The simplest known bacterium able to drive photosynthesis is found in muddy soils near hot springs. Heliobacterium modesticaldum is a sun-loving, soil-dwelling, thermophilic bacterium that photosynthesizes near-infrared light, unlike plants, which use different parts of visible light. The photosynthesis reaction centers (RCs) of H. modesticaldum are thought to resemble the earliest common ancestor of all photosynthesis complexes, which evolved around three billion years ago. Thus, a clear, detailed picture of the H. modesticaldum RC would provide valuable insight into the early evolution of photosynthesis. However, successfully purifying an RC protein and growing the crystals needed for X-ray crystallography can be a lengthy, difficult process.
Researchers successfully capped 7 years of work by obtaining a high-resolution (2.2 Å) structure of the membrane protein at the heart of the photosynthetic RC of H. modesticaldum. The structure revealed details such as the interactions between light-harvesting “antenna” molecules and the electron-transfer chain, where the initial steps required to convert photon energy into chemical energy occurs. With this data, the research team was able to make comparisons to other types of RCs, gaining new perspectives on the early evolution of photosynthesis and how nature optimized light-driven energy collection. This work will help unlock the secrets of photosynthesis, possibly leading to the development of cleaner, solar-based renewable energy.
Gisriel, C., et al. “Structure of a Symmetric Photosynthetic Reaction Center–Photosystem.” Science357(6355), 1021–1025 (2017). [DOI:10.1126/science.aan5611].
Instruments and Facilities: Beamline 8.2.1 at Advanced Light Source at Lawrence Berkeley National Laboratory; Advanced Photon Source at Argonne National Laboratory.
Funding Acknowledgements: Work funded by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences (OBES), U.S. Department of Energy (DOE) Office of Science, through grant DE-SC0010575 to K.E.R., R.F., and J.H.G. and supported with x-ray crystallographic equipment and infrastructure provided by P. Fromme of the Biodesign Center for Applied Structural Discovery at Arizona State University. Berkeley Center for Structural Biology supported in part by National Institutes of Health’s (NIH) National Institute of General Medical Sciences (NIGMS) and Howard Hughes Medical Institute (HHMI). The Advanced Light Source (ALS) is a DOE Scientific User Facility supported by OBES, Director, DOE Office of Science, and operated for DOE Office of Science by Lawrence Berkeley National Laboratory (LBNL). Results derived from work performed at Argonne National Laboratory’s (ANL) Structural Biology Center (SBC) at the Advanced Photon Source (APS). SBC funded by the Office of Biological and Environmental Research (OBER), DOE Office of Science. ANL is operated by University of Chicago Argonne, LLC, for the DOE Office of Science under contract DE-AC02-06CH11357.
A research team has uncovered a new mechanism for enzyme-mediated carbon dioxide (CO2) capture and conversion. They have revealed new and unique elements of carboxylation chemistry (CC) that could be used in catalytic strategies for converting CO2 into chemical feedstock or biomass. The crystal structure of acetone carboxylase (AC; enzyme involved in biodegradation by bacteria) was solved using X-ray crystallographic data. The inactive, unbound (apo) structure shows a substrate channel blocked off from the manganese (Mn) active site where CO2 conversion takes place. An adenosine monophosphate (AMP)–bound structure contains large conformational changes that open the channel to the active site. Highly reactive intermediates in the channel are protected from outside solvent as they are transported to the Mn active site for CO2 conversion. Stepwise mechanisms of carboxylation reactions differ in essential ways with respect to co-substrate, co-factor, and metal requirements. Knowledge of these mechanisms provides the basis for an increased fundamental understanding of CC, contributing to future strategies for CO2 capture and conversion to biomass. These in turn may mitigate the effects of increasing concentrations of CO2 on the global climate.
Mus, F., et al. “Structural Basis for the Mechanism of ATP-Dependent Acetone Carboxylation.” Nat. Sci. Rep. 7, Article 7234 (2017). [DOI:10.1038/s41598-017-06973-8].
