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130313-Thumbnail Image.png
Description
Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the

Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergent evolution.
ContributorsLi, Dianfan (Author) / Stansfeld, Phillip J. (Author) / Sansom, Mark S. P. (Author) / Keogh, Aaron (Author) / Vogeley, Lutz (Author) / Howe, Nicole (Author) / Lyons, Joseph A. (Author) / Aragao, David (Author) / Fromme, Petra (Author) / Fromme, Raimund (Author) / Basu, Shibom (Author) / Grotjohann, Ingo (Author) / Kupitz, Christopher (Author) / Rendek, Kimberley (Author) / Weierstall, Uwe (Author) / Zatsepin, Nadia (Author) / Cherezov, Vadim (Author) / Liu, Wei (Author) / Bandaru, Sateesh (Author) / English, Niall J. (Author) / Gati, Cornelius (Author) / Barty, Anton (Author) / Yefanov, Oleksandr (Author) / Chapman, Henry N. (Author) / Diederichs, Kay (Author) / Messerschmidt, Marc (Author) / Boutet, Sebastien (Author) / Williams, Garth J. (Author) / Seibert, M. Marvin (Author) / Caffrey, Martin (Author) / College of Liberal Arts and Sciences (Contributor) / School of Molecular Sciences (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / Department of Physics (Contributor)
Created2015-12-17
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Description
Phytochromes are a family of photoreceptors that control light responses of plants, fungi and bacteria. A sequence of structural changes, which is not yet fully understood, leads to activation of an output domain. Time-resolved serial femtosecond crystallography (SFX) can potentially shine light on these conformational changes. Here we report the

Phytochromes are a family of photoreceptors that control light responses of plants, fungi and bacteria. A sequence of structural changes, which is not yet fully understood, leads to activation of an output domain. Time-resolved serial femtosecond crystallography (SFX) can potentially shine light on these conformational changes. Here we report the room temperature crystal structure of the chromophore-binding domains of the Deinococcus radiodurans phytochrome at 2.1 Å resolution. The structure was obtained by serial femtosecond X-ray crystallography from microcrystals at an X-ray free electron laser. We find overall good agreement compared to a crystal structure at 1.35 Å resolution derived from conventional crystallography at cryogenic temperatures, which we also report here. The thioether linkage between chromophore and protein is subject to positional ambiguity at the synchrotron, but is fully resolved with SFX. The study paves the way for time-resolved structural investigations of the phytochrome photocycle with time-resolved SFX.
ContributorsEdlund, Petra (Author) / Takala, Heikki (Author) / Claesson, Elin (Author) / Henry, Leocadie (Author) / Dods, Robert (Author) / Lehtivuori, Heli (Author) / Panman, Matthijs (Author) / Pande, Kanupriya (Author) / White, Thomas (Author) / Nakane, Takanori (Author) / Berntsson, Oskar (Author) / Gustavsson, Emil (Author) / Bath, Petra (Author) / Modi, Vaibhav (Author) / Roy Chowdhury, Shatabdi (Author) / Zook, James (Author) / Berntsen, Peter (Author) / Pandey, Suraj (Author) / Poudyal, Ishwor (Author) / Tenboer, Jason (Author) / Kupitz, Christopher (Author) / Barty, Anton (Author) / Fromme, Petra (Author) / Koralek, Jake D. (Author) / Tanaka, Tomoyuki (Author) / Spence, John (Author) / Liang, Mengning (Author) / Hunter, Mark S. (Author) / Boutet, Sebastien (Author) / Nango, Eriko (Author) / Moffat, Keith (Author) / Groenhof, Gerrit (Author) / Ihalainen, Janne (Author) / Stojkovic, Emina A. (Author) / Schmidt, Marius (Author) / Westenhoff, Sebastian (Author) / College of Liberal Arts and Sciences (Contributor) / School of Molecular Sciences (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / Department of Physics (Contributor)
Created2016-10-19
Description
Microalgae-derived lipids are good sources of biofuel, but extracting them involves high cost, energy
expenditure, and environmental risk. Surfactant treatment to disrupt Scenedesmus biomass was evaluated
as a means to make solvent extraction more efficient. Surfactant treatment increased the recovery of fatty
acid methyl ester (FAME) by as much as 16-fold vs. untreated

