Structural basis for different substrate profiles of two closely related class D β-lactamases and their inhibition by halogens

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 that of 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 under another condition that included 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

The Drug-Resistant Variant P167S Expands the Substrate Profile of CTX-M β-Lactamases for Oxyimino-Cephalosporin Antibiotics by Enlarging the Active Site upon Acylation.

CTX-M β-lactamases provide resistance against the β-lactam antibiotic, cefotaxime, but not a related antibiotic, ceftazidime. β-Lactamases that carry the P167S substitution, however, provide ceftazidime resistance. In this study, CTX-M-14 was used as a model to study the structural changes caused by the P167S mutation that accelerate ceftazidime turnover. X-ray crystallography was used to determine the structures of the P167S apoenzyme along with the structures of the S70G/P167S, E166A/P167S, and E166A mutant enzymes complexed with ceftazidime as well as the E166A/P167S apoenzyme. The S70G and E166A mutations allow capture of the enzyme-substrate complex and the acylated form of ceftazidime, respectively. The results showed a large conformational change in the Ω-loop of the ceftazidime acyl-enzyme complex of the P167S mutant but not in the enzyme-substrate complex, suggesting the change occurs upon acylation. The change results in a larger active site that prevents steric clash between the aminothiazole ring of ceftazidime and the Asn170 residue in the Ω-loop, allowing accommodation of ceftazidime for hydrolysis. In addition, the conformational change was not observed in the E166A/P167S apoenzyme, suggesting the presence of acylated ceftazidime influences the conformational change. Finally, the E166A acyl-enzyme structure with ceftazidime did not exhibit the altered 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 toward a conformational change in the Ω-loop and that in CTX-M P167S enzymes found in drug-resistant bacteria this will lead to an increased level of ceftazidime hydrolysis

Structure of the catalytic domain of the colistin resistance enzyme MCR-1

Due 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.Results: The structure of the catalytic domain of MCR-1 at 1.32 Å reveals the active site is similar to that of related phosphoethanolamine transferases.Conclusions: The 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

Additional file 1: Figure S1. of Structure of the catalytic domain of the colistin resistance enzyme MCR-1

Amino acid sequence alignment of phosphoethanolamine transferases MCR-1, EptC (C. jejuni), and LptA (N. meningitidis). (TIF 10272 kb

Synergistic effects of functionally distinct substitutions in β-lactamase variants shed light on the evolution of bacterial drug resistance

The CTX-M β-lactamases have emerged as the most widespread extended-spectrum β-lactamases (ESBLs) in Gram-negative bacteria. These enzymes rapidly hydrolyze cefotaxime, but not the related cephalosporin, ceftazidime. ESBL variants have evolved, however, that provide enhanced ceftazidime resistance. We show here that a natural variant at a nonactive site, i.e. second-shell residue N106S, enhances enzyme stability but reduces catalytic efficiency for cefotaxime and ceftazidime and decreases resistance levels. However, when the N106S variant was combined with an active-site variant, D240G, that enhances enzyme catalytic efficiency, but decreases stability, the resultant double mutant exhibited higher resistance levels than predicted on the basis of the phenotypes of each variant. We found that this epistasis is due to compensatory effects, whereby increased stability provided by N106S overrides its cost of decreased catalytic activity. X-ray structures of the variant enzymes in complex with cefotaxime revealed conformational changes in the active-site loop spanning residues 103-106 that were caused by the N106S substitution and relieve steric strain to stabilize the enzyme, but also alter contacts with cefotaxime and thereby reduce catalytic activity. We noted that the 103-106 loop conformation in the N106S-containing variants is different from that of WT CTX-M but nearly identical to that of the non-ESBL, TEM-1 β-lactamase, having a serine at the 106 position. Therefore, residue 106 may serve as a "switch" that toggles the conformations of the 103-106 loop. When it is serine, the loop is in the non-ESBL, TEM-like conformation, and when it is asparagine, the loop is in a CTX-M-like, cefotaximase-favorable conformation

Removal of the Side Chain at the Active-Site Serine by a Glycine Substitution Increases the Stability of a Wide Range of Serine β-Lactamases by Relieving Steric Strain

Serine β-lactamases are bacterial enzymes that hydrolyze β-lactam antibiotics. They utilize an active-site serine residue as a nucleophile, forming an acyl-enzyme intermediate during hydrolysis. In this study, thermal denaturation experiments as well as X-ray crystallography were performed to test the effect of substitution of the catalytic serine by glycine on protein stability in serine β-lactamases. Six different enzymes comprising representatives from each of the three classes of serine β-lactamases were examined including TEM-1, CTX-M-14, and KPC-2 of class A, P99 of class C, and OXA-48 and OXA-163 of class D. For each enzyme, the wild type and a serine-to-glycine mutant were evaluated for stability. The glycine mutants all exhibited enhanced thermostability compared to the wild type. In contrast, alanine substitutions of the catalytic serine in TEM-1, OXA-48 and OXA-163 did not alter stability, suggesting removal of the Cβ atom is key to the stability increase associated with the glycine mutants. The X-ray crystal structures of P99 S64G, OXA-48 S70G and S70A, and OXA-163 S70G suggest that removal of the side chain of the catalytic serine releases steric strain to improve enzyme stability. Additionally, analysis of the torsion angles at the nucleophile position indicates that the glycine mutants exhibit improved distance and angular parameters of the intra-helical hydrogen bond network compared to the wild-type enzymes, which is also consistent with increased stability. The increased stability of the mutants indicates that the enzyme pays a price in stability for the presence of a side chain at the catalytic serine position but that the cost is necessary in that removal of the serine drastically impairs function. These findings support the stability-function hypothesis, which states that active-site residues are optimized for substrate binding and catalysis but that the requirements for catalysis are often not consistent with the requirements for optimal stability