Instruments and Facilities Used: X-ray crystallographic data from the Stanford Synchrotron Lightsource (SSLS) at SLAC National Accelerator Laboratory (SLAC). Structural data from macromolecular crystallography were measured on SSLS beamline 12-2; Advanced Photon Source (APS) at Argonne National Laboratory (ANL); and the Diamond Light Source, Oxfordshire, United Kingdom.
Funding Acknowledgements: Work supported by the Office of Basic Energy Sciences (OBES), U.S. Department of Energy (DOE) Office of Science, under Award Number DE-FG02-04ER15563. The Stanford Synchrotron Radiation Lightsource (SSRL) Structural Molecular Biology Program at the SLAC National Accelerator Laboratory (SLAC) supported by the DOE Office of Biological and Environmental Research (OBER) and by the National Institutes of Health’s (NIH) National Institute of General Medical Sciences (NIGMS; including P41GM103393). Argonne National Laboratory’s (ANL) Structural Biology Center at the Advanced Photon Source (APS). ANL is operated by University of Chicago Argonne, LLC, for DOE OBER under contract DE-AC02-06CH11357.
Bacterial DNA is characterized by regions of clustered regularly interspaced short palindromic repeats (CRISPRs) and associated Cas proteins (CRISPR-associated endonucleases). The CRISPR-Cas system has revolutionized gene editing by vastly simplifying the insertion of short snippets of new (“donor”) DNA into very specific locations of target DNA. Researchers in this study have discovered how Cas proteins recognize their target locations with such great specificity. They used x-ray crystallography to solve the structures of Cas1 and Cas2—responsible for DNA-snippet capture and integration—as the proteins were bound to synthesized DNA strands designed to mimic different stages of the process. The research also demonstrated how the system works in its native context as part of a bacterial immune system and how Cas proteins act as general-purpose molecular recording devices—tools for encoding information in genomes.
Cas1 appears to have evolved from a more “promiscuous” (less selective) type of enzyme that catalyzes the movement of DNA sequences from one position to another (a transposase). At some point, Cas1 acquired an unusual degree of specificity for a particular location in the bacterial genome, the CRISPR array. This specificity is critical to the bacteria, both for acquiring immunity and for avoiding genome damage caused by the insertion of viral fragments at the wrong location. The researchers wanted to learn how Cas1-Cas2 proteins recognize the target sequence to enable comparison with previously studied transposases and integrases (i.e., enzymes that catalyze the integration of donor DNA into target DNA) and to determine whether the proteins can be altered to recognize new sequences for custom applications.
The researchers crystallized Cas1-Cas2 in complex with preformed DNA strands that mimicked reaction intermediates and products. X-ray crystallography revealed that the structures showed substantial distortions in the target DNA, but there were surprisingly few sequence-specific contacts with the Cas1-Cas2 complex, and the DNA’s resulting flexibility produced disorder in the crystals. Attempts to model the DNA across the disordered sections showed that the DNA had to be even more distorted. Cryoelectron microscopy experiments, coupled with the crystallography data, confirmed that an accessor protein called the integration host factor (IHF) introduces an additional sharp bend in the DNA, bringing an upstream recognition sequence into contact with Cas1 to increase both the specificity and efficiency of integration. The architecture of the CRISPR integration complex suggests that subtle adjustment of the distance between Cas1 active sites could reprogram the system to recognize different target sites. Changes in its architecture could be exploited, thereby, for genome tagging applications and also may explain the natural divergence of CRISPR arrays in bacteria.
Wright, A. V., et al. “Structures of the CRISPR Genome Integration Complex,” Science357(6356), 1113–1118 (2017). [DOI:10.1126/science.aao0679].
Instruments and Facilities Used: X-ray macromolecular crystallography; beamline 8.3.1; protein crystallography (PX); and scattering/diffraction at the Advanced Light Source at Lawrence Berkeley National Laboratory; Stanford Synchrotron Radiation Light Source 9-2 beamline.
