Structural insights into the enhanced carbapenemase efficiency of OXA‐655 compared to OXA‐10

Leiros, Hanna‐Kirsti S.; Thomassen, Ane Molden; Samuelsen, Ørjan; Flach, Carl-Fredrik; Kotsakis, Stathis D.; Larsson, D. G. Joakim

doi:10.1002/2211-5463.12935

Cited by 11 publications

(11 citation statements)

References 23 publications

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“…Conformation III is observed in most (~93%) of the AEC structures lacking lysine carbamylation (Figure 3) (25,32). However, there are recently reported AEC structures arising from the reaction of OXA-10 with ertapenem and imipenem, and of OXA-10 V117L with meropenem, that manifest conformation III in the presence of a carbamylated active site lysine (33). The uncarbamylated structures are not expected to represent catalytically active complexes, as lysine carbamylation is critical for both hydrolysis and βlactone formation by class D SBLs (24,30).…”

Section: Conformations Of the C-6 Hydroxyethyl Side Chain Observed Bymentioning

confidence: 97%

Analysis of β-lactone formation by clinically observed carbapenemases informs on a novel antibiotic resistance mechanism

Aertker

Chan

Lohans

et al. 2020

Journal of Biological Chemistry

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An important mechanism of resistance to β-lactam antibiotics is via their β-lactamase catalyzed hydrolysis. Recent work has shown that, in addition to the established hydrolysis products, the reaction of the class D nucleophilic serine β-lactamases (SBLs) with carbapenems also produces β-lactones. We report studies on the factors determining βlactone formation by class D SBLs. We show that variations in hydrophobic residues at the active site of class D SBLs (i.e., Trp105, Val120, and Leu158, using OXA-48 numbering) impact on the relative levels of β-lactones and hydrolysis products formed. