Martin Bommer

Structural Basis for Organohalide Respiration.

How bacteria break down organohalides Anaerobic bacteria can break down a range of organohalide pollutants. To do so, they use unusual reductive dehalogenase enzymes that remove the halogen ion from the molecule, making the pollutants less toxic. Bommer et al. describe x-ray crystal structures of one such enzyme from Sulfurospirillum multivorans (see the Perspective by Edwards). Vitamin B12, present near the substrate binding site, catalyzes the reduction of trichloroethylene in concert with two iron-sulfur clusters. The structures provide mechanistic clues for how to engineer enzymes to recognize other pollutants. Science, this issue p. 455; see also p. 424 The crystal structure of a reductive dehalogenase shows how bacteria break down organohalide contaminants. [Also see Perspective by Edwards] Organohalide-respiring microorganisms can use a variety of persistent pollutants, including trichloroethene (TCE), as terminal electron acceptors. The final two-electron transfer step in organohalide respiration is catalyzed by reductive dehalogenases. Here we report the x-ray crystal structure of PceA, an archetypal dehalogenase from Sulfurospirillum multivorans, as well as structures of PceA in complex with TCE and product analogs. The active site harbors a deeply buried norpseudo-B12 cofactor within a nitroreductase fold, also found in a mammalian B12 chaperone. The structures of PceA reveal how a cobalamin supports a reductive haloelimination exploiting a conserved B12-binding scaffold capped by a highly variable substrate-capturing region.

Visualizing the substrate-, superoxo-, alkylperoxo-, and product-bound states at the nonheme Fe(II) site of homogentisate dioxygenase

Homogentisate 1,2-dioxygenase (HGDO) uses a mononuclear nonheme Fe2+ to catalyze the oxidative ring cleavage in the degradation of Tyr and Phe by producing maleylacetoacetate from homogentisate (2,5-dihydroxyphenylacetate). Here, we report three crystal structures of HGDO, revealing five different steps in its reaction cycle at 1.7–1.98 Å resolution. The resting state structure displays an octahedral coordination for Fe2+ with two histidine residues (His331 and His367), a bidentate carboxylate ligand (Glu337), and two water molecules. Homogentisate binds as a monodentate ligand to Fe2+, and its interaction with Tyr346 invokes the folding of a loop over the active site, effectively shielding it from solvent. Binding of homogentisate is driven by enthalpy and is entropically disfavored as shown by anoxic isothermal titration calorimetry. Three different reaction cycle intermediates have been trapped in different HGDO subunits of a single crystal showing the influence of crystal packing interactions on the course of enzymatic reactions. The observed superoxo:semiquinone-, alkylperoxo-, and product-bound intermediates have been resolved in a crystal grown anoxically with homogentisate, which was subsequently incubated with dioxygen. We demonstrate that, despite different folds, active site architectures, and Fe2+ coordination, extradiol dioxygenases can proceed through the same principal reaction intermediates to catalyze the O2-dependent cleavage of aromatic rings. Thus, convergent evolution of nonhomologous enzymes using the 2-His-1-carboxylate facial triad motif developed different solutions to stabilize closely related intermediates in unlike environments.

Acoustic Injectors for Drop-On-Demand Serial Femtosecond Crystallography

X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallization conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. We report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). Additional samples were screened to demonstrate that these methods can be applied to rare samples.

Determinants of substrate specificity and biochemical properties of the sn-glycerol-3-phosphate ATP binding cassette transporter (UgpB-AEC2 ) of Escherichia coli.

Under phosphate starvation conditions, Escherichia coli can utilize sn‐glycerol‐3‐phosphate (G3P) and G3P diesters as phosphate source when transported by an ATP binding cassette importer composed of the periplasmic binding protein, UgpB, the transmembrane subunits, UgpA and UgpE, and a homodimer of the nucleotide binding subunit, UgpC. The current knowledge on the Ugp transporter is solely based on genetic evidence and transport assays using intact cells. Thus, we set out to characterize its properties at the level of purified protein components. UgpB was demonstrated to bind G3P and glycerophosphocholine with dissociation constants of 0.68 ± 0.02 μM and 5.1 ± 0.3 μM, respectively, while glycerol‐2‐phosphate (G2P) is not a substrate. The crystal structure of UgpB in complex with G3P was solved at 1.8 Å resolution and revealed the interaction with two tryptophan residues as key to the preferential binding of linear G3P in contrast to the branched G2P. Mutational analysis validated the crucial role of Trp‐169 for G3P binding. The purified UgpAEC2 complex displayed UgpB/G3P‐stimulated ATPase activity in proteoliposomes that was neither inhibited by phosphate nor by the signal transducing protein PhoU or the phosphodiesterase UgpQ. Furthermore, a hybrid transporter composed of MalFG–UgpC could be functionally reconstituted while a UgpAE–MalK complex was unstable.