Microalgae-derived lipids are good sources of biofuel, but extracting them involves high cost, energy
expenditure, and environmental risk. Surfactant treatment to disrupt Scenedesmus biomass was evaluated
as a means to make solvent extraction more efficient. Surfactant treatment increased the recovery of fatty
acid methyl ester (FAME) by as much as 16-fold vs. untreated biomass using isopropanol extraction, and
nearly 100% FAME recovery was possible without any Folch solvent, which is toxic and expensive. Surfactant
treatment caused cell disruption and morphological changes to the cell membrane, as documented by
transmission electron microscopy and flow cytometry. Surfactant treatment made it possible to extract wet
biomass at room temperature, which avoids the expense and energy cost associated with heating
and drying of biomass during the extraction process. The best FAME recovery was obtained from highlipid
biomass treated with Myristyltrimethylammonium bromide (MTAB)- and 3-(decyldimethylammonio)-
propanesulfonate inner salt (3_DAPS)-surfactants using a mixed solvent (hexane : isopropanol = 1 : 1, v/v)
vortexed for just 1 min; this was as much as 160-fold higher than untreated biomass. The critical micelle
concentration of the surfactants played a major role in dictating extraction performance, but the growth
stage of the biomass had an even larger impact on how well the surfactants disrupted the cells and
improved lipid extraction. Surfactant treatment had minimal impact on extracted-FAME profiles and,
consequently, fuel-feedstock quality. This work shows that surfactant treatment is a promising strategy for
more efficient, sustainable, and economical extraction of fuel feedstock from microalgae.
Created2015-10-20
Description
Using a CH[subscript 4]-based membrane biofilm reactor (MBfR), we studied perchlorate (ClO[subscript 4]–) reduction by a biofilm performing anaerobic methane oxidation coupled to denitrification (ANMO-D). We focused on the effects of nitrate (NO[subscript 3]–) and nitrite (NO[subscript 2]–) surface loadings on ClO[subscript 4]– reduction and on the biofilm community’s mechanism

Using a CH[subscript 4]-based membrane biofilm reactor (MBfR), we studied perchlorate (ClO[subscript 4]–) reduction by a biofilm performing anaerobic methane oxidation coupled to denitrification (ANMO-D). We focused on the effects of nitrate (NO[subscript 3]–) and nitrite (NO[subscript 2]–) surface loadings on ClO[subscript 4]– reduction and on the biofilm community’s mechanism for ClO[subscript 4]– reduction. The ANMO-D biofilm reduced up to 5 mg/L of ClO[subscript 4]– to a nondetectable level using CH[subscript 4] as the only electron donor and carbon source when CH[subscript 4] delivery was not limiting; NO[subscript 3]– was completely reduced as well when its surface loading was ≤0.32 g N/m[superscript 2]-d. When CH[subscript 4] delivery was limiting, NO[subscript 3]– inhibited ClO[subscript 4]– reduction by competing for the scarce electron donor. NO[subscript 2]– inhibited ClO[subscript 4]– reduction when its surface loading was ≥0.10 g N/m[superscript 2]-d, probably because of cellular toxicity. Although Archaea were present through all stages, Bacteria dominated the ClO[subscript 4]–-reducing ANMO-D biofilm, and gene copies of the particulate methane mono-oxygenase (pMMO) correlated to the increase of respiratory gene copies. These pieces of evidence support that ClO[subscript 4]– reduction by the MBfR biofilm involved chlorite (ClO[subscript 2]–) dismutation to generate the O[subscript 2] needed as a cosubstrate for the mono-oxygenation of CH[subscript 4].
ContributorsLuo, Yi-Hao (Author) / Chen, Ran (Author) / Wen, Li-Lian (Author) / Meng, Fan (Author) / Zhang, Yin (Author) / Lai, Chun-Yu (Author) / Rittmann, Bruce (Author) / Zhao, He-Ping (Author) / Zheng, Ping (Author) / Biodesign Institute (Contributor) / Swette Center for Environmental Biotechnology (Contributor)
Created2015-02-17
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Description
UV photolysis was used to relieve inhibition of biomass growth by sulfadiazine (SD), a broad-spectrum anti-microbial. To investigate the effects of SD on biomass growth, three substrates—glucose alone (G), glucose plus sulfadiazine (G+SD), and glucose plus photolyzed SD (G+PSD)—were used to culture the bacteria acclimated to glucose. The biomass was

