Crystal Structure of <i>Bacillus cereus</i> Class Ib Ribonucleotide Reductase Di-iron NrdF in Complex with NrdI

Class Ib ribonucleotide reductases (RNRs) use a dimetal-tyrosyl radical (Y•) cofactor in their NrdF (β₂) subunit to initiate ribonucleotide reduction in the NrdE (α₂) subunit. Contrary to the diferric tyrosyl radical (Fe^III₂-Y•) cofactor, which can self-assemble from Fe^II₂-NrdF and O₂, generation of the Mn^III₂-Y• cofactor requires the reduced form of a flavoprotein, NrdI_hq, and O₂ for its assembly. Here we report the 1.8 Å resolution crystal structure of <i>Bacillus cereus</i> Fe₂-NrdF in complex with NrdI. Compared to the previously solved <i>Escherichia coli</i> NrdI-Mn^II₂-NrdF structure, NrdI and NrdF binds similarly in <i>Bacillus cereus</i> through conserved core interactions. This protein–protein association seems to be unaffected by metal ion type bound in the NrdF subunit. The <i>Bacillus cereus</i> Mn^II₂-NrdF and Fe₂-NrdF structures, also presented here, show conformational flexibility of residues surrounding the NrdF metal ion site. The movement of one of the metal-coordinating carboxylates is linked to the metal type present at the dimetal site and not associated with NrdI-NrdF binding. This carboxylate conformation seems to be vital for the water network connecting the NrdF dimetal site and the flavin in NrdI. From these observations, we suggest that metal-dependent variations in carboxylate coordination geometries are important for active Y• cofactor generation in class Ib RNRs. Additionally, we show that binding of NrdI to NrdF would structurally interfere with the suggested α₂β₂ (NrdE-NrdF) holoenzyme formation, suggesting the potential requirement for NrdI dissociation before NrdE-NrdF assembly after NrdI-activation. The mode of interactions between the proteins involved in the class Ib RNR system is, however, not fully resolved

How a Lytic Polysaccharide Monooxygenase Binds Crystalline Chitin

Lytic polysaccharide monooxygenases (LPMOs) are major players in biomass conversion, both in Nature and in the biorefining industry. How the monocopper LPMO active site is positioned relative to the crystalline substrate surface to catalyze powerful, but potentially self-destructive, oxidative chemistry is one of the major questions in the field. We have adopted a multidisciplinary approach, combining biochemical, spectroscopic, and molecular modeling methods to study chitin binding by the well-studied LPMO from <i>Serratia marcescens Sm</i>AA10A (or CBP21). The orientation of the enzyme on a single-chain substrate was determined by analyzing enzyme cutting patterns. Building on this analysis, molecular dynamics (MD) simulations were performed to study interactions between the LPMO and three different surface topologies of crystalline chitin. The resulting atomistic models showed that most enzyme–substrate interactions involve the polysaccharide chain that is to be cleaved. The models also revealed a constrained active site geometry as well as a tunnel connecting the bulk solvent to the copper site, through which only small molecules such as H₂O, O₂, and H₂O₂ can diffuse. Furthermore, MD simulations, quantum mechanics/molecular mechanics calculations, and electron paramagnetic resonance spectroscopy demonstrate that rearrangement of Cu-coordinating water molecules is necessary when binding the substrate and also provide a rationale for the experimentally observed C1 oxidative regiospecificity of <i>Sm</i>AA10A. This study provides a first, experimentally supported, atomistic view of the interactions between an LPMO and crystalline chitin. The confinement of the catalytic center is likely crucially important for controlling the oxidative chemistry performed by LPMOs and will help guide future mechanistic studies

