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Gretchen L W Heavner, Cresten B Mansfeldt, Garrett E Debs, Sage T Hellerstedt, Annette R Rowe, Ruth E Richardson
Using mRNA transcript levels for key functional enzymes as proxies for the organohalide respiration (OHR) rate, is a promising approach for monitoring bioremediation populations in situ at chlorinated solvent-contaminated field sites. However, to date, no correlations have been empirically derived for chlorinated solvent respiring, Dehalococcoides mccartyi (DMC) containing, bioaugmentation cultures. In the current study, genome-wide transcriptome and proteome data were first used to confirm the most highly expressed OHR-related enzymes in the bioaugmentation culture, KB-1TM, including several reductive dehalogenases (RDases) and a Ni-Fe hydrogenase, Hup...
February 8, 2018: Microorganisms
Shu-Ping Zou, Kai Gu, Yu-Guo Zheng
Halohydrin dehalogenase (HHDH)-mediated dehalogenation of 1,3-dichloro-2-propanol (1,3-DCP) is a key step in the chemoenzymatic synthesis of epichlorohydrin (ECH) from glycerol. In this study, a covalent immobilization strategy was employed to enhance the stability of Agrobacterium tumefaciens HHDH by using epoxy resin ES-103B as a carrier. Under optimal conditions, the activity recovery of ES-103B-immobilized HHDH (HHDH@ES-103B) was 62.4% and the specific activity was 1604 U/g. The HHDH@ES-103B exhibited excellent thermostability, with a half-life of 68...
February 1, 2018: Biotechnology Progress
Sebastian Keller, Cindy Kunze, Martin Bommer, Christian Paetz, Riya C Menezes, Aleš Svatoš, Holger Dobbek, Torsten Schubert
The organohalide-respiring bacterium Sulfurospirillum multivorans produces a unique cobamide, namely norpseudo-B12, which serves as cofactor of the tetrachloroethene (PCE) reductive dehalogenase (PceA). As previously reported, a replacement of the adeninyl-moiety, the lower base of the cofactor, by exogenously applied 5,6-dimethylbenzimidazole led to inactive PceA. To explore the general effect of benzimidazoles on the PCE metabolism, the susceptibility of the organism for guided biosynthesis of various singly substituted benzimidazolyl-norcobamides was investigated and their use as cofactor by PceA was analyzed...
January 29, 2018: Journal of Bacteriology
Bat-Erdene Jugder, Karl A P Payne, Karl Fisher, Susanne Bohl, Helene Lebhar, Mike Manefield, Matthew Lee, David Leys, Christopher P Marquis
Reductive dehalogenases (RDase) are key enzymes involved in the respiratory process of anaerobic organohalide respiring bacteria (ORB). Heterologous expression of respiratory RDases is desirable for structural and functional studies, however there are few reports of successful expression of these enzymes. Dehalobacter sp. strain UNSWDHB is an ORB, whose preferred electron acceptor is chloroform. This study describes efforts to express recombinant reductive dehalogenase (TmrA), derived from UNSWDHB, using the heterologous hosts Escherichia coli and Bacillus megaterium...
January 24, 2018: ACS Chemical Biology
Yayue Wang, Yanbin Feng, Xupeng Cao, Yinghui Liu, Song Xue
D-2-haloacid dehalogenases (D-DEXs) catalyse the hydrolytic dehalogenation of D-2-haloacids, releasing halide ions and producing the corresponding 2-hydroxyacids. A structure-guided elucidation of the catalytic mechanism of this dehalogenation reaction has not been reported yet. Here, we report the catalytic mechanism of a D-DEX, HadD AJ1 from Pseudomonas putida AJ1/23, which was elucidated by X-ray crystallographic analysis and the H218O incorporation experiment. HadD AJ1 is an α-helical hydrolase that forms a homotetramer with its monomer including two structurally axisymmetric repeats...
