BackgroundDue to the paucity of novel antibiotics, colistin has become a last resort antibiotic for treating multidrug resistant bacteria. Colistin acts by binding the lipid A component of lipopolysaccharides and subsequently disrupting the bacterial membrane. The recently identified plasmid-encoded MCR-1 enzyme is the first transmissible colistin resistance determinant and is a cause for concern for the spread of this resistance trait. MCR-1 is a phosphoethanolamine transferase that catalyzes the addition of phosphoethanolamine to lipid A to decrease colistin affinity.ResultsThe structure of the catalytic domain of MCR-1 at 1.32 Å reveals the active site is similar to that of related phosphoethanolamine transferases.ConclusionsThe putative nucleophile for catalysis, threonine 285, is phosphorylated in cMCR-1 and a zinc is present at a conserved site in addition to three zincs more peripherally located in the active site. As noted for catalytic domains of other phosphoethanolamine transferases, binding sites for the lipid A and phosphatidylethanolamine substrates are not apparent in the cMCR-1 structure, suggesting that they are present in the membrane domain.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0303-0) contains supplementary material, which is available to authorized users.
OXA-163 and OXA-48 are closely related class D β-lactamases that exhibit different substrate profiles. OXA-163 hydrolyzes oxyimino-cephalosporins, particularly ceftazidime, while OXA-48 prefers carbapenem substrates. OXA-163 differs from OXA-48 by one substitution (S212D) in the active site β5-strand and a four-amino acid deletion (214-RIEP-217) in the loop connecting the β5 and β6 strands. Although the structure of OXA-48 has been determined, the structure of OXA-163 is unknown. To further understand the basis for their different substrate specificities we performed enzyme kinetic analysis, inhibition assays, x-ray crystallography, and molecular modeling. The results confirm the carbapenemase nature of OXA-48 and the ability of OXA-163 to hydrolyze the oxyimino-cephalosporin ceftazidime. The crystal structure of OXA-163 determined at 1.72-Å resolution reveals an expanded active site compared to OXA-48, which allows the bulky substrate ceftazidime to be accommodated. The structural differences with OXA-48, which cannot hydrolyze ceftazidime, provide a rationale for the change in substrate specificity between the enzymes. OXA-163 also crystallized in another condition that contained iodide. The crystal structure determined at 2.87-Å resolution revealed iodide in the active site accompanied by several significant conformational changes including a distortion of the β5-strand, decarboxylation of Lys73, and distortion of the substrate-binding site. Further studies showed that both OXA-163 and OXA-48 are inhibited in the presence of iodide. In addition, OXA-10, which is not a member of the OXA-48-like family, is also inhibited by iodide. These findings provide a molecular basis for the hydrolysis of ceftazidime by OXA-163 and, more broadly, show how minor sequence changes can profoundly alter the active site configuration and thereby affect the substrate profile of an enzyme.
Background: TEM-1 -lactamase hydrolyzes penicillins and early cephalosporins but not oxyimino-cephalosporins. Results: A TEM-1 triple mutant, W165Y/E166Y/P167G, exhibits ceftazidime hydrolysis and a large active site conformational change. Conclusion:The mutant has an enlarged active site to accommodate ceftazidime and an alternative catalytic residue, Tyr-166. Significance: The study reveals plasticity in -lactamase structure and mechanism in the evolution of altered substrate specificity.
β-Lactamases are enzymes produced by bacterial cells that provide resistance to β-lactam antibiotics. The CTX-M class of β-lactamases provides resistance against the antibiotic, cefotaxime, but not a related oxyimino-cephalosporin antibiotic, ceftazidime. β-lactamases that carry the P167S substitution, however, have been reported to provide ceftazidime resistance. The mechanism by which the P167S substitution expands the substrate profile of CTX-M enzymes is not known. In this study, CTX-M-14 was used as the model enzyme to study the structural changes caused by the P167S mutation that may accelerate ceftazidime turnover. X-ray crystallography was used to determine the structures of the CTX-M-14 P167S apo-enzyme along with the structures of the S70G/P167S, E166A/P167S and E166A mutant enzymes complexed with ceftazidime as well as the E166A/P167S apo-enzyme. The S70G and E166A mutations allow the capture of the enzyme-substrate complex and acylated forms of the ceftazidime molecule, respectively. The results showed a large conformational change in the Ω-loop of the CTX-M-14 ceftazidime acyl-enzyme complex of the P167S mutant but not in the enzyme-substrate complex suggesting the conformational change occurs upon acylation. The conformational change results in a larger active site cavity that prevents steric clash between the aminothiazole ring of ceftazidime and the Asn170 residue in the Ω-loop, allowing for accommodation of ceftazidime for hydrolysis. In addition, the conformational change in the Ω-loop was not observed in the E166A/P167S apo-enzyme, suggesting the presence of acylated ceftazidime influences the conformational change. Finally, the E166A acyl-enzyme structure with ceftazidime did not exhibit the altered Ω-loop conformation, indicating the P167S substitution is required for the change. Taken together, the results reveal that the P167S substitution and the presence of acylated ceftazidime both drive the structure towards a conformational change of the Ω-loop and that in CTX-M P167S enzymes found in drug-resistant bacteria this will lead to increased ceftazidime hydrolysis. This study demonstrates how a naturally occurring substitution can dramatically alter the active site to expand the substrate profile of an enzyme due to antibiotic selective pressure.
Carbapenem-hydrolyzing class D β-lactamases (CHDLs) are an important source of resistance to these last resort β-lactam antibiotics. OXA-48 is a member of a group of CHDLs named OXA-48-like enzymes. On the basis of sequence similarity, OXA-163 can be classified as an OXA-48-like enzyme, but it has altered substrate specificity. Compared to OXA-48, it shows impaired activity for carbapenems but displays an enhanced hydrolysis of oxyimino-cephalosporins. Here, we address the mechanistic and structural basis for carbapenem hydrolysis by OXA-48-like enzymes. Pre-steady-state kinetic analysis indicates that the rate-limiting step for OXA-48 and OXA-163 hydrolysis of carbapenems is deacylation and that the greatly reduced carbapenemase activity of OXA-163 compared to that of OXA-48 is due entirely to a slower deacylation reaction. Furthermore, our structural data indicate that the positioning of the β5-β6 loop is necessary for carbapenem hydrolysis by OXA-48. A major difference between the OXA-48 and OXA-163 complexes with carbapenems is that the 214-RIEP-217 deletion in OXA-163 creates a large opening in the active site that is absent in the OXA-48/carbapenem structures. We propose that the larger active site results in less constraint on the conformation of the 6α-hydroxyethyl group in the acyl-enzyme. The acyl-enzyme intermediate assumes multiple conformations, most of which are incompatible with rapid deacylation. Consistent with this hypothesis, molecular dynamics simulations indicate that the most stable complex is formed between OXA-48 and imipenem, which correlates with the OXA-48 hydrolysis of imipenem being the fastest observed. Furthermore, the OXA-163 complexes with imipenem and meropenem are the least stable and show significant conformational fluctuations, which correlates with the slow hydrolysis of these substrates.
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