The inner workings of α-amino ester hydrolases and crystallization experiments with an integral membrane transporter

+172hlm.;24c

An unexpected reactivity of the P460 cofactor in hydroxylamine oxidoreductase

Hydroxylamine oxidoreductases (HAOs) contain a unique haem cofactor called P460 that consists of a profoundly ruffled c-type haem with two covalent bonds between the haem porphyrin and a conserved tyrosine. This cofactor is exceptional in that it abstracts electrons from a ligand bound to the haem iron, whereas other haems involved in redox chemistry usually inject electrons into their ligands. The effects of the tyrosine cross-links and of the haem ruffling on the chemistry of this cofactor have been investigated theoretically but are not yet clear. A new crystal structure of an HAO from Candidatus Kuenenia stuttgartiensis, a model organism for anaerobic ammonium oxidation, now shows that its P460 cofactor has yet another unexpected reactivity: when ethylene glycol was used as a cryoprotectant, the 1.8 Å resolution electron-density maps showed additional density which could be interpreted as an ethylene glycol molecule covalently bound to the C16 atom of the haem ring, opposite the covalent links to the conserved tyrosine. Possible causes for this unexpected reactivity are discussed

Structure of a neodymium-containing, XoxF1-type methanol dehydrogenase

Growth of Methylacidimicrobium thermophilum AP8 and medium composition.Methylacidimicrobium thermophilum AP8 was isolated from geothermal soil on the island of Pantelleria and grown in a chemostat batch culture on methane at maximum growth rate (μmax) (see Fig. S1 in the supplemental material) with Pantelleria medium, as described previously (51). Neodymium was the only lanthanide that was supplemented, to a final concentration of 0.5 μM Nd2O3. The biomass used for purification of XoxF was harvested after a batch phase of about 7 days. NH4Cl and trace element solutions were manually added to the culture to reach an optical density at 600 (OD600) of 10. Purification of Nd-XoxF1 from M. thermophilum AP8.To purify native Nd-XoxF1 from M. thermophilum AP8 biomass, cells from the above-described culture were harvested by centrifugation (15 min at 5,000 × g and 4°C). The supernatant was discarded and the cell pellet was resuspended in 10 mM piperazine-N,N′-bis(2-ethanesulfonic acid (PIPES) buffer (pH 7.2) supplemented with 30 mg · liter−1 DNase I. To lyse the cells, the concentrated cell suspension was passed three times through a French pressure cell press at 120 MPa (American Instrument Company, Silver Spring, MD). To separate the soluble proteins from the membrane proteins, the crude extract was centrifuged in an ultracentrifuge (1 h at 140,000 × g and 4°C). The supernatant was taken and centrifuged again at the same speed to pellet any remaining membranes. Subsequently, the supernatant was used for protein purification. Nd-XoxF1 was purified using an isolation procedure described previously (28). The buffer used during the procedure contained 1 mM methanol, which is necessary to minimize loss of enzymatic activity. SDS-PAGE.The purity of Nd-XoxF1 was assessed on home-made 10% SDS polyacrylamide gels. As a molecular weight reference, 3 μl PageRuler Plus prestained protein ladder (Thermo Fisher Scientific, Walthman, MA) was used. A 2-μg aliquot of purified Nd-XoxF1 was incubated in SDS sample buffer (39) for 10 min at 100°C before being loaded on the gel. After running, the gels were stained in Coomassie brilliant blue for 1 h and destained afterwards. Inductively coupled plasma mass spectrometry.To determine the occupancy of neodymium (Nd3+) in purified XoxF1, inductively coupled plasma mass spectrometry (ICP-MS) was performed. Protein concentrations were determined as described previously (33). Fractions of 450 μl containing 16.6 μM isolated MDH were concentrated to 45 μl in Vivaspin 500 centrifugal spin filters with a 30-kDa cutoff value (Sartorius, Germany). The concentrated protein sample was mixed with 455 μl of 11% nitric acid, and samples were destructed by heating at 90°C for 1 h. Subsequently, samples were mixed with 4.5 ml of 1% nitric acid and measured for metal content. Calibrations of 0 to 200 ppb were used for Ca, Mn, Cu, Zn, Sr, Ba, La, Ce, Pr, Nd, Sm, Eu, and Gd. Crystal structure determination.Initial attempts at determining the crystal structure of Nd-XoxF1 resulted in structures without PQQ bound to the active site. We attributed this to the extensive buffer exchange performed prior to crystallization, as well as to the presence of acetate in the crystallization procedure, which we found to lead to dissociation of PQQ from the protein. We therefore performed new crystallization screens using Nd-XoxF1 (A2801 cm = 27.4) in 250 mM PIPES/NaOH (pH 6.7) protein supplemented with 1 mM NdCl3 and 1 mM PQQ sodium salt. Optimal crystallization conditions were identified by screening in 100- + 100-nl sitting drop setups pipetted from commercial crystallization screens onto XTL low-profile plates (Greiner Bio One, Frickenhausen, Germany) by a Mosquito pipetting robot (TTP Labtech, Jena, Germany). This resulted in several hits, including a condition from the JCSG Core II screen containing 0.01 M nickel chloride, 20% (wt/vol) polyethylene glycol (PEG) 2000 monomethyl ether and 0.1 M Tris-HCl (pH 8.5). From this condition, thick plate-like crystals grew in 1 day. These crystals were cryoprotected in reservoir solution supplemented with 15% (vol/vol) ethylene glycol and flash-cooled in liquid nitrogen. A 2.3-Å resolution data set was collected