January 23, 2018: Scientific Reports
Yukihiro Kitade, Ryoma Hashimoto, Masako Suda, Kazumi Hiraga, Masayuki Inui
Corynebacterium glutamicum was metabolically engineered to produce 4-hydroxybenzoic acid (4-HBA), a valuable aromatic compound used as a raw material for the production of liquid crystal polymers and paraben. C. glutamicum was found to have a higher tolerance to 4-HBA toxicity than previously reported hosts used for the production of genetically engineered 4-HBA. To obtain higher titers of 4-HBA, we employed a stepwise overexpression of all seven target genes in the shikimate pathway in C. glutamicum Specifically, multiple chromosomal integrations of a mutated aroG from Escherichia coli, encoding a DAHP synthase, and wild-type aroCKB from C...
January 5, 2018: Applied and Environmental Microbiology
Andrew T Quaile, Peter J Stogios, Olga Egorova, Elena Evdokimova, Dylan Valleau, Boguslaw Nocek, Purnima S Kompella, Sergio Peisajovich, Alexander F Yakunin, Alexander W Ensminger, Alexei Savchenko
Host colonization by Gram negative pathogens often involves delivery of bacterial proteins called ″effectors″ into the host cell. The pneumonia-causing pathogen Legionella pneumophila delivers more than 330 effectors into the host cell via its type IVB Dot/Icm secretion system. The collective functions of these proteins is the establishment of a replicative niche from which Legionella can recruit cellular materials to grow while evading lysosomal fusion inhibiting its growth. Using a combination of structural, biochemical, and in vivo approaches, we show that one of these translocated effector proteins, Ceg4, is a phosphotyrosine phosphatase harboring a haloacid dehalogenase hydrolase domain...
January 4, 2018: Journal of Biological Chemistry
Shangwei Zhang, Lorenz Adrian, Gerrit Schüürmann
The bacterium Dehalococcoides, strain CBDB1, transforms aromatic halides through reductive dehalogenation. So far, however, the structures of its vitamin B12-containing dehalogenases are unknown, hampering clarification of the catalytic mechanism and substrate specificity as basis for targeted remediation strategies. This study employs a quantum chemical donor-acceptor approach for the Co(I)-substrate electron transfer. Computational characterization of the substrate electron affinity at carbon-halogen bonds enables discriminating aromatic halides ready for dehalogenation by strain CBDB1 (active substrates) from non-dehalogenated (inactive) counterparts with 92% accuracy, covering 86 of 93 bromobenzenes, chlorobenzenes, chlorophenols, chloroanilines, polychlorinated biphenyls, and dibenzo-p-dioxins...
December 28, 2017: Environmental Science & Technology
Lifeng Chen, Ed S Krol, Meena K Sakharkar, Haseeb A Khan, Abdullah S Alhomida, Jian Yang
Maleylacetate reductase (PcpE), the last enzyme in the pentachlorophenol biodegradation pathway in Sphingobium chlorophenolicum L-1, catalyzes two consecutive reductive reactions, reductive dehalogenation of 2-chloromaleylacetate (2-CMA) to maleylacetate (MA) and subsequent reduction of MA to 3-oxoadipate (3-OXO). In each reaction, one molecule of NADH is consumed. To better understand its catalytic function, we undertook a structural model-based site-directed mutagenesis and steady-state kinetics study of PcpE...
December 22, 2017: Scientific Reports
Qian Liao, Xuan Du, Wei Jiang, Yapei Tong, Zhipeng Zhao, Ruiqin Fang, Juan Feng, Lixia Tang
Halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) shows great potential to produce valuable optically pure epoxides and β-substituted alcohols. However, this enzyme has been reported to be very sensitive and less stable under oxidative conditions. Enzyme immobilization represents a powerful means to overcome this limitation and provides the enzyme characteristics of a biocatalyst. In this study, the crude extract of HheC was directly subjected to enzyme immobilization using a carrier-free cross-linked enzyme aggregates (CLEAs) method...
December 19, 2017: Journal of Biotechnology
Holly L Sewell, Anne-Kristin Kaster, Alfred M Spormann
The deep marine subsurface is one of the largest unexplored biospheres on Earth and is widely inhabited by members of the phylum Chloroflexi In this report, we investigated genomes of single cells obtained from deep-sea sediments of the Peruvian Margin, which are enriched in such Chloroflexi 16S rRNA gene sequence analysis placed two of these single-cell-derived genomes (DscP3 and Dsc4) in a clade of subphylum I Chloroflexi which were previously recovered from deep-sea sediment in the Okinawa Trough and a third (DscP2-2) as a member of the previously reported DscP2 population from Peruvian Margin site 1230...