UV photolysis was used to relieve inhibition of biomass growth by sulfadiazine (SD), a broad-spectrum anti-microbial. To investigate the effects of SD on biomass growth, three substrates—glucose alone (G), glucose plus sulfadiazine (G+SD), and glucose plus photolyzed SD (G+PSD)—were used to culture the bacteria acclimated to glucose. The biomass was strongly inhibited when SD was added into the glucose solution, but inhibition was relieved to a significant degree when the SD was treated with UV irradiation as a pretreatment. The biomass growth kinetics were described well by the Monod model when glucose was used as a substrate alone, but the kinetics followed a hybrid Aiba model for non-competitive inhibition when SD was added to the solution. When photolyzed SD was added to glucose solution to replace original SD, the growth still followed Aiba inhibition, but inhibition was significantly relieved: the maximum specific growth rate (μ[subscript max]) increased by 17 %, and the Aiba inhibition concentration increased by 60 %. Aniline, a major product of UV photolysis, supported the growth of the glucose-biodegrading bacteria. Thus, UV photolysis of SD significantly relieved inhibition by lowering the SD concentration and by generating a biodegradable product.
ContributorsPan, Shihui (Author) / Yan, Ning (Author) / Zhang, Yongming (Author) / Rittmann, Bruce (Author) / Biodesign Institute (Contributor) / Swette Center for Environmental Biotechnology (Contributor)
Created2015-05-01
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Description
Sustainable production of microalgae for biofuel requires efficient phosphorus (P) utilization, which is a limited resource and vital for global food security. This research tracks the fate of P through biofuel production and investigates P recovery from the biomass using the cyanobacterium Synechocystis sp. PCC 6803. Our results show that

Sustainable production of microalgae for biofuel requires efficient phosphorus (P) utilization, which is a limited resource and vital for global food security. This research tracks the fate of P through biofuel production and investigates P recovery from the biomass using the cyanobacterium Synechocystis sp. PCC 6803. Our results show that Synechocystis contained 1.4% P dry weight. After crude lipids were extracted (e.g., for biofuel processing), 92% of the intracellular P remained in the residual biomass, indicating phospholipids comprised only a small percentage of cellular P. We estimate a majority of the P is primarily associated with nucleic acids. Advanced oxidation using hydrogen peroxide and microwave heating released 92% of the cellular P into orthophosphate. We then recovered the orthophosphate from the digestion matrix using two different types of anion exchange resins. One resin impregnated with iron nanoparticles adsorbed 98% of the influent P through 20 bed volumes, but only released 23% during regeneration. A strong-base anion exchange resin adsorbed 87% of the influent P through 20 bed volumes and released 50% of it upon regeneration. This recovered P subsequently supported growth of Synechocystis. This proof-of-concept recovery process reduced P demand of biofuel microalgae by 54%.
Created2015-03-01
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Description

X-ray free-electron lasers provide novel opportunities to conduct single particle analysis on nanoscale particles. Coherent diffractive imaging experiments were performed at the Linac Coherent Light Source (LCLS), SLAC National Laboratory, exposing single inorganic core-shell nanoparticles to femtosecond hard-X-ray pulses. Each facetted nanoparticle consisted of a crystalline gold core and a

X-ray free-electron lasers provide novel opportunities to conduct single particle analysis on nanoscale particles. Coherent diffractive imaging experiments were performed at the Linac Coherent Light Source (LCLS), SLAC National Laboratory, exposing single inorganic core-shell nanoparticles to femtosecond hard-X-ray pulses. Each facetted nanoparticle consisted of a crystalline gold core and a differently shaped palladium shell. Scattered intensities were observed up to about 7 nm resolution. Analysis of the scattering patterns revealed the size distribution of the samples, which is consistent with that obtained from direct real-space imaging by electron microscopy. Scattering patterns resulting from single particles were selected and compiled into a dataset which can be valuable for algorithm developments in single particle scattering research.