How a Lytic Polysaccharide Monooxygenase Binds Crystalline Chitin

Lytic polysaccharide monooxygenases (LPMOs) are major players in biomass conversion, both in Nature and in the biorefining industry. How the monocopper LPMO active site is positioned relative to the crystalline substrate surface to catalyze powerful, but potentially self-destructive, oxidative chemistry is one of the major questions in the field. We have adopted a multidisciplinary approach, combining biochemical, spectroscopic, and molecular modeling methods to study chitin binding by the well-studied LPMO from <i>Serratia marcescens Sm</i>AA10A (or CBP21). The orientation of the enzyme on a single-chain substrate was determined by analyzing enzyme cutting patterns. Building on this analysis, molecular dynamics (MD) simulations were performed to study interactions between the LPMO and three different surface topologies of crystalline chitin. The resulting atomistic models showed that most enzyme–substrate interactions involve the polysaccharide chain that is to be cleaved. The models also revealed a constrained active site geometry as well as a tunnel connecting the bulk solvent to the copper site, through which only small molecules such as H₂O, O₂, and H₂O₂ can diffuse. Furthermore, MD simulations, quantum mechanics/molecular mechanics calculations, and electron paramagnetic resonance spectroscopy demonstrate that rearrangement of Cu-coordinating water molecules is necessary when binding the substrate and also provide a rationale for the experimentally observed C1 oxidative regiospecificity of <i>Sm</i>AA10A. This study provides a first, experimentally supported, atomistic view of the interactions between an LPMO and crystalline chitin. The confinement of the catalytic center is likely crucially important for controlling the oxidative chemistry performed by LPMOs and will help guide future mechanistic studies

Homology modelling of crucian carp RNR subunits with mammalian RNR as templates.

<p>Panel (A) shows R1 subunits superimposed on human structure (PDB code 3HCN), panel (B) shows R2 subunits superimposed on mouse R2 structure (PDB code 1W68), and panel (C) shows p53R2 subunits superimposed on human p53R2 structure (PDB code 3HF1). Blue colour signifies strong homology in structure, green to yellow signifies intermediate homology, and red signifies low homology in structure. The tyrosyl radical site in each of the R2/p53R2 subunits is marked with a red star.</p

mRNA levels of cell division markers and RNR subunits at different oxygen levels.

<p>Panels (A–D) show heart mRNA levels and panels (E–H) show brain mRNA levels in normoxia (N), hypoxia (H), anoxia (A) and reoxygenation (R). All qPCR data are normalized to the external standard mw2060. d = duration of exposure (days). Significant differences from the normoxic group are indicated by asterisk (<i>P</i><0.05). n = 6–10 in each group. Note that y-axis scales vary between panels.</p

Mixed-valent EPR spectra of crucian carp R2ii and p53R2i compared to mammalian homologs.

<p>Mixed-valent EPR spectra of crucian carp R2ii and p53R2i compared to mammalian homologs.</p

Primers used for cloning, RACE, qPCR and in vitro expression.

<p>Mean qPCR primer pair efficiencies (E) are given in brain (B) and hearts (H).</p>*<p>Start and terminal codons are highlighted in bold. Restriction sites (¹NdeI, ²SalI, ³HindIII) are underlined.</p

g-values for the tyrosyl radical in class Ia RNR.

<p>g-values for the tyrosyl radical in class Ia RNR.</p

g-values for the mixed-valent form of RNR.

<p>g-values for the mixed-valent form of RNR.</p

Binding of Cu²⁺ to MopE under equilibrium conditions.

<p>(A) MopE (10 µM, 500 µl) was dialysed overnight at 4°C against 100 ml of 20 mM Tris pH 7.5, 80 mM NaCl and 1 mM CaCl₂ containing from 0 to 100 µM CuCl₂. MopE bound Cu²⁺ was determined by ICP-MS (subtracting the Cu(II) concentrations inside and outside the dialysis cassette). The molar ratio (<i>r</i>) of bound Cu(II) to MopE* has been plotted against the concentration of CuCl₂ in the dialysis buffer. The data were adjusted for copper bound to MopE* at no addition of CuCl₂.</p