6DJA

ZN-DEPENDENT 5/B/6 METALLO-BETA-LACTAMASE FROM BACILLUS CEREUS

Summary for 6DJA

• Entry DOI: 10.2210/pdb6dja/pdb
• Descriptor: Metallo-beta-lactamase type 2, ZINC ION (3 entities in total)
• Functional Keywords: hydrolase, zinc, lactamase
• Biological source: Bacillus cereus
• Total number of polymer chains: 1
• Total formula weight: 24586.80

Authors

Bui, A.A.,Khan, N.H.,Shaw, R.W.,Sutton, R.B. (deposition date: 2018-05-24, release date: 2019-05-29, Last modification date: 2023-10-11)

Primary citation

Khan, N.H.,Bui, A.A.,Xiao, Y.,Sutton, R.B.,Shaw, R.W.,Wylie, B.J.,Latham, M.P.
A DNA aptamer reveals an allosteric site for inhibition in metallo-beta-lactamases.
Plos One, 14:e0214440-e0214440, 2019

PubMed Abstract: The hydrolysis of β-lactam antibiotics by β-lactamase enzymes is the most prominent antibiotic resistance mechanism for many pathogenic bacteria. Out of this broad class of enzymes, metallo-β-lactamases are of special clinical interest because of their broad substrate specificities. Several in vitro inhibitors for various metallo-β-lactamases have been reported with no clinical efficacy. Previously, we described a 10-nucleotide single stranded DNA aptamer (10-mer) that inhibits Bacillus cereus 5/B/6 metallo-β-lactamase very effectively. Here, we find that the aptamer shows uncompetitive inhibition of Bacillus cereus 5/B/6 metallo-β-lactamase during cefuroxime hydrolysis. To understand the mechanism of inhibition, we report a 2.5 Å resolution X-ray crystal structure and solution-state NMR analysis of the free enzyme. Chemical shift perturbations were observed in the HSQC spectra for several residues upon titrating with increasing concentrations of the 10-mer. In the X-ray crystal structure, these residues are distal to the active site, suggesting an allosteric mechanism for the aptamer inhibition of the enzyme. HADDOCK molecular docking simulations suggest that the 10-mer docks 26 Å from the active site. We then mutated the three lysine residues in the basic binding patch to glutamine and measured the catalytic activity and inhibition by the 10-mer. No significant inhibition of these mutants was observed by the 10-mer as compared to wild type. Interestingly, mutation of Lys50 (Lys78; according to standard MBL numbering system) resulted in reduced enzymatic activity relative to wild type in the absence of inhibitor, further highlighting an allosteric mechanism for inhibition.

PubMed: 31009467
DOI: 10.1371/journal.pone.0214440

Experimental method

X-RAY DIFFRACTION (2.48 Å)