Serial macromolecular crystallography at ALBA Synchrotron Light Source

12 pags., 4 figs., 2 tabs. -- Addenda and errata: https://journals.iucr.org/s/issues/2022/03/00/rv5160/rv5160.pdfThe increase in successful adaptations of serial crystallography at synchrotron radiation sources continues. To date, the number of serial synchrotron crystallography (SSX) experiments has grown exponentially, with over 40 experiments reported so far. In this work, we report the first SSX experiments with viscous jets conducted at ALBA beamline BL13-XALOC. Small crystals (15-30 μm) of five soluble proteins (lysozyme, proteinase K, phycocyanin, insulin and α-spectrin-SH3 domain) were suspended in lipidic cubic phase (LCP) and delivered to the X-ray beam with a high-viscosity injector developed at Arizona State University. Complete data sets were collected from all proteins and their high-resolution structures determined. The high quality of the diffraction data collected from all five samples, and the lack of specific radiation damage in the structures obtained in this study, confirm that the current capabilities at the beamline enables atomic resolution determination of protein structures from microcrystals as small as 15 μm using viscous jets at room temperature. Thus, BL13-XALOC can provide a feasible alternative to X-ray free-electron lasers when determining snapshots of macromolecular structures.The following funding is acknowledged: Ayuda de Atracciony Retencion de Talento Investigador" from the Community of Madrid (scholarship No. 2019-T1/BMD-15552); STC Programof the National Science Foundation through BioXFEL (awardNo. 1231306); the Centre for Applied Structural Discovery(CASD) at the Biodesign Institute at Arizona State University; the Spanish Ministry of Science and Innovation, grants EQC2021-007532-P, PID2020-117028GB-I00, BIO2016-77883-C2-2-

Modulation of heme orientation and binding by a single residue in catalase HPII of Escherichia coli

Heme-containing catalases have been extensively studied, revealing the roles of many residues, the existence of two heme orientations, flipped 180° relative to one another along the propionate-vinyl axis, and the presence of both heme b and heme d. The focus of this report is a residue, situated adjacent to the vinyl groups of the heme at the entrance of the lateral channel, with an unusual main chain geometry that is conserved in all catalase structures so far determined. In Escherichia coli catalase HPII, the residue is Ile274, and replacing it with Gly, Ala, and Val, found at the same location in other catalases, results in a reduction in catalytic efficiency, a reduced intensity of the Soret absorbance band, and a mixture of heme orientations and species. The reduced turnover rates and higher H2O2 concentrations required to attain equivalent reaction velocities are explained in terms of less efficient containment of substrate H2O2 in the heme cavity arising from easier escape through the more open entrance to the lateral channel created by the smaller side chains of Gly and Ala. Inserting a Cys at position 274 resulted in the heme being covalently linked to the protein through a Cys-vinyl bond that is hypersensitive to X-ray irradiation being largely degraded within seconds of exposure to the X-ray beam. Two heme orientations, flipped along the propionate-vinyl axis, are found in the Ala, Val, and Cys variants. © 2011 American Chemical Society.Este trabajo fue financiado por Discovery Grant 9600 del Natural Sciences and Engineering Research Council of Canada (para P.C.L.), por el Canada Research Chair Program (para P.C.L.), y por la subvención BFU2009-09268 del Ministerio de España de Ciencia e Innovación (para I.F.).Peer Reviewe

Influence of main channel structure on H2O2 access to the heme cavity of catalase KatE of Escherichia coli

The main channel for H 2O 2 access to the heme cavity in large subunit catalases is twice as long as in small subunit catalases and is divided into two distinct parts. Like small subunit catalases, the 15 of the channel adjacent to the heme has a predominantly hydrophobic surface with only weak water occupancy, but the next 15 extending to the protein surface is hydrophilic and contains a complex water matrix in multiple passages. At the approximate junction of these two sections are a conserved serine and glutamate that are hydrogen bonded and associated with H 2O 2 in inactive variants. Mutation of these residues changed the dimensions of the channel, both enlarging and constricting it, and also changed the solvent occupancy in the hydrophobic, inner section of the main channel. Despite these structural changes and the prominent location of the residues in the channel, the variants exhibited less than a 2-fold change in the k cat and apparent K M kinetic constants. These results reflect the importance of the complex multi-passage structure of the main channel. Surprisingly, mutation of either the serine or glutamate to an aliphatic side chain interfered with heme oxidation to heme d. © 2012 Elsevier Inc. All rights reserved.This work was supported by a Discovery Grant 9600 from the Natural Sciences and Engineering Research Council of Canada and by the Canada Research Chair Program (to P.C.L.).Peer Reviewe

