broadest cargo scope, and hence even their topology remains elusive. Here, we present structures and complementary functional analyses of an archetypal PIB-4-ATPase, sCoaT from Sulfitobacter sp. NAS14-1. The data disclose the architecture, devoid of classical so-called heavy-metal-binding domains (HMBDs), and provide fundamentally new insights into the mechanism and diversity of heavy-metal transporters. We reveal several novel P-type ATPase features, including a dual role in heavy-metal release and as an internal counter ion of an invariant histidine. We also establish that the turnover of PIB-ATPases is potassium independent, contrasting to many other P-type ATPases. Combined with new inhibitory compounds, our results open up for efforts in for example drug discovery, since PIB-4-ATPases function as virulence factors in many pathogens.

Heavy metals such as zinc and cobalt are toxic at high levels, yet most organisms need tiny amounts for their cells to work properly. As a result, proteins studded through the cell membrane act as gatekeepers to finetune import and export. These proteins are central to health and disease; their defect can lead to fatal illnesses in humans, and they also help bacteria infect other organisms. Despite their importance, little is known about some of these metal-export proteins. This is particularly the case for P_IB-4-ATPases, a subclass found in plants and bacteria and which includes, for example, a metal transporter required for bacteria to cause tuberculosis. Intricate knowledge of the three-dimensional structure of these proteins would help to understand how they select metals, shuttle the compounds in and out of cells, and are controlled by other cellular processes. To reveal this three-dimensional organisation, Grønberg et al. used X-ray diffraction, where high-energy radiation is passed through crystals of protein to reveal the positions of atoms. They focused on a type of PIB-4-ATPases found in bacteria as an example. The work showed that the protein does not contain the metal-binding regions seen in other classes of metal exporters; however, it sports unique features that are crucial for metal transport such as an adapted pathway for the transport of zinc and cobalt across the membrane. In addition, Grønberg et al. tested thousands of compounds to see if they could block the activity of the protein, identifying two that could kill bacteria. This better understanding of how PIB-4-ATPases work could help to engineer plants capable of removing heavy metals from contaminated soils, as well as uncover new compounds to be used as antibiotics.

Acrylate protects a marine bacterium from grazing by a ciliate predator.

Cleavage of dimethylsulfoniopropionate (DMSP) can deter herbivores in DMSP-producing eukaryotic algae; however, it is unclear whether a parallel defence mechanism operates in marine bacteria. Here we demonstrate that the marine bacterium Puniceibacterium antarcticum SM1211, which does not use DMSP as a carbon source, has a membrane-associated DMSP lyase, DddL. At high concentrations of DMSP, DddL causes an accumulation of acrylate around cells through the degradation of DMSP, which protects against predation by the marine ciliate Uronema marinum. The presence of acrylate can alter the grazing preference of U. marinum to other bacteria in the community, thereby influencing community structure.

Mangrovicoccus algicola sp. nov., an alginate lyase - producing marine bacterium.

A Gram-stain-negative, non-motile, ellipsoid bacterium, designated HB182678T, was isolated from brown alga collected from Hainan province, PR China. Growth was observed at 10-50 °C (optimum 37-40 °C), at pH 6-10 (optimum pH 8) and in the presence of 0.5-13% (w/v) NaCl (optimum, 2-4%). The predominant isoprenoid quinone was Q-10 and the major fatty acids were C18 : 1 ω7c, C16 : 0, C18 : 0 and C19 : 0 cyclo ω8c. The polar lipids contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine, an unidentified phospholipid, two unidentified glycolipids and three unidentified aminophospholipids. The size of the draft genome was 4.40 Mbp with G+C content 68.8 mol%. Phylogenetic analysis of 16S rRNA gene sequence indicated that strain HB182678T belonged to the genus Mangrovicoccus, and the closest phylogenetically related species was Mangrovicoccus ximenensis T1lg56T (with the similarity of 96.3%). Whole genome average nucleotide identity (ANI) value between them was 84.3% and in silico DNA-DNA hybridization value was 27.2%. The combined phylogenetic relatedness, phenotypic and genotypic features supported the conclusion that strain HB182678T represents a novel species of the genus Mangrovicoccus, for which the name Mangrovicoccus algicola sp. nov. is proposed. The type strain is HB182678T (=MCCC 1K04624T=KCTC 82318T).

Characterisation and optimisation of a novel laccase from Sulfitobacter indolifex for the decolourisation of organic dyes.