Cobamide-mediated enzymatic reductive dehalogenation via long-range electron transfer.

The capacity of metal-containing porphyrinoids to mediate reductive dehalogenation is implemented in cobamide-containing reductive dehalogenases (RDases), which serve as terminal reductases in organohalide-respiring microbes. RDases allow for the exploitation of halogenated compounds as electron acceptors. Their reaction mechanism is under debate. Here we report on substrate–enzyme interactions in a tetrachloroethene RDase (PceA) that also converts aryl halides. The shape of PceA’s highly apolar active site directs binding of bromophenols at some distance from the cobalt and with the hydroxyl substituent towards the metal. A close cobalt–substrate interaction is not observed by electron paramagnetic resonance spectroscopy. Nonetheless, a halogen substituent para to the hydroxyl group is reductively eliminated and the path of the leaving halide is traced in the structure. Based on these findings, an enzymatic mechanism relying on a long-range electron transfer is concluded, which is without parallel in vitamin B12-dependent biochemistry and represents an effective mode of RDase catalysis.

Improvements in serial femtosecond crystallography of photosystem II by optimizing crystal uniformity using microseeding procedures

In photosynthesis, photosystem II (PSII) is the multi-subunit membrane protein complex that catalyzes photo-oxidation of water into dioxygen through the oxygen evolving complex (OEC). To understand the water oxidation reaction, it is important to get structural information about the transient and intermediate states of the OEC in the dimeric PSII core complex (dPSIIcc). In recent times, femtosecond X-ray pulses from the free electron laser (XFEL) are being used to obtain X-ray diffraction (XRD) data of dPSIIcc microcrystals at room temperature that are free of radiation damage. In our experiments at the XFEL, we used an electrospun liquid microjet setup that requires microcrystals less than 40 μm in size. In this study, we explored various microseeding techniques to get a high yield of monodisperse uniform-sized microcrystals. Monodisperse microcrystals of dPSIIcc of uniform size were a key to improve the stability of the jet and the quality of XRD data obtained at the XFEL. This was evident by an improvement of the quality of the datasets obtained, from 6.5 Å, using crystals grown without the micro seeding approach, to 4.5 Å using crystals generated with the new method.

ATP-induced electron transfer by redox-selective partner recognition

Thermodynamically unfavourable electron transfers are enabled by coupling to an energy-supplying reaction. How the energy is transduced from the exergonic to the endergonic process is largely unknown. Here we provide the structural basis for an energy transduction process in the reductive activation of B12-dependent methyltransferases. The transfer of one electron from an activating enzyme to the cobalamin cofactor is energetically uphill and relies on coupling to an ATPase reaction. Our results demonstrate that the key to coupling is, besides the oxidation state-dependent complex formation, the conformational gating of the electron transfer. Complex formation induces a substitution of the ligand at the electron-accepting Co ion. Addition of ATP initiates electron transfer by provoking conformational changes that destabilize the complex. We show how remodelling of the electron-accepting Co(2+) promotes ATP-dependent electron transfer; an efficient strategy not seen in other electron-transferring ATPases.

Crystal structure of reduced MsAcg, a putative nitroreductase from Mycobacterium smegmatis and a close homologue of Mycobacterium tuberculosis Acg.

