The Methylococcus capsulatus (Bath) Secreted Protein, MopE*, Binds Both Reduced and Oxidized Copper

Under copper limiting growth conditions the methanotrophic bacterium Methylococcus capsulatus (Bath) secrets essentially only one protein, MopE*, to the medium. MopE* is a copper-binding protein whose structure has been determined by X-ray crystallography. The structure of MopE* revealed a unique high affinity copper binding site consisting of two histidine imidazoles and one kynurenine, the latter an oxidation product of Trp130. In this study, we demonstrate that the copper ion coordinated by this strong binding site is in the Cu(I) state when MopE* is isolated from the growth medium of M. capsulatus. The conclusion is based on X-ray Near Edge Absorption spectroscopy (XANES), and Electron Paramagnetic Resonance (EPR) studies. EPR analyses demonstrated that MopE*, in addition to the strong copper-binding site, also binds Cu(II) at two weaker binding sites. Both Cu(II) binding sites have properties typical of non-blue type II Cu (II) centres, and the strongest of the two Cu(II) sites is characterised by a relative high hyperfine coupling of copper (

Structural and Functional Characterization of a Lytic Polysaccharide Monooxygenase with Broad Substrate Specificity

The recently discovered lytic polysaccharide monooxygenases (LPMOs) carry out oxidative cleavage of polysaccharides and are of major importance for efficient processing of biomass. NcLPMO9C from Neurospora crassa acts both on cellulose and on non-cellulose β-glucans, including cellodextrins and xyloglucan. The crystal structure of the catalytic domain of NcLPMO9C revealed an extended, highly polar substrate-binding surface well suited to interact with a variety of sugar substrates. The ability of NcLPMO9C to act on soluble substrates was exploited to study enzyme-substrate interactions. EPR studies demonstrated that the Cu2+ center environment is altered upon substrate binding, whereas isothermal titration calorimetry studies revealed binding affinities in the low micromolar range for polymeric substrates that are due in part to the presence of a carbohydrate-binding module (CBM1). Importantly, the novel structure of NcLPMO9C enabled a comparative study, revealing that the oxidative regioselectivity of LPMO9s (C1, C4, or both) correlates with distinct structural features of the copper coordination sphere. In strictly C1-oxidizing LPMO9s, access to the solvent-facing axial coordination position is restricted by a conserved tyrosine residue, whereas access to this same position seems unrestricted in C4-oxidizing LPMO9s. LPMO9s known to produce a mixture of C1- and C4-oxidized products show an intermediate situation

Human gut Faecalibacterium prausnitzii deploy a highly efficient conserved system to cross-feed on β-mannan-derived oligosaccharides

ACKNOWLEDGMENTS We are grateful for support from The Research Council of Norway (FRIPRO program to P.B.P.: 250479; BIONÆR program to B.W.: 244259), the European Research Commission Starting Grant Fellowship (awarded to P.B.P.; 336355 MicroDE), and the Scottish Government Rural and Environmental Sciences and Analytical Services (RESAS) (for P.L. and S.H.D.). S.L.L.R. generated constructs and performed recombinant protein production and purification and functional characterizations of the binding protein and GHs. L.J.L., S.L., and L.M. expressed, purified, and performed functional characterization of FpCE2 and FpCE17. Growth experiments on mannans and SCFA quantifications were performed by G.L. ITC was performed by Å.K.R., Z.L., and L.S.M. G.V.P. and S.L.L.R. conducted the human metagenomic analysis. S.L.L.R., P.B.P., and B.W. conceived the study and supervised research. The manuscript was written primarily by S.L.L.R. with contributions from P.B.P., S.H.D., G.L, L.M., S.L., G.V.P., E.C.M., L.S.M., B.W., and L.J.L. Figures were prepared by S.L.L.R. We declare that we have no competing interests. Funding Information: We are grateful for support from The Research Council of Norway (FRIPRO program to P.B.P.: 250479; BIONÆR program to B.W.: 244259), the European Research Commission Starting Grant Fellowship (awarded to P.B.P.; 336355 MicroDE), and the Scottish Government Rural and Environmental Sciences and Analytical Services (RESAS) (for P.L. and S.H.D.).Peer reviewedPublisher PD

