The long axial cobalt-nitrogen bond first observed in the structure of MCM appears to result from a contribution of the species without upper ligand. The tight binding of the tartrate ion conforms to the requirements of tight control of the reactive intermediates and suggests how the enzyme might use the substrate-binding energy to initiate cleavage of the cobalt-carbon bond. The cofactor does not appear to have a participating role during the radical rearrangement reaction.

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Esterases form a diverse class of enzymes of largely unknown physiological role. Because many drugs and pesticides carry ester functions, the hydrolysis of such compounds forms at least one potential biological function. Carboxylesterases catalyze the hydrolysis of short chain aliphatic and aromatic carboxylic ester compounds. Esterases, D-alanyl-D-alanine-peptidases (DD-peptidases) and ␤-lactamases can be grouped into two distinct classes of hydrolases with different folds and topologically unrelated catalytic residues, the one class comprising of esterases, the other one of ␤-lactamases and DD-peptidases. The chemical reactivities of esters and ␤-lactams towards hydrolysis are quite similar, which raises the question of which factors prevent esterases from displaying ␤-lactamase activity and vice versa. Here we describe the crystal structure of EstB, an esterase isolated from Burkholderia gladioli. It shows the protein to belong to a novel class of esterases with homology to Penicillin binding proteins, notably DD-peptidase and class C ␤-lactamases. Site-directed mutagenesis and the crystal structure of the complex with diisopropyl-fluorophosphate suggest Ser75 within the "␤-lactamase" Ser-x-x-Lys motif to act as catalytic nucleophile. Despite its structural homology to ␤-lactamases, EstB shows no ␤-lactamase activity. Although the nature and arrangement of active-site residues is very similar between EstB and homologous ␤-lactamases, there are considerable differences in the shape of the active site tunnel. Modeling studies suggest steric factors to account for the enzyme's selectivity for ester hydrolysis versus ␤-lactam cleavage.

Growing cells of Propionibacterium shermanii, when supplemented with imidazole, produced a "complete" corrinoid, as reported from a similar (but also cobinamide supplemented) setup by Müller and co-workers (Eberhard, G.; et al. Biol. Chem. Hoppe-Seyler 1988, 369,1091. The corrinoid isolate from workup with added potassium cyanide was confirmed to consist mainly (84%) of the suggested Co/3-cyanoimidazolylcobamide (1), the vitamin Bn analogue, in which the cobalt-coordinating 5,6-dimethylbenzimidazole base of the vitamin is replaced by imidazole. An extensive NMR spectroscopic analysis of aqueous solutions of 1 was carried out: all and 13C signals in the NMR spectra of 1 were assigned unambiguously, exploiting two-dimensional gradient enhanced homonuclear and heteronuclear experiments, including a new variant of the two-dimensional H-relayed , -Overhauser experiment at natural abundance, called 2D-HSQC-ROESY, which combines the merits of heteronuclear single quantum coherence (HSQC) and rotating frame Overhauser spectroscopy (ROESY). The structural analyses by NMR, UV, and CD spectroscopy of 1 in aqueous solution did not unravel any gross structural differences of the corrin ligand and of the nucleotide loop of 1 and of vitamin B]2 (2). Cryo-temperature single-crystal structure analyses have been carried out for 1 and 2, the latter one to obtain reference data more accurate than the ones available from Hodgkin's analysis (Brink-Shoemaker, C. et al. Proc. R. Soc. London, A 1964,278,1). The imidazolylcobamide 1 crystallized at room temperature from aqueous acetone in the orthorhombic space group P2{2{2\, with Z = 4. Diffraction data were collected at 92 K (a = 15.335( 16) Á, b = 21.974(9) Á, c = 25.501(11) Á, V = 8705(2) Á3), the structure was solved by the Patterson method and refined to a final R value of 0.1017 for 6105 reflections with F0 > 4