Solution structures of the Bacillus cereus metallo-ß-lactamase BcII and its complex with the broad spectrum inhibitor R-thiomandelic acid.

Metallo-ß-lactamases, enzymes which inactivate ß-lactam antibiotics, are of increasing biological and clinical significance as a source of antibiotic resistance in pathogenic bacteria. In the present study we describe the high-resolution solution NMR structures of the Bacillus cereus metallo-ß-lactamase BcII and of its complex with R-thiomandelic acid, a broad-spectrum inhibitor of metallo-ß-lactamases. This is the first reported solution structure of any metallo-ß-lactamase. There are differences between the solution structure of the free enzyme and previously reported crystal structures in the loops flanking the active site, which are important for substrate and inhibitor binding and catalysis. The binding of R-thiomandelic acid and the roles of active-site residues are defined in detail. Changes in the enzyme structure upon inhibitor binding clarify the role of the mobile ß3-ß4 loop. Comparisons with other metallo-ß-lactamases highlight the roles of individual amino-acid residues in the active site and the ß3-ß4 loop in inhibitor binding and provide information on the basis of structure-activity relationships among metallo-ß-lactamase inhibitors.

Synthesis of a simplified bryostatin C-ring analogue that binds to the CRD2 of human PKC-alpha and construction of a novel BC-analogue by an unusual Julia olefination process.

[structure: see text] The synthesis of two truncated bryostatin analogues 2 and 3 is described. High-field NMR measurements on the C-ring analogue 3 in C(2)H(3)CN containing 25% (2)H(2)O have shown that it binds to the CRD2 of human PKC-alpha at virtually the same position as phorbol-13-acetate (PA) and bryostatin 1 (1). NMR titration studies have also revealed that 3 binds to the CRD2 with a potency similar in magnitude to PA but much less potently than 1.