at the PX-II beam line of the Swiss Light Source at the Paul Scherrer Institute in Villigen (Switzerland), which was processed using XDS software (73). These data were phased by molecular replacement with PHASER (74, 75) using the structure of the cerium-containing XoxF2-type MDH from Methylacidiphilum fumariolicum SolV (28) (PDB entry 4MAE). The final model was obtained by iterative rebuilding in COOT (76) and refinement in PHENIX (77, 78). To ensure that the metal ion observed in the active site is not a nickel ion from the crystallization solution, we also refined the structure with nickel ions in the metal binding site, using B-factor and occupancy refinement. This resulted in strong electron difference density around the nickel ions, B-factors that were much lower than those of the surrounding atoms, and occupancies higher than those of the PQQ molecule in the A molecule. When neodymium ions were placed in these sites, only minimal difference density was observed around the Nd ions, their B-factors were close to those of the surrounding atoms, and their occupancy refined to a value very close to that obtained for the PQQ molecule. We therefore conclude that the ions in the metal binding sites are neodymium ions. Data and model statistics are reported in Table S1 and have been submitted to the PDB under accession number 7O6Z. UV-visible spectroscopy.Spectra were collected using a Cary 60 spectrophotometer in a quartz microcuvette with 10-mm path length. To assess the effect of washing on dissociation of the PQQ cofactor, Nd-XoxF1 was washed with 20 mM PIPES buffer (pH 7.2Enzyme (10 μl) was diluted in buffer at a ratio of 1:10. The diluted protein sample was transferred to an equilibrated Vivaspin 500 centrifugal spin filter with a 30-kDa cutoff value (Sartorius) and centrifuged at 4,000 × g and 4°C for 15 min. This procedure was repeated six times, and spectra were recorded. Data were baseline corrected to 600 nm and then normalized to the absorbance measured at 280 nm, representing the total protein peak. Circular dichroism spectroscopy.To assess the stability of Nd-XoxF1 at different temperatures, circular dichroism (CD) spectroscopy was performed using a J-810 CD spectrometer (Jasco, Oklahoma City, OK). The enzyme was diluted in 20 mM potassium phosphate buffer (pH 7) to 11.9 μM. Potassium phosphate buffer was used because it displays significantly less background absorption compared to PIPES buffer at a wavelength of 200 to 250 nm. In a 2-mm-path-length cuvette, the spectrum was monitored at different temperatures using an external water bath connected to the circular dichroism cell compartment. Scanning settings were performed as described previously (33). Dye-coupled enzyme kinetics.Methanol dehydrogenase activity tests were performed in 100 mM multicomponent buffer (25 mM citric acid, 25 mM bis-Tris, 25 mM Tris, and 25 mM N-cyclohexyl-2-aminoethanesulfonic acid [CHES]), 1 mM phenazine ethosulfate (PES), and 100 μM 2,6-dichlorophenolindophenol (DCPIP) at pH 7 and 45°C in an Epoch 2 plate reader and 96-well plates (71). Each well contained 200 μl total volume. To minimize background reactions, an assay premixture containing 2 mM PES and 200 μM DCPIP in buffer was prepared and heated to 45°C for 15 min and then stored on ice in an amber Falcon tube to prevent light-induced degradation. 100 μl of this assay mixture was placed in each well, followed by addition of 90 μl Nd-XoxF1 in buffer to yield a final concentration of 200 nM enzyme in the assay. Methanol dilutions (10 μl) were added after the background activity had been monitored for 2 min at 45°C in the plate reader at 600 nm. The obtained data were path length corrected to 1 cm using the protocol in the Gen5 software. The activity was then monitored for 10 min, and data were fitted separately using the first 2 min, min 4 to 6, and min 8 to 10. A 1 M formaldehyde stock was prepared by dissolving paraformaldehyde powder in multicomponent buffer (pH 7.5), followed by addition of a few drops of 1 M NaOH to help with dissolution. The resulting suspension was sonicated at 50°C for 30 min until the solution was clear. The pH was then readjusted to 7.0 with 1 M HCl. Dilutions were prepared using Millipore water. In addition, to assess temperature dependence, enzyme activity was assessed at pH 7 at 30 to 60°C in 5°C steps in duplicates. To determine the enzymatic activity at different temperatures and pH values, the above-mentioned 100 mM multicomponent buffer was used. The pH dependence was assessed both with and without 15 mM NH4Cl. The MDH activity assay was also performed in a 4-ml stirred glass cuvette and placed in a Cary spectrophotometer heated at 45°C. The reaction mixture was prepared as described above. The Nd-XoxF1 used in this essay was stored in 10 mM phosphate buffer supplied with 1 mM methanol. Several washing steps using 30-kDa Vivaspin centrifugal spin filters (Sartorius) were necessary to eliminate the residual methanol before the essay could be performed. The enzyme was resuspended in 100 mM multicomponent buffer and 1 mM cyanide. Phylogenetic analysis.The XoxF amino acid sequences of Methylacidimicrobium thermophilum AP8 were used in BLASTP searches against the GenBank database. Representative homologous protein sequences were downloaded and combined with representatives from the different types of methanol dehydrogenases (31). Sequences were aligned by the ClustalW tool available in MEGA7 (79). MEGA7 was also used to infer the evolutionary history of the representative protein sequences using the neighbor-joining method