Tuning of Thioredoxin Redox Properties by Intramolecular Hydrogen Bonds

Thioredoxin-like proteins contain a characteristic C-x-x-C active site motif and are involved in a large number of biological processes ranging from electron transfer, cellular red-ox level maintenance, and regulation of cellular processes. The mechanism for deprotonation of the buried C-terminal active site cysteine in thioredoxin, necessary for dissociation of the mixeddisulfide intermediate that occur under thiol/disulfide mediated electron transfer, is not well understood for all thioredoxin superfamily members. Here we have characterized a 8.7 kD thioredoxin (BC3987) from Bacillus cereus that unlike the typical thioredoxin appears to use the conserved Thr8 side chain near the unusual C-P-P-C active site to increase enzymatic activity by forming a hydrogen bond to the buried cysteine. Our hypothesis is based on biochemical assays and thiolate pKa titrations where the wild type and T8A mutant are compared, phylogenetic analysis of related thioredoxins, and QM/MM calculations with the BC3987 crystal structure as a precursor for modeling of reduced active sites. We suggest that our model applies to other thioredoxin subclasses with similar active site arrangements

Structure of BC3987 active site.

<p>(A) The active site and immediate surroundings in BC3987. Thr8 and Thr53 can possibly form hydrogen bonds to the S_γ-atom of Cys15 upon reduction of the disulfide bridge. The |2F_o–F_c| map is contoured at 1.5 σ. (B) The wild type (in brown) and T8A (in blue) crystal structures have CPPC active sites that superimpose with a RMS value of 0.053 Å. This verifies that the mutation does not disturb the CPPC active site. (C) Comparison of C-P-P-C motifs in oxidized BC3987 (in brown), reduced Tryparedoxin, TXN-II, (in green), and reduced Trx h1 (in grey). From this superimposition, it is suggested that the cysteine side chains and not the backbone undergo the largest structural rearrangement upon reduction of the active site.</p

Overview of operon organization of Cp9-redoxins, NrdH-redoxins, and BC3987.

<p>(A) The gene encoding the Cp9-redoxin is located between a thioredoxin reductase (cp34) and a peroxiredoxin (cp20). (B) The classical class Ib RNR operon where the NrdH-redoxin is found in front of the genes encoding the flavodoxin-like protein NrdI, the catalytic subunit NrdE and the radical/metal cofactor containing NrdF protein. (C) The organization of class Ib RNR genes in the <i>Bacillus cereus</i> group where the putative NrdH-redoxin (BC3987) is located elsewhere in the genome.</p

QM/MM models of the BC3987 active site.

<p>(A) QM/MM geometry optimized structure of BC3987 showing the quantum mechanical and molecular mechanics regions in ball-and-stick and cartoon representation, respectively. (B) Active site (Model I) where the Thr8 residue has the rotamer observed in the crystal structure. The Cys12 side chain is hydrogen bonded to the Cys15 amide proton. The Gln9 amide proton is a possible hydrogen bond donor to the buried Cys15 thiolate. (C) In this structure (Model II) the Thr8 side chain was rotated to resemble the rotamer observed in <i>E. coli</i> and <i>C. ammoniagenes</i> NrdH-redoxins prior to geometry optimization. The Thr8 hydroxyl group and Gln9 amide proton appear to form hydrogen bonds to the buried Cys15 side chain while the Cys12 side chain is hydrogen bonded to the Cys15 amide proton. The link atoms connecting the MM and QM layer in B and C are colored pink.</p

Insulin reduction by wild type and mutant BC3987.

<p>In the insulin reduction assay, the rate of insulin disulfide reduction by DTT is enhanced using 10 µM thioredoxin as a catalyst. The wild type BX3987 thioredoxin (▪) is more efficient than the T8A mutant (•). The control experiment without thioredoxin shows that the uncatalyzed reaction with DTT is very slow (▴). The concentration of insulin and DTT used in the assay was 160 µM and 1 mM, respectively. The turbidity of the assay solution was monitored at 580 nm (light scattering).</p

Controlled depolymerization of cellulose by light-driven lytic polysaccharide oxygenases

Lytic polysaccharide (mono)oxygenases (LPMOs) perform oxidative cleavage of polysaccharides, and are key enzymes in biomass processing and the global carbon cycle. It has been shown that LPMO reactions may be driven by light, using photosynthetic pigments or photocatalysts, but the mechanism behind this highly attractive catalytic route remains unknown. Here, prompted by the discovery that LPMOs catalyze a peroxygenase reaction more efficiently than a monooxygenase reaction, we revisit these light-driven systems, using an LPMO from Streptomyces coelicolor (ScAA10C) as model cellulolytic enzyme. By using coupled enzymatic assays, we show that H2O2 is produced and necessary for efficient light-driven activity of ScAA10C. Importantly, this activity is achieved without addition of reducing agents and proportional to the light intensity. Overall, the results highlight the importance of controlling fluxes of reactive oxygen species in LPMO reactions and demonstrate the feasibility of light-driven, tunable enzymatic peroxygenation to degrade recalcitrant polysaccharides