Laccases are multi­copper oxidases that possess the potential for industrial wastewater treatments. In this study, a putative laccase from Sulfitobacter indolifex was recombinantly produced and characterised. The enzyme was found to be stable and active at low to ambient temperature and across a range of pH conditions. The ability of the putative bacterial laccase to catalyse the decolourisation of seven common industrial dyes was also examined. Our results showed that the putative laccase could efficiently decolourise Indigo Carmine, Coomassie Brilliant Blue R-250, Congo Red, Malachite Green and Alizarin in the presence of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as a redox mediator. Furthermore, the use of enzyme immobilisation technology to improve the operational stability and reusability of the putative laccase was also investigated. We found that immobilising the enzyme through the cross-linked enzyme aggregate method significantly improved its tolerance towards extreme pH as well as the presence of organic solvents. This work expands the arsenal of bacterial laccases available for the bioremediation of dye-containing wastewater.

Cloning and characterization of a novel thermostable amidase, Xam, from Xinfangfangia sp. DLY26.

OBJECTIVE: Identification and characterization of a novel thermostable amidase (Xam) with wide pH tolerance and broad-spectrum substrate specificity. RESULTS: Xam was identified from non-thermophilic Xinfangfangia sp. DLY26 and its acyl transfer activity was investigated. Recombinant Xam was optimally active at 60 °C and pH 9.0. The enzyme had a half life of 18 h at 55 °C and maintained more than 60 % of its maximum activity in the range of pH 3.0-11.0. Additionally, Xam exhibited broad substrate specificity towards aliphatic, aromatic, and heterocyclic amides. CONCLUSIONS: These unique properties make Xam a promising biocatalyst for production of important hydroxamic acids at elevated temperatures.

PpCas9 from Pasteurella pneumotropica - a compact Type II-C Cas9 ortholog active in human cells.

CRISPR-Cas defense systems opened up the field of genome editing due to the ease with which effector Cas nucleases can be programmed with guide RNAs to access desirable genomic sites. Type II-A SpCas9 from Streptococcus pyogenes was the first Cas9 nuclease used for genome editing and it remains the most popular enzyme of its class. Nevertheless, SpCas9 has some drawbacks including a relatively large size and restriction to targets flanked by an 'NGG' PAM sequence. The more compact Type II-C Cas9 orthologs can help to overcome the size limitation of SpCas9. Yet, only a few Type II-C nucleases were fully characterized to date. Here, we characterized two Cas9 II-C orthologs, DfCas9 from Defluviimonas sp.20V17 and PpCas9 from Pasteurella pneumotropica. Both DfCas9 and PpCas9 cleave DNA in vitro and have novel PAM requirements. Unlike DfCas9, the PpCas9 nuclease is active in human cells. This small nuclease requires an 'NNNNRTT' PAM orthogonal to that of SpCas9 and thus potentially can broaden the range of Cas9 applications in biomedicine and biotechnology.

Crystal structure of gluconate 5-dehydrogenase from Lentibacter algarum.

Gluconate 5-dehydrogenase (Ga5DH; EC 1.1.1.69) from Lentibacter algarum (LaGa5DH) was recombinantly expressed in Escherichia coli and purified to homogeneity. The protein was crystallized and the crystal structure was solved at 2.1 Šresolution. The crystal belonged to the monoclinic system, with space group P1 and unit-cell parameters a = 55.42, b = 55.48, c = 79.16 Å, α = 100.51, ß = 105.66, γ = 97.99°. The structure revealed LaGaDH to be a tetramer, with each subunit consisting of six α-helices and three antiparallel ß-hairpins. LaGa5DH has high structural similarity to other Ga5DH proteins, demonstrating that this enzyme is highly conserved.

Molecular Characterization of a Novel Cold-Active Hormone-Sensitive Lipase (HaHSL) from Halocynthiibacter Arcticus.

Bacterial hormone-sensitive lipases (bHSLs), which are homologous to the catalytic domains of human HSLs, have received great interest due to their uses in the preparation of highly valuable biochemicals, such as drug intermediates or chiral building blocks. Here, a novel cold-active HSL from Halocynthiibacter arcticus (HaHSL) was examined and its enzymatic properties were investigated using several biochemical and biophysical methods. Interestingly, HaHSL acted on a large variety of substrates including tertiary alcohol esters and fish oils. Additionally, this enzyme was highly tolerant to high concentrations of salt, detergents, and glycerol. Furthermore, immobilized HaHSL retained its activity for up to six cycles of use. Homology modeling suggested that aromatic amino acids (Trp23, Tyr74, Phe78, Trp83, and Phe245) in close proximity to the substrate-binding pocket were important for enzyme activity. Mutational analysis revealed that Tyr74 played an important role in substrate specificity, thermostability, and enantioselectivity. In summary, the current study provides an invaluable insight into the novel cold-active HaHSL from H. arcticus, which can be efficiently and sustainably used in a wide range of biotechnological applications.