Structure of the 4-hydroxy-tetrahydrodipicolinate synthase from the thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV and the phylogeny of the aminotransferase pathway

Contains fulltext : 219180.pdf (publisher's version ) (Open Access

A 60-heme reductase complex from an anammox bacterium shows an extended electron transfer pathway

Contains fulltext : 202760.pdf (publisher's version ) (Closed access

Similar but Not the Same: First Kinetic and Structural Analyses of a Methanol Dehydrogenase Containing a Europium Ion in the Active Site

Contains fulltext : 193464.pdf (publisher's version ) (Open Access

Subtomogram average structure of anammoxosomal nitrite oxidoreductase

Nitrate is an abundant nutrient and electron acceptor throughout Earth's biosphere. Virtually all nitrate in nature is produced by the oxidation of nitrite by the nitrite oxidoreductase (NXR) multiprotein complex. NXR is a crucial enzyme in the global biological nitrogen cycle, and is found in nitrite-oxidizing bacteria (including comammox organisms), which generate the bulk of the nitrate in the environment, and in anaerobic ammonium-oxidizing (anammox) bacteria which produce half of the dinitrogen gas in our atmosphere. However, despite its central role in biology and decades of intense study, no structural information on NXR is available. Here, we present a structural and biochemical analysis of the NXR from the anammox bacterium Kuenenia stuttgartiensis, integrating X-ray crystallography, cryo-electron tomography, helical reconstruction cryo-electron microscopy, interaction and reconstitution studies and enzyme kinetics. We find that NXR catalyses both nitrite oxidation and nitrate reduction, and show that in the cell, NXR is arranged in tubules several hundred nanometres long. We reveal the tubule architecture and show that tubule formation is induced by a previously unidentified, haem-containing subunit, NXR-T. The results also reveal unexpected features in the active site of the enzyme, an unusual cofactor coordination in the protein's electron transport chain, and elucidate the electron transfer pathways within the complex

Helical reconstruction of nitrite oxidoreductase from anammox bacteria

Nitrate is an abundant nutrient and electron acceptor throughout Earth's biosphere. Virtually all nitrate in nature is produced by the oxidation of nitrite by the nitrite oxidoreductase (NXR) multiprotein complex. NXR is a crucial enzyme in the global biological nitrogen cycle, and is found in nitrite-oxidizing bacteria (including comammox organisms), which generate the bulk of the nitrate in the environment, and in anaerobic ammonium-oxidizing (anammox) bacteria which produce half of the dinitrogen gas in our atmosphere. However, despite its central role in biology and decades of intense study, no structural information on NXR is available. Here, we present a structural and biochemical analysis of the NXR from the anammox bacterium Kuenenia stuttgartiensis, integrating X-ray crystallography, cryo-electron tomography, helical reconstruction cryo-electron microscopy, interaction and reconstitution studies and enzyme kinetics. We find that NXR catalyses both nitrite oxidation and nitrate reduction, and show that in the cell, NXR is arranged in tubules several hundred nanometres long. We reveal the tubule architecture and show that tubule formation is induced by a previously unidentified, haem-containing subunit, NXR-T. The results also reveal unexpected features in the active site of the enzyme, an unusual cofactor coordination in the protein's electron transport chain, and elucidate the electron transfer pathways within the complex