December 19, 2017: MBio
Muhammad Farhan Ul Haque, Ludovic Besaury, Thierry Nadalig, Françoise Bringel, Jérôme Mutterer, Hubert Schaller, Stéphane Vuilleumier
Chloromethane (CH3Cl) is a toxic gas mainly produced naturally, in particular by plants, and its emissions contribute to ozone destruction in the stratosphere. Conversely, CH3Cl can be degraded and used as the sole carbon and energy source by specialised methylotrophic bacteria, isolated from a variety of environments including the phyllosphere, i.e. the aerial parts of vegetation. The potential role of phyllospheric CH3Cl-degrading bacteria as a filter for plant emissions of CH3Cl was investigated using variants of Arabidopsis thaliana with low, wild-type and high expression of HOL1 methyltransferase previously shown to be responsible for most of CH3Cl emissions by A...
December 14, 2017: Scientific Reports
Won-Hee Jung, Ye-Eun Son, Sang-Hun Oh, Ci Fu, Hye Shin Kim, Jin-Hwan Kwak, Maria E Cardenas, Joseph Heitman, Hee-Soo Park
Calcineurin modulates environmental stress survival and virulence of the human fungal pathogen Cryptococcus neoformans Previously, we identified 44 putative calcineurin substrates and proposed that the calcineurin pathway is branched to regulate targets including Crz1, Pbp1, and Puf4 in C. neoformans In this study we characterized Had1, which is one of the putative calcineurin substrates and belongs to a ubiquitously conserved haloacid dehalogenase β-phosphoglucomutase protein super-family. Growth of the had1∆ mutant was found to be compromised at 38°C or higher...
December 12, 2017: G3: Genes—Genomes—Genetics
Jennifer Gadkari, Tobias Goris, Christian L Schiffmann, Raffael Rubick, Lorenz Adrian, Torsten Schubert, Gabriele Diekert
Reductive dehalogenation of organohalides is carried out by organohalide-respiring bacteria (OHRB) in anoxic environments. The tetrachloroethene (PCE)-respiring Epsilonproteobacterium Sulfurospirillum multivorans is one of few OHRB able to respire oxygen. Therefore, we investigated the organism's capacity to dehalogenate PCE in the presence of oxygen, which would broaden the applicability to use S. multivorans, unlike other commonly oxygen-sensitive OHRB, for bioremediation, e.g. at oxic/anoxic interphases...
December 5, 2017: FEMS Microbiology Ecology
Rebecca Davidson, Bert-Jan Baas, Eyal Akiva, Gemma L Holliday, Benjamin J Polacco, Jake A LeVieux, Collin R Pullara, Yan Jessie Zhang, Christian P Whitman, Patricia C Babbitt
The tautomerase superfamily (TSF) consists of more than 11,000 non-redundant sequences present throughout the biosphere. Characterized members have attracted much attention because of the unusual and key catalytic role of an N-terminal proline. These few characterized members catalyze a diverse range of chemical reactions, but the full scale of their chemical capabilities and biological functions remains unknown. To gain new insight into TSF structure-function relationships, we performed a global analysis of similarities across the entire superfamily and computed a sequence-similarity network to guide classification into distinct subgroups...
