Structure‐Based Demystification of Radical Catalysis by a Coenzyme B12 Dependent Enzyme—Crystallographic Study of Glutamate Mutase with Cofactor Homologues

Abstract Catalysis by radical enzymes dependent on coenzyme B12 (AdoCbl) relies on the reactive primary 5′‐deoxy‐5′adenosyl radical, which originates from reversible Co−C bond homolysis of AdoCbl. This bond homolysis is accelerated roughly 1012‐fold upon binding the enzyme substrate. The structural basis for this activation is still strikingly enigmatic. As revealed here, a displaced firm adenosine binding cavity in substrate‐loaded glutamate mutase (GM) causes a structural misfit for intact AdoCbl that is relieved by the homolytic Co−C bond cleavage. Strategically interacting adjacent adenosine‐ and substrate‐binding protein cavities provide a tight caged radical reaction space, controlling the entire radical path. The GM active site is perfectly structured for promoting radical catalysis, including “negative catalysis”, a paradigm for AdoCbl‐dependent mutases.

The electron density in the cofactor regions indicated an intact cobalt carbon bond in all B12molecules.To prevent unwanted van-der-Waals repulsion between these two atoms during structure refinement, an additional geometric restraint was specified for the Co-C bond with an ideal value of 2.0 Å but a large standard deviation of 1.0 Å, thereby allowing an unbiased estimate of its length.We employed the same strategy for treating the interaction between the cobalt atom and the imidazole ring of H16 from the small s-subunit.Throughout the refinement, the structures were validated using the program Molprobity. [11]ta on the diffraction data collection and structure refinement are summarised in Table S1.Coordinates and structure factors have been deposited in the Protein Data Bank (PDB) under accession numbers 6H9E and 6H9F.
Active site cavities around the Ado-ligands and the tartrate ion were analysed using the program CavMan employing a modified LIGSITE algorithm [12] with a cutoff-value of 5.The program is available as a PyMOL-plugin from Innophore (www.innophore.com).
All structural figures were prepared using the program PyMOL (www.pymol.org).

Supplementary Figures
Figure S1: (A) Overall structure of GM with the AdoCbl-homologue AdoMeCbl and tartrate bound.The two larger e-subunits are shown in a cyan cartoon representation, and the smaller s-subunits are shown in light green.(B) Binding of the cobalamin moiety in a "baseoff/His-on mode" to the s-subunit of GM with residue H16 coordinating to the cobalt centre.

Figure S4 :
Figure S4:Polder omit-maps[13] contoured at 3s, calculates around AdoMe (A) and AdoEt (B) in the respective GM structures.Both structures are shown in two orientations roughly related to each other by a 90°-rotation around the vertical axis.The ligands are shown as sticks, and the difference electron density is depicted as the usual light blue "chicken wire".

Figure S5 :
Figure S5: Structures of AdoCbl [2c] (PDB-code: 1I9C, ra-conf, green), AdoMeCbl (PDB-code: 6H9E, orange) and AdoEtCbl (PDB-code: 6H9F, pink) bound to GM.The four corrin N-atoms were used for the superposition.The molecules are shown in a sticks-representation.The two views are related by an approximately 90°-rotation around the vertical axis.

Figure S6 :
Figure S6: Stereo representation of the active sites of GM reconstituted with CNCbl [2b] (A, PDB-code: 1CCW) or MeCbl [2b] (B, PDB-code: 1CB7).Display styles and colouring schemes: Amino acid side chains, cofactors, and ligands are shown as sticks.Portions of the main chain of GM are shown as a ribbons-representation.Residues from the eand the ssubunits are shown with light cyan or light green carbon atoms, respectively.The cobalamin is shown in dark red, with the "upper" ligand shown in orange.Tartrate and methylaspartate molecules are coloured yellow.Water molecules are depicted as small red spheres.Hydrogen bonding interactions are shown as green dashed lines (as also used in Figure S3 and Figure 4 in the main manuscript).

Figure S8 :
Figure S8: (A) Cylinder projection of the corrin ring of B12 as observed in the structures of GM reconstituted with CNCbl [2b] (black) and AdoMeCbl (red).The positions of C1, C10, and C19 and the names of the pyrrole rings are given as references.(B) Structural superposition (using the four corrin nitrogen atoms) of the cofactor molecules in the structures of GM reconstituted with CNCbl [2b] (grey) and AdoMeCbl (dark red).The H-bond between N3 of the adenine moiety and the amide-NH2 of the corrin c-sidechain is indicated as a green dashed line.

Figure S13a :
Figure S13a: Stereo-views of active sites of acyl-CoA mutases (for details, see next page).

Table S1 :
Summary of crystallographic data.
a Statistics for the highest-resolution shell are shown in parentheses.

Table S2 :
Geometric parameters of B12-derivatives bound to glutamate mutase.Values are averages of measurements obtained from the two crystallographically independent molecules in the asymmetric unit.