Funding Acknowledgements: Advanced Light Source (ALS) 8.3.1 beamline, Lawrence Berkeley National Laboratory (LBNL), and Stanford Synchrotron Radiation Lightsource (SSRL) 9-2 beamline, SLAC National Accelerator Laboratory (SLAC), for assistance with data collection. ALS Beamline 8.3.1, is operated by University of California Office of the President, Multicampus Research Programs and Initiatives (grant MR-15-328599), and Program for Breakthrough Biomedical Research, partially funded by the Sandler Foundation. Use of SSRL supported by the Office of Basic Energy Sciences (OBES), U.S. Department of Energy (DOE) Office of Science, under contract no. DE-AC02-76SF00515. Electron microscopy (EM) data collected in Howard Hughes Medical Institute (HHMI) EM facility located at University of California, Berkeley. SSRL Structural Molecular Biology Program supported by DOE Office of Biological and Environmental Research (OBER) and the National Institutes of Health’s (NIH) National Institute of General Medical Sciences (NIGMS; including grant no. P41GM103393). Project funded by U.S. National Science Foundation (NSF) grant no. 1244557 (to J.A.D.) and NIGMS grant no. 1P50GM102706-01 (to J. H. Cate). A.V.W. and K.W.D. support: NSF Graduate Research Fellowship; G.J.K. funding: HHMI. J.A.D. and E.N.: HHMI investigators and members of the Center for RNA Systems Biology. Atomic coordinates and structure factors for the reported crystal structures deposited in the Protein Data Bank under accession codes 5VVJ (half-site–bound), 5VVK (pseudo–full-site–bound), and 5VVL (pseudo–full-site–bound with Ni2+). Cryo-EM structure and map deposited in the Protein Data Bank under accession code 5WFE and the Electron Microscopy Data Bank under accession code EMD-8827.
A research study has identified and characterized a new, functionally distinct member of the orange carotenoid protein (OCP) family. The OCP complex enables chromatically acclimating blue-green algae to avoid cellular damage and growth inhibition in conditions of high light or nutrient stress. In a recent bioinformatic analysis of all available cyanobacterial genomes, the group found that many of these ecophysiologically diverse organisms encode more than one copy of the full-length OCP. The study’s focus was the filamentous blue-green algae Tolypothrix, which encodes two OCPs.
One copy was determined to be functionally equivalent to the well-characterized OCP of Synechocystis cyanobacteria, dubbed OCP1. But the second, OCP2, was distinct in several key aspects. The researchers hypothesize that OCP2 and another bioinformatically identified protein, OCPx, reflect intermediate stages in the evolution of photoprotection in cyanobacteria.
Bao, H., et al. “Additional Families of Orange Carotenoid Proteins in the Photoprotective System of Cyanobacteria.” Nature Plants3, Article 17089 (2017). [DOI:10.1038/nplants.2017.89].
Instruments and Facilities: X-ray macromolecular crystallography and diffraction at Advanced Light Source at Lawrence Berkeley National Laboratory.
Funding Acknowledgements: Work supported by the National Science Foundation (NSF; IOS 1557324). Advanced Light Source supported by the Office of Basic Energy Sciences (OBES), Director, U.S. Department of Energy (DOE) Office of Science, under Contract No. DE-AC02-05CH11231.
Each year in the United States, more than 57,000 children younger than 5 years old are hospitalized due to respiratory syncytial virus (RSV) infection, and about 14,000 adults older than 65 die from it. By age 2, most children have been infected with RSV, experiencing usually only mild cold symptoms. People with weakened immune systems, however, such as infants and the elderly, can face serious complications, including pneumonia and, in some cases, death. Now, scientists studying the virus using bright X-rays have found clues to how RSV causes disease. They mapped the molecular structure of an RSV protein that interferes with the body’s ability to fight off the virus. Knowing the structure of this protein will help them understand how the virus impedes immune response, potentially leading to a vaccine or treatment for this common infection.