Characterization of a novel thermostable (S)-amine-transaminase from an Antarctic moderately-thermophilic bacterium Albidovulum sp. SLM16.

Amine-transaminases (ATAs) are enzymes that catalyze the reversible transfer of an amino group between primary amines and carbonyl compounds. They have been widely studied in the last decades for their application in stereoselective synthesis of chiral amines, which are one of the most valuable building blocks in pharmaceuticals manufacturing. Their excellent enantioselectivity, use of low-cost substrates and no need for external cofactors has turned these enzymes into a promising alternative to the chemical synthesis of chiral amines. Nevertheless, its application at industrial scale remains limited mainly because most of the available ATAs are scarcely tolerant to harsh reaction conditions such as high temperatures and presence of organic solvents. In this work, a novel (S)-ATA was discovered in a thermophilic bacterium, Albidovulum sp. SLM16, isolated from a geothermal Antarctic environmental sample, more specifically from a shoreline fumarole in Deception Island. The transaminase-coding gene was identified in the genome of the microorganism, cloned and overexpressed in Escherichia coli for biochemical characterization. The activity of the recombinant ATA was optimal at 65 °C and pH 9.5. Molecular mass estimates suggest a 75 kDa homodimeric structure. The enzyme turned out to be highly thermostable, maintaining 80% of its specific activity after 5 days of incubation at 50 °C. These results indicate that ATA_SLM16 is an excellent candidate for potential applications in biocatalytic synthesis. To the best of our knowledge, this would be the first report of the characterization of a thermostable (S)-ATA discovered by means of in vivo screening of thermophilic microorganisms.

Systematic characterization of sorbose/sorbosone dehydrogenases and sorbosone dehydrogenases from Ketogulonicigenium vulgare WSH-001.

2-Keto-L-gulonic acid (2-KLG) is the direct precursor of vitamin C in industrial synthesis. 2-KLG is mainly produced via the classical two-step fermentation route. In the two-step fermentation process, 2-KLG can be synthesized from L-sorbose by Ketogulonicigenium vulgare aided by Bacillus megaterium. There are five sorbose/sorbosone dehydrogenases (SSDHs), SSDA1, SSDA1-P, SSDA2, SSDA3 and SSDB, and two sorbosone dehydrogenases (SNDHs), glucose/sorbosone dehydrogenase (GSNDH) and sorbosone dehydrogenase (SNDH), in K. vulgare, which could play crucial roles in transforming L-sorbose or L-sorbosone to 2-KLG. However, confusion about the catalytic characteristics of the individual SSDHs and SNDHs makes construction of a recombinational strain for the purpose of enhancing 2-KLG production difficult. In this study, the five SSDHs and two SNDHs from K. vulgare WSH-001 were purified, and their optimal pH values and reaction temperatures, kinetic properties, thermostabilities, substrate spectra and effects of electron acceptors on their performances were systematically determined. Among these dehydrogenases, only SSDA1 and SSDA3 have high activity for catalyzing L-sorbose to 2-KLG directly. These data provide more clues for ways to achieve enhanced conversion of L-sorbose in K. vulgare, which could facilitate both the construction of a more efficient one-step fermentation 2-KLG producer and the reconstruction of a one-step fermentation process.

Cloning, expression, and characterization of a novel nitrilase, PaCNit, from Pannonibacter carbonis Q4.6.

OBJECTIVE: Identification of a heavy metal ion-stimulated nitrilase with broad-spectrum substrate specificity. RESULTS: A novel nitrilase, PaCNit, was identified from Pannonibacter carbonis Q4.6 and its enzymatic properties were investigated. The maximum activity of PaCNit was observed at 65 °C and pH 7.0. PaCNit showed broad substrate specificity towards aliphatic, aromatic, and heterocyclic nitriles, and was tolerant to different organic solvents. Remarkably, PaCNit activity was highly stimulated by metal ions, particularly by Ag+ and Hg2+. CONCLUSION: PaCNit nitrilase has a broad range of substrate specificity and can be activated by heavy metal ions. This specific characteristic makes it have a great potential for industrial application.

Isolation and partial characterization of a new moderate thermophilic Albidovulum sp. SLM16 with transaminase activity from Deception Island, Antarctica.