ContributorsLi, Xuanxuan (Author) / Chiu, Chun-Ya (Author) / Wang, Hsiang-Ju (Author) / Kassemeyer, Stephan (Author) / Botha, Sabine (Author) / Shoeman, Robert L. (Author) / Lawrence, Robert (Author) / Kupitz, Christopher (Author) / Kirian, Richard (Author) / James, Daniel (Author) / Wang, Dingjie (Author) / Nelson, Garrett (Author) / Messerschmidt, Marc (Author) / Boutet, Sebastien (Author) / Williams, Garth J. (Author) / Hartman, Elisabeth (Author) / Jafarpour, Aliakbar (Author) / Foucar, Lutz M. (Author) / Barty, Anton (Author) / Chapman, Henry (Author) / Liang, Mengning (Author) / Menzel, Andreas (Author) / Wang, Fenglin (Author) / Basu, Shibom (Author) / Fromme, Raimund (Author) / Doak, R. Bruce (Author) / Fromme, Petra (Author) / Weierstall, Uwe (Author) / Huang, Michael H. (Author) / Spence, John (Author) / Schlichting, Ilme (Author) / Hogue, Brenda (Author) / Liu, Haiguang (Author) / ASU Biodesign Center Immunotherapy, Vaccines and Virotherapy (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / College of Liberal Arts and Sciences (Contributor) / School of Molecular Sciences (Contributor) / Department of Physics (Contributor) / School of Life Sciences (Contributor)
Created2017-04-11
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Description

Inhibition by ammonium at concentrations above 1000 mgN/L is known to harm the methanogenesis phase of anaerobic digestion. We anaerobically digested swine waste and achieved steady state COD-removal efficiency of around 52% with no fatty-acid or H[subscript 2] accumulation. As the anaerobic microbial community adapted to the gradual increase of total

Inhibition by ammonium at concentrations above 1000 mgN/L is known to harm the methanogenesis phase of anaerobic digestion. We anaerobically digested swine waste and achieved steady state COD-removal efficiency of around 52% with no fatty-acid or H[subscript 2] accumulation. As the anaerobic microbial community adapted to the gradual increase of total ammonia-N (NH[subscript 3]-N) from 890 ± 295 to 2040 ± 30 mg/L, the Bacterial and Archaeal communities became less diverse. Phylotypes most closely related to hydrogenotrophic Methanoculleus (36.4%) and Methanobrevibacter (11.6%), along with acetoclastic Methanosaeta (29.3%), became the most abundant Archaeal sequences during acclimation. This was accompanied by a sharp increase in the relative abundances of phylotypes most closely related to acetogens and fatty-acid producers (Clostridium, Coprococcus, and Sphaerochaeta) and syntrophic fatty-acid Bacteria (Syntrophomonas, Clostridium, Clostridiaceae species, and Cloacamonaceae species) that have metabolic capabilities for butyrate and propionate fermentation, as well as for reverse acetogenesis. Our results provide evidence countering a prevailing theory that acetoclastic methanogens are selectively inhibited when the total ammonia-N concentration is greater than ~1000 mgN/L. Instead, acetoclastic and hydrogenotrophic methanogens coexisted in the presence of total ammonia-N of ~2000 mgN/L by establishing syntrophic relationships with fatty-acid fermenters, as well as homoacetogens able to carry out forward and reverse acetogenesis.

Created2016-08-11
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Description

Anaerobic oxidation of methane (AOM) is an important process for understanding the global flux of methane and its relation to the global carbon cycle. Although AOM is known to be coupled to reductions of sulfate, nitrite, and nitrate, evidence that AOM is coupled with extracellular electron transfer (EET) to conductive