With no approved vaccine and limited treatment for RSV, doctors prescribe the antiviral drug ribavirin only in the most severe cases because it is expensive and not very effective. Thus, most people with RSV receive only supportive care to make them more comfortable while their bodies fight off the virus. People with weakened immune systems face a tough fight with RSV. The researchers say that solving the structure of this elusive protein will enable them to see what the protein looks like and help them define what it does and how it does it. Ultimately, this capability and finding could lead to new targets for vaccine or drug development.
Scientists have long known that a nonstructural RSV protein (known as NS1) is key to the virus’s ability to evade the body’s immune response. However, its structure was unknown, so scientists were unable to determine exactly how the enigmatic NS1 interfered with the immune system. Using X-ray crystallography, the scientists determined the three-dimensional (3D) structure of NS1, and, in a detailed analysis of the structure, they identified a piece of the protein known as the alpha 3 helix, possibly critical for suppressing the immune response.
The researchers created different versions of this NS1 protein, with some having an intact and some with a mutated alpha 3 helix region. They tested the functional impact of helix 3 and created a set of viruses containing either the original or the mutant NS1 genes, measuring the effect on the immune response when they infected cells with these viruses. They found that viruses with the mutated helix region did not suppress the immune response, while the ones with the intact helix region did, globally modulating the immune response.
The findings also showed that the protein’s alpha 3 helix region was necessary for the virus to dial down the body’s immune response, giving the virus a better chance of surviving and multiplying—in other words, causing disease. Thus, a vaccine or treatment that targets the alpha 3 helix may supply what is needed to prevent immune suppression.
Chatterjee, S., et al. “Structural Basis for Human Respiratory Syncytial Virus NS1-Mediated Modulation of Host Responses.” Nat. Microbiol. 2, Article 17101 (2017). [DOI:10.1038/nmicrobiol.2017.101].
Instruments and Facilities Used: X-ray crystallography at the Advanced Photon Source at Argonne National Laboratory.
Funding Acknowledgements: Laboratory work support in part: National Institutes of Health (NIH) grants (R01AI107056 (to D.W.L.), R01AI123926 (to G.K.A.), R01AI114654 (to C.F.B.), U191099565 (G.K.A. is PI of the subaward from a U19 grant for which Ting is the PI), U19AI109945 (to C.F.B.), U19AI109664 (to C.F.B.), U19AI070489 (to M.J.H.), R01AI111605 (to M.J.H.), R01 AI130591 (to M.J.H.), R01AI087798 (to M.L.M.), U19AI095227 (to M.L.M.) and T32-CA09547-37 (D.S.J. is recipient of training award from T32 grant for which Allen is the PI)), the U.S. Department of Defense’s (DoD) Defense Threat Reduction Agency grants HDTRA1-16-0033 (to C.F.B.) and HDTRA1-16-0033 (to C.F.B.), the National Science Foundation (NSF) MCB-1121867 (to R.V.P.) and Children’s Discovery Institute PD-II-2013-272 (to G.K.A.). S.C. funding in part: American Heart Association Postdoctoral Fellowship (15POST25140009). Amarasinghe, Leung, Basler, Artyomov, and Holtzman laboratories and S. Ginell, N. Duke, R. Alkire, K. Lazarski, M. Ficner-Radford, Y. Kim, and A. Joachimiak: aid at Argonne National Laboratory (ANL) Structural Biology Center (SBC) Sector 19. Use of ANL SBC beam lines at Advanced Photon Source (APS) support: U.S. Department of Energy (DOE) under contract DE-AC02-06CH11357.
Plants caught in the act, changing chemistry thought to be immutable because necessary for life
Plants are the chemists of the living world, producing hundreds of thousands of small molecules that provide protection—to screen sunrays, to poison plant eaters, to scent the air, to color flowers, and for much other vegetative business.