Crystallization of the lipoxygenase of Pseudomonas aeruginosa 42A2, evolution and phylogenetic study of the subfamilies of the lipoxygenases

Editor: Diego Muñoz-Torrero.Lipoxygenases are non-heme iron enzymes essential in eukaryotes, where they catalyze the formation of the fatty acid hydroperoxides that are required by a large diversity of biological and pathological processes. In prokaryotes, most of them totally lacking in polyunsaturated fatty acids, the possible biological roles of lipoxygenases have remained obscure. In this study, it is reported the crystallization of a lipoxygenase of Pseudomonas aeruginosa (Pa_LOX), the first from a prokaryote. High resolution data has been acquired which is expected to yield structural clues to the questions adressed. Besides, a preliminar phylogenetic analysis using 14 sequences has confirmed the existence of this subfamily of bacterial lipoxygenases, on one side, and a greater diversity than in the corresponding eukaryotic ones, on the other. Finally, an evolutionary study of bacterial lipoxygenases on the same set of lipoxygenases, show a selection pressure of a basically purifying or neutral character except for a single aminoacid, which would have been selected after a positive selection event.Peer Reviewe

Crystallization and preliminary X-ray analysis of the catalase-peroxidase KatG from Burkholderia pseudomallei

The bifunctional catalase-peroxidase KatG encoded by the katG gene of Burkholderia pseudomallei has a predicted subunit size of 81.6 kDa. It shows high sequence similarity to other catalase-peroxidases of bacterial, archaebacterial and fungal origin, including 64% identity to KatG from Mycobacterium tuberculosis and lesser sequence similarity to members of the plant peroxidase family. Crystals from this protein were grown in 16-20% PEG 4000, 20% 2-methyl-2,4-pentanediol and 0.1 M sodium citrate pH 5.6 by the hanging-drop vapour-diffusion method at 293 K. These crystals diffracted beyond 1.8 Å resolution and belong to space group P212121, with unit-cell parameters a = 100.9, b = 115.6, c = 175.2 Å. The data are consistent with either a monomer or a dimer in the crystal asymmetric unit.This work was supported by grants BIO099-0865 from DGICYT to IF and OGP9600 from the Natural Sciences and Engineering Research Council of Canada (NSERC) to PCLPeer Reviewe

Eukaryotic Catalase-Peroxidase: The Role of the Trp-Tyr-Met Adduct in Protein Stability, Substrate Accessibility, and Catalysis of Hydrogen Peroxide Dismutation

Bernhard Gasselhuber et al.© 2015 American Chemical Society. Recently, it was demonstrated that bifunctional catalase-peroxidases (KatGs) are found not only in archaea and bacteria but also in lower eukaryotes. Structural studies and preliminary biochemical data of the secreted KatG from the rice pathogen Magnaporthe grisea (MagKatG2) suggested both similar and novel features when compared to those of the prokaryotic counterparts studied so far. In this work, we demonstrate the role of the autocatalytically formed redox-active Trp140-Tyr273-Met299 adduct of MagKatG2 in (i) the maintenance of the active site architecture, (ii) the catalysis of hydrogen peroxide dismutation, and (iii) the protein stability by comparing wild-type MagKatG2 with the single mutants Trp140Phe, Tyr273Phe, and Met299Ala. The impact of disruption of the covalent bonds between the adduct residues on the spectral signatures and heme cavity architecture was small. By contrast, loss of its integrity converts bifunctional MagKatG2 to a monofunctional peroxidase of significantly reduced thermal stability. It increases the accessibility of ligands due to the increased flexibility of the KatG-typical large loop 1 (LL1), which contributes to the substrate access channel and anchors at the adduct Tyr. We discuss these data with respect to those known from prokaryotic KatGs and in addition present a high-resolution structure of an oxoiron compound of MagKatG2.This project was supported by the Austrian Science Foundation, FWF [Doctoral program BioToP-Biomolecular Technology of Proteins (W1224) and Projects P23855 and P25270]Peer Reviewe

Structure and interaction with phospholipids of a prokaryotic lipoxygenase from Pseudomonas aeruginosa