Background: Acg proteins are up-regulated during dormancy in tuberculosis. Results: Acg proteins bind flavin mononucleotide like nitroreductases but with the active site closed by a lid. They are not reduced by NADPH or NADH. Conclusion: Acg proteins may have evolved from active nitroreductases to sequester FMN instead. Significance: Turning off a flavin-dependent pathway may be important in tuberculosis dormancy. This paper presents the structure of MsAcg (MSMEG_5246), a Mycobacterium smegmatis homologue of Mycobacterium tuberculosis Acg (Rv2032) in its reduced form at 1.6 Å resolution using x-ray crystallography. Rv2032 is one of the most induced genes under the hypoxic model of tuberculosis dormancy. The Acg family turns out to be unusual flavin mononucleotide (FMN)-binding proteins that have probably arisen by gene duplication and fusion from a classical homodimeric nitroreductase such that the monomeric protein resembles a classical nitroreductase dimer but with one active site deleted and the other active site covered by a unique lid. The FMN cofactor is not reduced by either NADH or NADPH, but the chemically reduced enzyme is capable of reduction of nitro substrates, albeit at no kinetic advantage over free FMN. The reduced enzyme is rapidly oxidized by oxygen but without any evidence for a radical state commonly seen in oxygen-sensitive nitroreductases. The presence of the unique lid domain, the lack of reduction by NAD(P)H, and the slow rate of reaction of the chemically reduced protein raises a possible alternative function of Acg proteins in FMN storage or sequestration from other biochemical pathways as part of the bacterias adaptation to a dormancy state.

Structural insights into the light-driven auto-assembly process of the water-oxidizing Mn4CaO5-cluster in photosystem II

In plants, algae and cyanobacteria, Photosystem II (PSII) catalyzes the light-driven splitting of water at a protein-bound Mn4CaO5-cluster, the water-oxidizing complex (WOC). In the photosynthetic organisms, the light-driven formation of the WOC from dissolved metal ions is a key process because it is essential in both initial activation and continuous repair of PSII. Structural information is required for understanding of this chaperone-free metal-cluster assembly. For the first time, we obtained a structure of PSII from Thermosynechococcus elongatus without the Mn4CaO5-cluster. Surprisingly, cluster-removal leaves the positions of all coordinating amino acid residues and most nearby water molecules largely unaffected, resulting in a pre-organized ligand shell for kinetically competent and error-free photo-assembly of the Mn4CaO5-cluster. First experiments initiating (i) partial disassembly and (ii) partial re-assembly after complete depletion of the Mn4CaO5-cluster agree with a specific bi-manganese cluster, likely a di-µ-oxo bridged pair of Mn(III) ions, as an assembly intermediate. DOI: http://dx.doi.org/10.7554/eLife.26933.001

Crystallographic and Computational Analysis of the Barrel Part of the Psbo Protein of Photosystem II -Carboxylate-Water Clusters as Putative Proton Transfer Relays and Structural Switches

In all organisms that employ oxygenic photosynthesis, the membrane-extrinsic PsbO protein is a functionally important component of photosystem II. To study the previously proposed proton antenna function of carboxylate clusters at the protein-water interface, we combined crystallography and simulations of a truncated cyanobacterial (Thermosynechococcus elongatus) PsbO without peripheral loops. We expressed the PsbO β-barrel heterologously and determined crystal structures at resolutions of 1.15-1.5 Å at 100 K at various pH values and at 297 K and pH 6. (1) Approximately half of the 177 surface waters identified at 100 K are resolved at 297 K, suggesting significant occupancy of specific water sites at room temperature, and loss of resolvable occupancy for other sites. (2) Within a loop region specific to cyanobacterial PsbO, three residues and four waters coordinating a calcium ion are well ordered even at 297 K; the ligation differs for manganese. (3) The crystal structures show water-carboxylate clusters that could facilitate fast Grotthus-type proton transfer along the protein surface and/or store protons. (4) Two carboxylate side chains, which are part of a structural motif interrupting two β-strands and connecting PsbO to photosystem II, are within hydrogen bonding distance at pH 6 (100 K). Simulations indicate coupling between protein structure and carboxylate protonation. The crystal structure determined at 100 K and pH 10 indicates broken hydrogen bonding between the carboxylates and local structural change. At pH 6 and 297 K, both conformations were present in the crystal, suggesting conformational dynamics in the functionally relevant pH regime. Taken together, crystallography and molecular dynamics underline a possible mechanism for pH-dependent structural switching.