Called “secondary metabolites,” these chemicals are distinguished from “primary metabolites,” which are the essential building blocks of proteins, fats, sugars, and DNA. Secondary metabolites just smooth the way in life, but failure to make primary metabolites correctly and efficiently is fatal. Genes for enzymes in the molecular assembly lines of primary metabolism have duplicates, allowing more tolerance of mutations that might have destabilized the primary pathways because the originals were still on the job. With evolutionary constraints thus relaxed, synthetic machinery was able to accumulate enough mutations to do new chemistry.
Widely conserved, primary metabolism it was thought to remain unchanged across many different groups of organisms because it operates correctly and efficiently and because its products are necessary for life. But now, a collaborative team of scientists has caught primary metabolism in the act of evolving. In a comprehensive study of a primary-metabolism assembly line in plants, they discovered a key enzyme evolving from a canonical form possessed by most plants, through noncanonical forms in tomatoes, to a switch-hitting form found in peanuts, and finally committing to the novel form in some strains of soybeans. This feat is comparable to pulling the tablecloth out from under the dishes without breaking any of them. A collaborative study of this biochemical pathway resulted in the crystallization of the soybean enzyme to reveal how nature changed the way the protein works, also capturing plants “building a pathway that links the primary to the secondary metabolism,” to reveal evolutionary machinery that creates new molecules.
A new pathway discovered for making tyrosine is much less constrained than the old one, raising the possibility that carbon flow could be directed away from lignin to increase the yields of drugs or nutrients to levels that would allow them to be produced in commercial quantities. Though the scientists have found two different assembly lines for tyrosine, they have not determined why except in general terms. This work is important because it demonstrates that primary metabolism does evolve.
Schenck, C. A., et al. “Molecular Basis of the Evolution of Alternative Tyrosine Biosynthetic Routes in Plants.” Nat. Chem. Biol.13, 1029–1035 (2017). [DOI:10.1038/nchembio.2414].
Instruments and Facilities Used: X-ray macromolecular crystallography; diffraction data collected at beamline 19-ID of the Advanced Photon Source at Argonne National Laboratory Structural Biology Center.
Funding Acknowledgements: Work supported by the National Science Foundation (NSF; IOS-1354971 to H.A.M. and MCB-1614539 to J.M.J.). C.K.H. support: NSF Graduate Research Fellowship Program (DGE-1143954). Portions of research carried out at Argonne National Laboratory (ANL) Structural Biology Center (SBC) of the Advanced Photon Source (APS), a national user facility operated by the University of Chicago for the Office of Biological and Environmental Research (OBER), U.S. Department of Energy (DOE) Office of Science (DE-AC02-06CH11357).
Structure of microcompartment’s protein shell could help research in bioenergy, pathogenesis, and biotechnology
Scientists are providing the clearest view yet of an intact bacterial microcompartment, revealing the structure and assembly of the organelle’s protein shell at atomic-level resolution. They studied the “photogenic” organelle shell of an ocean-dwelling slime bacteria Haliangium ochraceum. Providing the first view of the shell of an intact bacterial organelle membrane, this full structural view can help provide important information for beneficial use in fighting pathogens or bioengineering bacterial organelles. The research team said these organelles, or bacterial microcompartments (BMCs), are used by some bacteria to fix carbon dioxide. Thus, understanding how the microcompartment membrane is assembled, as well as how it lets some compounds pass through while impeding others, could contribute to research in enhancing carbon fixation and, more broadly, bioenergy. This class of organelles also helps many types of pathogenic bacteria metabolize compounds that are not available to normal, nonpathogenic microbes, giving the pathogens a competitive advantage.
Sutter, M., et al. “Assembly Principles and Structure of a 6.5-MDa Bacterial Microcompartment Shell.” Science23(6344), 1293–1297 (2017). [DOI:10.1126/science.aan3289].
Instruments and Facilities Used: Michigan State University–DOE Plant Research Laboratory and the Molecular Biophysics and Integrated Bioimaging Division at Lawrence Berkeley National Laboratory; Stanford Synchrotron Radiation Lightsource.