Albert Garreta et al.Lipoxygenases (LOXs), which are essential in eukaryotes, have no confirmed function in prokaryotes that are devoid of polyunsaturated fatty acids. The structure of a secretable LOX from Pseudomonas aeruginosa (Pa-LOX), the first available from a prokaryote, presents significant differences with respect to eukaryotic LOXs, including a cluster of helices acting as a lid to the active center. The mobility of the lid and the structural variability of the N-terminal region of Pa-LOX was confirmed by comparing 2 crystal forms. The binding pocket contains a phosphatidylethanolamine phospholipid with branches of 18 (sn-1) and 14/16 (sn-2) carbon atoms in length. Carbon atoms from the sn-1 chain approach the catalytic iron in a manner that sheds light on how the enzymatic reaction might proceed. The findings in these studies suggest that Pa-LOX has the capacity to extract and modify unsaturated phospholipids from eukaryotic membranes, allowing this LOX to play a role in the interaction of P. Aeruginosa with host cells.Garreta, A., Val-Moraes, S. P., García-Fernández, Q., Montserrat Busquets, C. J., Oliver, A., Ortiz, A., Gaffney, B. J., Fita, I., Manresa, A., Carpena, X. Structure and interaction with phospholipids of a prokaryotic lipoxygenase from Pseudomonas aeruginosa. © FASEB.This work was supported by grants CTQ2010-21283-C02/01/PPQ, HBP2006-0027 from the Ministerio de Ciencia e Innovacion (MICINN) and 2009SGR819 from the Generalitat de Catalunya to A.M.; grants BFU2012-36827 from MICINN and SGR2009-00327 from the Generalitat de Catalunya to I.F.; grant R01GM065268 from the U.S. National Institutes of Health to B.J.G.; and grants SAF2012-38539, CP12/03324, and RD12/0015 from the Ministerio de Economia y Competitividad-Instituto de Salud Carlos III of Spain to C.J. and A.Ol.Peer Reviewe

Thirty years of heme catalases structural biology

About thirty years ago the crystal structures of the heme catalases from Penicillium vitale (PVC) and, a few months later, from bovine liver (BLC) were published. Both enzymes were compact tetrameric molecules with subunits that, despite their size differences and the large phylogenetic separation between the two organisms, presented a striking structural similarity for about 460 residues. The high conservation, confirmed in all the subsequent structures determined, suggested a strong pressure to preserve a functional catalase fold, which is almost exclusively found in these mono-functional heme catalases. However, even in the absence of the catalase fold an efficient catalase activity is also found in the heme containing catalase-peroxidase proteins. The structure of these broad substrate range enzymes, reported for the first time less than ten years ago from the halophilic archaebacterium Haloarcula marismortui (HmCPx) and from the bacterium Burkholderia pseudomallei (BpKatG), showed a heme pocket closely related to that of plant peroxidases, though with a number of unique modifications that enable the catalase reaction. Despite the wealth of structural information already available, for both monofunctional catalases and catalase-peroxidases, a number of unanswered major questions require continuing structural research with truly innovative approaches.This work was supported in part by the Spanish Ministerio de Ciencia e Innovación (Grant BFU2009-09268, to I.F.), the Natural Sciences and Engineering Research Council of Canada (to P.C.L.), the Canada Research Chair Program (to P.C.L) and the Generalitat de Catalunya (BP-B2008-00239, to X.C.)Peer Reviewe

An electrical potential in the access channel of catalases enhances catalysis

Substrate H2O2 must gain access to the deeply buried active site of catalases through channels of 30-50 Å in length. The most prominent or main channel approaches the active site perpendicular to the plane of the heme and contains a number of residues that are conserved in all catalases. Changes in Val169, 8 Å from the heme in catalase HPII from Escherichia coli, introducing smaller, larger or polar side chains reduces the catalase activity. Changes in Asp181, 12 Å from the heme, reduces activity by up to 90% if the negatively charged side chain is removed when Ala, Gln, Ser, Asn, or Ile are the substituted residues. Only the D181E variant retains wild type activity. Determination of the crystal structures of the Glu181, Ala181, Ser181, and Gln181 variants of HPII reveals lower water occupancy in the main channel of the less active variants, particularly at the position forming the sixth ligand to the heme iron and in the hydrophobic, constricted region adjacent to Val169. It is proposed that an electrical potential exists between the negatively charged aspartate (or glutamate) side chain at position 181 and the positively charged heme iron 12 Å distant. The potential field acts upon the electrical dipoles of water generating a common orientation that favors hydrogen bond formation and promotes interaction with the heme iron. Substrate hydrogen peroxide would be affected similarly and would enter the active site oriented optimally for interaction with active site residues.This work was supported by Grant BIO2002-04419 from Direccion General de Investigacion Ciencia y Technologia (to I. F.) and Grant OGP9600 from the Natural Sciences and Engineering Research Council of Canada (to P. C. L.)Peer Reviewe