BACKGROUND: A moderately thermophilic, slightly halophilic, aerobic, Gram-stain negative, bacterial strain, SLM16, was isolated from a mixed of seawater-sand-sediment sample collected from a coastal fumarole located in Whalers Bay, Deception Island, Antarctica. The aim was to screen for thermophilic microorganisms able to degrade primary amines and search for amine transaminase activity for potential industrial application. RESULTS: Identification and partial characterization of the microorganism SLM16 were carried out by means of morphological, physiological and biochemical tests along with molecular methods. Cells of strain SLM16 were non-motile irregular rods of 1.5-2.5 µm long and 0.3-0.45 µm wide. Growth occurred in the presence of 0.5-5.5% NaCl within temperature range of 35-55 °C and pH range of 5.5-9.5, respectively. The DNA G+C composition, estimated from ftsY gene, was 66% mol. Phylogenetic analysis using de 16S rRNA gene sequence showed that strain SLM16 belongs to the marine bacterial genus Albidovulum. CONCLUSION: Strain SLM16 is a moderate thermophilic Gram negative microorganisms which belongs to the marine bacterial genus Albidovulum and is closely related to Albidovulum inexpectatum species based on phylogenetic analysis. Additionally, amine-transaminase activity towards the arylaliphatic amine α-methylbenzylamine was detected.

A New Microbial Pathway for Organophosphonate Degradation Catalyzed by Two Previously Misannotated Non-Heme-Iron Oxygenases.

The assignment of biochemical functions to hypothetical proteins is challenged by functional diversification within many protein structural superfamilies. This diversification, which is particularly common for metalloenzymes, renders functional annotations that are founded solely on sequence and domain similarities unreliable and often erroneous. Definitive biochemical characterization to delineate functional subgroups within these superfamilies will aid in improving bioinformatic approaches for functional annotation. We describe here the structural and functional characterization of two non-heme-iron oxygenases, TmpA and TmpB, which are encoded by a genomically clustered pair of genes found in more than 350 species of bacteria. TmpA and TmpB are functional homologues of a pair of enzymes (PhnY and PhnZ) that degrade 2-aminoethylphosphonate but instead act on its naturally occurring, quaternary ammonium analogue, 2-(trimethylammonio)ethylphosphonate (TMAEP). TmpA, an iron(II)- and 2-(oxo)glutarate-dependent oxygenase misannotated as a γ-butyrobetaine (γbb) hydroxylase, shows no activity toward γbb but efficiently hydroxylates TMAEP. The product, ( R)-1-hydroxy-2-(trimethylammonio)ethylphosphonate [( R)-OH-TMAEP], then serves as the substrate for the second enzyme, TmpB. By contrast to its purported phosphohydrolytic activity, TmpB is an HD-domain oxygenase that uses a mixed-valent diiron cofactor to enact oxidative cleavage of the C-P bond of its substrate, yielding glycine betaine and phosphate. The high specificities of TmpA and TmpB for their N-trimethylated substrates suggest that they have evolved specifically to degrade TMAEP, which was not previously known to be subject to microbial catabolism. This study thus adds to the growing list of known pathways through which microbes break down organophosphonates to harvest phosphorus, carbon, and nitrogen in nutrient-limited niches.

Isolation and partial characterization of a new moderate thermophilic Albidovulum sp. SLM16 with transaminase activity from Deception Island, Antarctica

BACKGROUND: A moderately thermophilic, slightly halophilic, aerobic, Gram-stain negative, bacterial strain, SLM16, was isolated from a mixed of seawater-sand-sediment sample collected from a coastal fumarole located in Whalers Bay, Deception Island, Antarctica. The aim was to screen for thermophilic microorganisms able to degrade primary amines and search for amine transaminase activity for potential industrial application. RESULTS: Identification and partial characterization of the microorganism SLM16 were carried out by means of morphological, physiological and biochemical tests along with molecular methods. Cells of strain SLM16 were non-motile irregular rods of 1.5-2.5 µm long and 0.3-0.45 µm wide. Growth occurred in the presence of 0.5-5.5% NaCl within temperature range of 35-55 °C and pH range of 5.5-9.5, respectively. The DNA G+C composition, estimated from ftsY gene, was 66% mol. Phylogenetic analysis using de 16S rRNA gene sequence showed that strain SLM16 belongs to the marine bacterial genus Albidovulum. CONCLUSION: Strain SLM16 is a moderate thermophilic Gram negative microorganisms which belongs to the marine bacterial genus Albidovulum and is closely related to Albidovulum inexpectatum species based on phylogenetic analysis. Additionally, amine-transaminase activity towards the arylaliphatic amine α-methylbenzylamine was detected.