Funding Acknowledgements: Support: National Institutes of Health’s (NIH) National Institute of Allergy and Infectious Diseases (NIAID) grant 1R01AI114975-01 and the Office of Basic Energy Sciences (OBES), U.S. Department of Energy (DOE) Office of Science, under contract no. DE-FG02-91ER20021. Advanced Light Source (ALS), Lawrence Berkeley National Laboratory (LBNL) support: OBES, Director, DOE Office of Science, under contract no. DE-AC02-05CH11231. B.G. support: advanced postdoctoral mobility fellowship from Swiss National Science Foundation (NSF; project P300PA_160983). Use of Stanford Synchrotron Radiation Lightsource (SSRL), SLAC National Accelerator Laboratory (SLAC) support: OBES, DOE Office of Science, under contract no. DE-AC02-76SF00515. M.S. and C.A.K.: inventors on patent application 62509553 submitted by LBNL that covers strategies for scaling the shell-protein system described in this work. Cryo-EM map of complete shell deposited at Electron Microscopy Data Bank (EMDB) with accession code EMD-8747. X-ray crystallographic coordinates and structure-factor files deposited in Protein Data Bank (PDB) under the following accession numbers: 5V74 (complete shell), 5V75 (BMC-T2), and 5V76 (BMC-T3).
Joint BioEnergy Institute study targets LigM for its role in breaking down aromatic pollutants
A protein used by common soil bacteria is providing new clues in the effort to convert aryl compounds, a common waste product from industrial and agricultural practices, into something of value. The protein structure of the enzyme LigM was determined using X-ray crystallography, revealing novel structural elements (red in figure) and a conserved tetrahydrofolate-binding domain (gray), with LigM binding to its substrates (green) using internal binding cavities.
Researchers used have resolved the soil bacterium Sphingomonas to metabolize aryl compounds derived from lignin, the stiff, organic material that gives plants their structure. In biofuel production, aryl compounds are a byproduct of the breakdown of lignin, some pathways of which involve demethylation, an often critical precursor to additional modification steps of lignin-derived aryl compounds. The simple, single-enzyme system of LigM, as well as its functionality over a broad temperature range, makes it an attractive demethylase for use in aromatic conversion. Other findings included: half the LigM enzyme was homologous to known structures with a tetrahydrofolate-binding domain that is found in both simple and complex organisms; the other half of LigM’s structure is completely unique, providing a starting point for determining where its aryl substrate-binding site is located; and LigM is a tyrosine-dependent demethylase. This research provides groundwork needed to aid in developing an enzyme-based system for converting aromatic waste into useful products.
Kohlera, A. C., et al. “Structure of Aryl O-Demethylase Offers Molecular Insight into a Catalytic Tyrosine-Dependent Mechanism.” PNAS114(16), E3205–E3214 (2017). [DOI:10.1073/pnas.1619263114].
Instruments and Facilities Used: Beam line 8.2.2 and X-ray macromolecular crystallography at Berkeley Center for Structural Biology Advanced Light Source at Lawrence Berkeley National Laboratory.
Funding Acknowledgements: Crystallographic work: Berkeley Center for Structural Biology (BCSB) Advanced Light Source (ALS) beam line 8.2.2. BCSB ALS staff: technical support; J. H. Pereira: assistance in early stages of crystallographic work. BCSB support in part: National Institutes of Health’s (NIH) National Institute of General Medical Sciences (NIGMS) and Howard Hughes Medical Institute (HHMI). ALS support: Office of Basic Energy Sciences (OBES), Director, U.S. Department of Energy (DOE) Office of Science, under Contract DE-AC02-05CH11231. Work conducted by Joint BioEnergy Institute (JBEI) and supported by Office of Biological and Environmental Research (OBER), DOE Office of Science, under Contract DE-AC02-05CH11231.