Anaerobic oxidation of methane (AOM) is an important process for understanding the global flux of methane and its relation to the global carbon cycle. Although AOM is known to be coupled to reductions of sulfate, nitrite, and nitrate, evidence that AOM is coupled with extracellular electron transfer (EET) to conductive solids is relatively insufficient. Here, we demonstrate EET-dependent AOM in a biofilm anode dominated by Geobacter spp. and Methanobacterium spp. using carbon-fiber electrodes as the terminal electron sink. The steady-state current density was kept at 11.0 ± 1.3 mA/m[superscript 2] in a microbial electrochemical cell, and isotopic experiments supported AOM-EET to the anode. Fluorescence in situ hybridization images and metagenome results suggest that Methanobacterium spp. may work synergistically with Geobacter spp. to allow AOM, likely by employing intermediate (formate or H[subscript 2])-dependent inter-species electron transport. Since metal oxides are widely present in sedimentary and terrestrial environments, an AOM-EET niche would have implications for minimizing the net global emissions of methane.

ContributorsGao, Yaohuan (Author) / Lee, Jangho (Author) / Neufeld, Josh D. (Author) / Park, Joonhong (Author) / Rittmann, Bruce (Author) / Lee, Hyung-Sool (Author) / Biodesign Institute (Contributor) / Swette Center for Environmental Biotechnology (Contributor)
Created2017-07-11
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Description
CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1), and has previously been shown to induce the production of anti-HIV-1 antibodies with antiviral functions. To further improve the design

CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1), and has previously been shown to induce the production of anti-HIV-1 antibodies with antiviral functions. To further improve the design of this candidate vaccine, X-ray crystallography experiments were performed to obtain structural information about this fusion protein. Several variants of CTB-MPR were designed, constructed and recombinantly expressed in Escherichia coli. The first variant contained a flexible GPGP linker between CTB and MPR, and yielded crystals that diffracted to a resolution of 2.3 Å, but only the CTB region was detected in the electron-density map. A second variant, in which the CTB was directly attached to MPR, was shown to destabilize pentamer formation. A third construct containing a polyalanine linker between CTB and MPR proved to stabilize the pentameric form of the protein during purification. The purification procedure was shown to produce a homogeneously pure and monodisperse sample for crystallization. Initial crystallization experiments led to pseudo-crystals which were ordered in only two dimensions and were disordered in the third dimension. Nanocrystals obtained using the same precipitant showed promising X-ray diffraction to 5 Å resolution in femtosecond nanocrystallography experiments at the Linac Coherent Light Source at the SLAC National Accelerator Laboratory. The results demonstrate the utility of femtosecond X-ray crystallography to enable structural analysis based on nano/microcrystals of a protein for which no macroscopic crystals ordered in three dimensions have been observed before.
ContributorsLee, Ho-Hsien (Author) / Cherni, Irene (Author) / Yu, HongQi (Author) / Fromme, Raimund (Author) / Doran, Jeffrey (Author) / Grotjohann, Ingo (Author) / Mittman, Michele (Author) / Basu, Shibom (Author) / Deb, Arpan (Author) / Dorner, Katerina (Author) / Aquila, Andrew (Author) / Barty, Anton (Author) / Boutet, Sebastien (Author) / Chapman, Henry N. (Author) / Doak, R. Bruce (Author) / Hunter, Mark (Author) / James, Daniel (Author) / Kirian, Richard (Author) / Kupitz, Christopher (Author) / Lawrence, Robert (Author) / Liu, Haiguang (Author) / Nass, Karol (Author) / Schlichting, Ilme (Author) / Schmidt, Kevin (Author) / Seibert, M. Marvin (Author) / Shoeman, Robert L. (Author) / Spence, John (Author) / Stellato, Francesco (Author) / Weierstall, Uwe (Author) / Williams, Garth J. (Author) / Yoon, Chun Hong (Author) / Wang, Dingjie (Author) / Zatsepin, Nadia (Author) / Hogue, Brenda (Author) / Matoba, Nobuyuki (Author) / Fromme, Petra (Author) / Mor, Tsafrir (Author) / ASU Biodesign Center Immunotherapy, Vaccines and Virotherapy (Contributor) / Department of Chemistry and Biochemistry (Contributor) / College of Liberal Arts and Sciences (Contributor) / School of Life Sciences (Contributor) / Biodesign Institute (Contributor) / Infectious Diseases and Vaccinology (Contributor) / Department of Physics (Contributor)
Created2014-08-20