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Bacterial SBP56 Identified As a Cu-Dependent Methanethiol Oxidase Widely Distributed in the Biosphere Özge Eyice, Nataliia My

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Bacterial SBP56 Identified As a Cu-Dependent Methanethiol Oxidase Widely Distributed in the Biosphere Özge Eyice, Nataliia My Bacterial SBP56 identified as a Cu-dependent methanethiol oxidase widely distributed in the biosphere Özge Eyice, Nataliia Myronova, Arjan Pol, Ornella Carrión, Jonathan Todd, Tom J. Smith, Stephen J. Gurman, Adam Cuthbertson, Sophie Mazard, Monique A.S.H. Mennink-Kersten, Timothy D.H. Bugg, K. Kristoffer Andersson, Andrew W.B. Johnston, Huub J.M. Op den Camp, Hendrik Schäfer Supplementary Information This document contains supplementary information including additional detail concerning methods and results of methanethiol oxidase protein purification and characterization, Electron Paramagnetic Resonance Spectroscopy (EPR) analysis, X-ray spectroscopy analyses (EXAFS), genome sequencing of Hyphomicrobium sp. VS (Supplementary Figures S1-S12, Supplementary Tables S1-S7). Protein purification An initial protocol for purification of methanethiol oxidase from Hyphomicrobium sp. VS was developed as shown below in Supplementary Table S1. This resulted in a protein preparation that revealed a single dominant polypeptide in SDS-PAGE analysis with a molecular weight of approximately 46 kDa (Kertens-Mennink, Pol, Op den Camp, unpublished results). The activity of methanethiol oxidase in this preparation was enriched 36-fold in this fraction. Supplementary Table S1. Purification Table for preliminary MTO purification. MTO activity Protein Purification volume (µmol MT/min) [mg] fold Recovery crude extract 5.0 4.8 135.0 1.0 100.0 Q-sepharose 7.4 3.5 12.7 7.8 73.3 Amicon filtration 2.0 4.1 10.3 10.9 84.0 Superose 12 12.0 4.7 3.3 40.2 98.0 Amicon filtration 6.1 1.8 3.9 12.8 36.7 TSK-DEAE 7.0 0.5 0.4 36.0 10.8 Notes: after the second Amicon filtration, only 26.3% of the material was purified further, leading to a recovery of 10.8%. The theoretical recovery would therefore be 40% if all material had been further purified. The above purification procedure (Supplementary Table S1) resulted in an active enzyme preparation showing a single dominant polypeptide on SDS-PAGE of a molecular weight of ~45-46 kDa (result not shown). The final purification protocol adopted for MTO was as described in Materials and Methods of the main text, consisted of sequential steps of anion exchange chromatography on a MonoQ 10/100 column followed by size exclusion chromatography using a Superdex-75 column. This also resulted in a protein preparation that was dominated by a 46 kDa polypeptide. The fraction with MTO activity eluting from the anion-chromatography run again showed a single dominant polypeptide of 46 kDa, which was up to 50-fold enriched in specific activity in neighbouring fractions compared to the cleared supernatant (Supplementary Table S2). This demonstrated that the enzyme activity responsible for MT degradation in Hyphomicrobium sp. VS was successfully enriched. Supplementary Table S2. Modified purification scheme, comparison of specific enzyme activity in active fraction recovered after MonoQ anion exchange chromatography with initial soluble enzyme extract after removal of cell membranes specific MT degraded activity protein in [µmol] in 30 (µmol/min mg fold- assay [mg] min protein) purification Supernatant 1.56 10 0.21 1.0 MonoQ fraction 18 0.17 54 10.58 50.4 A B C ' M S 18 19 20 M MTO kDa 18 669 1 16 440 2 14 66 232 12 45 10 3 d 140 ' 36 K 8 29 6 24 4 4 20 5 2 14 67 0 1 10 100 molecular weight [kDa] Supplementary Figure S1. (A) SDS-PAGE analysis of protein fractions of Hyphomicrobium sp. VS. M, molecular weight marker (weights in kDa); S, soluble fraction after removal of membrane fraction; 18, 19 and 20 are fraction numbers of the Mono-Q run. (B) Native (10% w/v polyacrylamide) gel electrophoresis analysis of MTO purified from Hyphomicrobium VS. Lanes: MTO, methanethiol oxidase; lane M, molecular size standards (Mw given in kDa). (C) Calibration plot of the analytical gel filtration showing for estimation of the molecular weight of MTO. Standards used for column calibration: 1, ovalbumin (43 kDa); 2, conalbumin (75 kDa); 3, aldolase (158 kDa); 4, ferritin (440 kDa); 5, thyroglobulin (669 kDa), the relative elution of MTO is indicated giving an estimated Mw of approX 200 kDa. Supplementary Figure S2. Electrospray ionisation mass spectrometry analysis of purified MTO shows molecular weight of the subunit to be approXimately 46.2 kDa. Kinetics MTO 18 16 14 12 10 8 µmol MT/mg/min 6 4 2 0 0 2 4 6 8 10 µM MT Supplementary Figure S3. Kinetic analysis of methanethiol oxidase from Hyphomicrobium sp. VS. Based on the data shown in the plot, the estimated Km for MT was 0.2-0.3µM -1 -1 and the VmaX was approXimately 16µmol mg protein min . Electron Paramagnetic Resonance Spectroscopy – Results In resting and oxidized samples we observed EPR signals at temperatures of 7 and 13 K (Supplementary Fig S4). The resting oxidized MTO EPR signals did not have well-resolved Cu(II) EPR signals from an isolated mono-nuclear Cu(II) site(s), suggesting that the Cu-ions are coupled/interacting between Cu ions and/or with a radical in some redox state (Supplementary Fig S4). We could observe trace amounts of a radical with a very narrow signal centered around 2.0 (as it is a first derivative of a peak it has both positive and negative features). These broad signals were probably from two magnetically-interacting Cu(II) centres, with positive signals before 2.05, somewhat similar to CuA in cytochrome c oxidase or nitrous-oxide reductase, both binuclear copper centres, as well as Cu model complex possibly also without bridging sulfur (Antholine et al 1992, Monzani et al 1998, Solomon et al 1996, Kaim et al, 2013). These could be species from a Cu(II)Cu(I)/ Cu(1.5)Cu(1.5) cluster. The oxidized samples also had a signal around g=4.8 (see Supplementary Fig S5 at 7 and 13 K) indicating spin-coupled or spin-interacting species at this high g-value. The EPR spectra (Supplementary Figures S4 and S5) have several features of a Cu protein that cannot come from a single Cu site as the positive signals at g = 2.46 lack other resolved hyperfine interactions from a single isolated Cu (it is also a very weak Cu(II) at higher g-values visible with 4 hyperfine couplings resolved). The g= 2.39 and 2.24 could be linked to the 2.46 signal but all these three features did not look like normal hyperfines but could possibly indicate that sulfur may be a ligand. The g=2.053 is a zero crossing signal (like the radical) with both positive and negative signal and might be linked to any of the 2.46, 2.39 or 2.24 signals, but at this point of time it is unclear which. The last negative feature with g= 1.905 has unusually low g-value that might be seen in spin-interacting signals (Andersson et al 2003). In summary, the changes in features in the EPR spectra indicate changes in coordination of Cu when substrate binds, which could indicate direct interaction of the substrate with the Cu centre. Although at this point the exact nature of the Cu environment and status cannot be fully resolved, it is likely to be a binuclear site, as the data do not support a single atom Cu centre. g=2.05 | g=4.8 | Electron Paramagnetic Resonance first derivative Field, mTesla Supplementary Figure S4. Oxidized MTO at 7 K (blue) and 13 K (orange). MTO 9.2 mg/ml, 10 mM TRICINE, pH 8.2, 1 mM benzamidine, 1.8 mM sodium heXachloroiridate (V). 2.24 2.053 2.309 -6000 2.46 -7000 -8000 -9000 -10000 intensity -11000 -12000 1.905 -13000 Electron Paramagnetic Resonance first derivative -14000 2000 2500 3000 3500 4000 4500 Field, field,gaussmTesla Supplementary Figure S5. Electron Paramagnetic Resonance Spectroscopy analysis of oXidized MTO (MTO with ethanethiol and oXygen) at 15 K and microwave power 200 μW. X-ray Spectroscopy Analysis of Methanethiol Oxidase Detailed Methods X-ray absorption spectra were obtained in fluorescent mode on station B18 of the Diamond Light Source (Didcot, UK). This uses the technique of quick EXAFS (QuEXAFS), where the monochromator rotates at a constant rate during data acquisition. The fluorescence was detected using a nine element germanium solid state detector. Data were obtained at the Cu K edge for a variety of samples and standards. All data were obtained with the samples at 77K in a cryostat. To minimize radiation damage the beam was rastered across the sample, which was moved between each scan. Each scan took about 20 minutes to acquire. The fluorescence signals were merged and normalized using the Athena program. The output from this program is used in studies of the absorption edge shape and the chemical shifts. The background was then subtracted using the Pyspline program to produce the oscillatory EXAFS spectrum. This was analysed using the EXCURV program. This employs the rapid curved wave theory, including multiple scattering when necessary (Gurman et al. 1986). Scattering properties of the atoms were calculated ab initio within the program. The structure surrounding the Cu atom is described in terms of shells of atoms: a shell is a set of atoms of the same chemical type at the same average distance. The structural parameters fitted are the number and type of atom within a shell, their average distance from the Cu atom and the mean square deviation in this distance (the Debye-Waller factor). Copper metal (foil), CuO and CuS were used as reference samples. The copper- containing enzyme tyrosinase (Sigma Aldrich, Gillingham, UK) was used as additional reference.
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