In the Ebola virus one of the proteins, VP40, has the ability to refold to achieve new functions; three structural forms of VP40 have been determined, with each structure conferring a separate and essential function in the virus life cycle. The image illustrates (top) a butterfly-shaped dimer critical for membrane trafficking; (middle) a rearranged hexameric structure essential for building and releasing nascent virions; (bottom) an RNA-binding octameric ring that controls transcription in infected cells
Bornholdt, T. Noda, D.M. Abelson, P. Halfmann, M.R. Wood, Y. Kawaoka, E. Ollmann Saphire, Cell154, 763 (2013) [DOI: 10.1016/j.cell.2013.07.015]
Funding Acknowledgements: Beamlines at Argonne National Laboratory’s (ANL) Advanced Photon Source (APS): 19-ID and GM/CA 23-ID; SLAC National Accelerator Laboratory’s (SLAC) Stanford Synchrotron Radiation Lightsource (SSRL): 12-2; and Lawrence Berkeley National Laboratory’s (LBNL) Advanced Light Source (ALS) 5.0.2. E.O.S. support: Career Award in the Biomedical Sciences and an Investigator in Pathogenesis of Infectious Disease Award from Burroughs Welcome Fund, as well as The Skaggs Institute of Chemical Biology and a National Institutes of Health’s (NIH) National Institute of Allergy and Infectious Disease (NIAID) award (R43 AI1088843). Z.A.B. support: grant (2T32AI007244) to The Scripps Research Institute (TSRI; manuscript #21649) Department of Immunology and Microbial Science. Y.K.: membership within and support from the Region V “Great Lakes” Regional Center for Excellence (RCE) for Biodefense and Emerging Infectious Disease Research Program (NIH award U54 AI057153). T.N. support: Grant-in-Aid for Young Scientists from Japan Society for the Promotion of Science and Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science, and Technology.
Fungal lytic polysaccharide monooxygenases (LPMOs) are known to enhance the efficiency of cellulose-hydrolyzing enzymes through oxidative cleavage of the glycosidic bonds. For this study, PMO-2 from Neurospora crassa was heterologously expressed from Pichia pastoris, purified, and crystallized for high-resolution X-ray crystal structures that revealed “prebound” molecular oxygen in the resting state and a dioxo species in complex with the catalytic copper (Cu2+) ion, which is the first structural description of molecular oxygen (O2) activation by a LPMO. In addition, neutron diffraction studies and density functional theory calculations have identified a role for a conserved histidine in promoting oxygen activation. Extension of these studies to the enzyme-substrate complex could provide a complete picture of the enzymatic mechanism for the potential benefit of applications such as bioethanol production.
O’Dell, W. B., et al. “Oxygen Activation at the Active Site of a Fungal Lytic Polysaccharide Monooxygenase.” Angew. Chem. Int. Ed.129(3), 785–788 (2017). [DOI:10.1002/anie.201610502].
Instruments and Facilities Used: Neutron crystallography. Joint X-ray/neutron refinement at Center for Structural Molecular Biology at Oak Ridge National Laboratory (ORNL). Diffraction data were collected at SER-CAT 22-ID at the Advanced Photon Source at Argonne National Laboratory and at CG-4D IMAGINE (NSF MRI 09229719) at the High Flux Isotope Reactor at ORNL.
Funding Acknowledgements: Protein expression and purification experiments at Center for Structural Molecular Biology (CSMB), a User Facility of the Office of Biological and Environmental Research (OBER), U.S. Department of Energy (DOE) Office of Science. Diffraction data collected at SER‐CAT 22‐ID at Argonne National Laboratory’s (ANL) Advanced Photon Source (APS) and at CG‐4D IMAGINE (National Science Foundation [NSF] magnetic resonance imaging [MRI] 09229719) at Oak Ridge National Laboratory’s (ORNL) High Flux Isotope Reactor (HFIR), both DOE Office of Biological Energy Sciences (OBES) User Facilities. W.B.O. support: NSF IGERT 1069091. F.M. support: U.S. Department of Agriculture (USDA National Institutes of Food and Agriculture (NIFA) Hatch 211001. P.K.A. support: NIH GM105978.