November 28, 2017: Journal of Biological Chemistry
Monica Martínez-Martínez, Cristina Coscolín, Gerard Santiago, Jennifer Chow, Peter J Stogios, Rafael Bargiela, Christoph Gertler, José Navarro-Fernández, Alexander Bollinger, Stephan Thies, Celia Méndez-García, Ana Popovic, Greg Brown, Tatyana N Chernikova, Antonio García-Moyano, Gro Ek Bjergah, Pablo Pérez-García, Tran Hai, Mercedes V Del Pozo, Runar Stokke, Ida H Steen, Hong Cui, Xiaohui Xu, Boguslaw P Nocek, María Alcaide, Marco Distaso, Victoria Mesa, Ana I Pelaez, Jesús Sánchez, Patrick C F Buchholz, Juergen Pleiss, Antonio Fernández-Guerra, Frank O Glöckner, Olga V Golyshina, Michail M Yakimov, Alexei Savchenko, Karl-Erich Jaeger, Alexander F Yakunin, Wolfgang R Streit, Peter N Golyshin, Victor Guallar, Manuel Ferrer
Esterases receive special attention because their wide distribution in biological systems and environments and their importance for physiology and chemical synthesis. The prediction of esterases substrate promiscuity level from sequence data and the molecular reasons why certain such enzymes are more promiscuous than others, remain to be elucidated. This limits the surveillance of the sequence space for esterases potentially leading to new versatile biocatalysts and new insights into their role in cellular function...
November 28, 2017: ACS Chemical Biology
Stanislav Mazurenko, Antonin Kunka, Koen Beerens, Christopher M Johnson, Jiri Damborsky, Zbynek Prokop
Studies of protein unfolding mechanisms are critical for understanding protein functions inside cells, de novo protein design as well as defining the role of protein misfolding in neurodegenerative disorders. Calorimetry has proven indispensable in this regard for recording full energetic profiles of protein unfolding and permitting data fitting based on unfolding pathway models. While both kinetic and thermodynamic protein stability are analysed by varying scan rates and reheating, the latter is rarely used in curve-fitting, leading to a significant loss of information from experiments...
November 24, 2017: Scientific Reports
S A Cuozzo, P E Sineli, J Davila Costa, G Tortella
Actinobacteria are well-known degraders of toxic materials that have the ability to tolerate and remove organochloride pesticides; thus, they are used for bioremediation. The biodegradation of organochlorines by actinobacteria has been demonstrated in pure and mixed cultures with the concomitant production of metabolic intermediates including γ-pentachlorocyclohexene (γ-PCCH); 1,3,4,6-tetrachloro-1,4-cyclohexadiene (1,4-TCDN); 1,2-dichlorobenzene (1,2-DCB), 1,3-dichlorobenzene (1,3-DCB), or 1,4-dichlorobenzene (1,4-DCB); 1,2,3-trichlorobenzene (1,2,3-TCB), 1,2,4-trichlorobenzene (1,2,4-TCB), or 1,3,5-trichlorobenzene (1,3,5-TCB); 1,3-DCB; and 1,2-DCB...
November 10, 2017: Critical Reviews in Biotechnology
Hang T Dam, John Vollmers, Anne-Kristin Kaster, Max M Häggblom
Polychlorinated dibenzo-p-dioxin (PCDD) contaminated sites are widespread and associated with a variety of anthropogenic sources. PCDDs and other organohalide pollutants can serve as terminal electron acceptors for anaerobic respiration by specialized bacteria containing reductive dehalogenases (RdhA). These microorganisms, therefore, play an important role in bioremediation of PCDD contaminated sites. Two anaerobic enrichment cultures established using sediments collected from the PCDD polluted Hackensack (USA) and Kymijoki (Finland) rivers showed robust reductive dechlorination of 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TeCDD)...
November 3, 2017: FEMS Microbiology Ecology
Jake A LeVieux, Bert-Jan Baas, Tamer S Kaoud, Rebecca Davidson, Patricia C Babbitt, Yan Jessie Zhang, Christian P Whitman
A Pseudomonas sp. UW4 protein (UniProt K9NIA5) of unknown function was identified as similar to 4-oxalocrotonate tautomerase (4-OT)-like and cis-3-chloroacrylic acid dehalogenase (cis-CaaD)-like subgroups of the tautomerase superfamily (TSF). This protein lacks only Tyr-103 of the amino acids critical for cis-CaaD activity (Pro-1, His-28, Arg-70, Arg-73, Tyr-103, Glu-114). As it may represent an important variant of these enzymes, its kinetic and structural properties have been determined. The protein shows tautomerase activity with phenylenolpyruvate, but lacks native 4-OT activity and dehalogenase activity with the isomers of 3-chloroacrylic acid...
October 27, 2017: Archives of Biochemistry and Biophysics
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