A fundamental catalytic difference between zinc and manganese dependent enzymes revealed in a bacterial isatin hydrolase.

The catalytic mechanism of the cyclic amidohydrolase isatin hydrolase depends on a catalytically active manganese in the substrate-binding pocket. The Mn2+ ion is bound by a motif also present in other metal dependent hydrolases like the bacterial kynurenine formamidase. The crystal structures of the isatin hydrolases from Labrenzia aggregata and Ralstonia solanacearum combined with activity assays allow for the identification of key determinants specific for the reaction mechanism. Active site residues central to the hydrolytic mechanism include a novel catalytic triad Asp-His-His supported by structural comparison and hybrid quantum mechanics/classical mechanics simulations. A hydrolytic mechanism for a Mn2+ dependent amidohydrolases that disfavour Zn2+ as the primary catalytically active site metal proposed here is supported by these likely cases of convergent evolution. The work illustrates a fundamental difference in the substrate-binding mode between Mn2+ dependent isatin hydrolase like enzymes in comparison with the vast number of Zn2+ dependent enzymes.

Discerning three novel chromate reduce and transport genes of highly efficient Pannonibacter phragmitetus BB: From genome to gene and protein.

Here, Pannonibacter phragmitetus BB was investigated at genomic, genetic and protein levels to explore molecular mechanisms of chromium biotransformation, respectively. The results of Miseq sequencing uncovered that a high-qualified bacterial genome draft was achieved with 5.07 Mb in length. Three novel genes involved in chromate reduce and transport, named nitR, chrA1 and chrA2, were identified by alignment, annotation and phylogenetic tree analyses, which encode a chromate reductase (NitR) and two chromate transporters (ChrA1 and ChrA2). Reverse transcription real-time polymerase chain reaction (RT-qPCR) analyses showed that the relative quantitative transcription of the three genes as the maximum reduction rate of Cr(VI) were significantly up-regulated with the increasing initial Cr(VI) concentrations. However, at the maximum cell growth points nitR was in a low transcription level, while the transcription of chrA1 and chrA2 were hold at a relatively high level and decreased with the increasing initial Cr(VI) concentrations. The ex-situ chromate reducing activity of NitR was revealed a Vmax of 34.46 µmol/min/mg enzyme and Km of 14.55 µmol/L, suggesting feasibility of the reaction with Cr(VI) as substrate. The multiple alignment demonstrates that NitR is potentially a nicotinamide adenine dinucleotide phosphate (NADPH) dependent flavin mononucleotide (FMN) reductase of Class I chromate reductases. Our results will prompt a large-scaled bioremediation on the contaminated soils and water by Pannonibacter phragmitetus BB, taking advantage of uncovering its molecular mechanisms of chromium biotransformation.

The Dimethylsulfoniopropionate (DMSP) Lyase and Lyase-Like Cupin Family Consists of Bona Fide DMSP lyases as Well as Other Enzymes with Unknown Function.

Marine organisms release dimethylsulfide (DMS) via cleavage of dimethylsulfoniopropionate (DMSP). Different genes encoding proteins with DMSP lyase activity are known, yet these exhibit highly variable levels of activity. Most assigned bacterial DMSP lyases, including DddK, DddL, DddQ, DddW, and DddY, appear to belong to one, cupin-like superfamily. Here, we attempted to define and map this superfamily dubbed cupin-DLL (DMSP lyases and lyase-like). To this end, we have pursued the characterization of various recombinant DMSP lyases belonging to this superfamily of metalloenzymes, and especially of DddY and DddL that seem to be the most active DMSP lyases in this superfamily. We identified two conserved sequence motifs that characterize this superfamily. These motifs include the metal-ligating residues that are absolutely essential and other residues including an active site tyrosine that seems to play a relatively minor role in DMSP lysis. We also identified a transition metal chelator, N, N, N', N'-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN), that selectively inhibits all known members of the cupin-DLL superfamily that exhibit DMSP lyase activity. A phylogenetic analysis indicated that the known DMSP lyase families are sporadically distributed suggesting that DMSP lyases evolved within this superfamily multiple times. However, unusually low specific DMSP lyase activity and genome context analysis suggest that DMSP lyase is not the native function of most cupin-DLL families. Indeed, a systematic profiling of substrate selectivity with a series of DMSP analogues indicated that some members, most distinctly DddY and DddL, are bona fide DMSP lyases, while others, foremost DddQ, may only exhibit promiscuous DMSP lyase activity.