Molecular Mechanisms of Substrate Recognition and Specificity of New Delhi Metallo-β-Lactamase

Abstract

Carbapenems are one of the last lines of defense for Gram-negative pathogens, such as members of the Enterobacteriaceae. Despite the fact that most carbapenems are resistant to extended-spectrum ␤-lactamase (ESBL), emerging metallo-␤-lactamases (MBLs), including New Delhi metallo-␤-lactamase 1 (NDM-1), that can hydrolyze carbapenems have become prevalent and are frequently associated with the so-called "superbugs," for which treatments are extremely limited. Crystallographic study sheds light on the modes of antibiotic binding to NDM-1, yet the mechanisms governing substrate recognition and specificity are largely unclear. This study provides a connection between crystallographic study and the functional significance of NDM-1, with an emphasis on the substrate specificity and catalysis of various ␤-lactams. L1 loop residues L 59 , V 67 , and W 87 were important for the activity of NDM-1, most likely through maintaining the partial folding of the L1 loop or active site conformation through hydrophobic interaction with the R groups of ␤-lactams or the ␤-lactam ring. Substitution of alanine for L 59 showed greater reduction of MICs to ampicillin and selected cephalosporins, whereas substitutions of alanine for V 67 had more impact on the MICs of carbapenems. K 224 and N 233 on the L3 loop played important roles in the recognition of substrate and contributed to substrate hydrolysis. These data together with the structure comparison of the B1 and B2 subclasses of MBLs revealed that the broad substrate specificity of NDM-1 could be due to the ability of its wide active site cavity to accommodate a wide range of ␤-lactams. This study provides insights into the development of efficient inhibitors for NDM-1 and offers an efficient tactic with which to study the substrate specificities of other ␤-lactamases.

Molecular Basis of Substrate Recognition and Product Release by the Klebsiella pneumoniae Carbapenemase (KPC-2)

“…They are mirrored by similar observations in metallo-β-lactamases that also harbor expanded active sites with a large number of hydrophobic residues. 36 Such features may enable carbapenemases to hydrolyze nearly all β-lactam antibiotics, while also exposing a weakness that can facilitate the engineering of high affinity inhibitors against these enzymes.…”

Bisthiazolidines: A Substrate-Mimicking Scaffold as an Inhibitor of the NDM-1 Carbapenemase

“…Mutagenesis studies on NDM-1 suggest that loop L3 is important in substrate recognition. 42 Indeed, improvement in IMP-1 inhibition by a series of succinic acid derivatives was achieved by optimizing hydrophobic interactions in the active site. 15 It is notable that the conformation of loop L3 varies in the different NDM-1 structures, with the meropenem EP and captopril complexes in particular differing from the native structure.…”

Crystal Structure of Escherichia coli originated MCR-1, a phosphoethanolamine transferase for Colistin Resistance

“…Analysis of the metal content in the MCR-1 protein was performed as previously reported16. Briefly, purified MCR-1 protein was dissolved in 20 mM Tris, pH 8.0, and 200 mM NaCl.…”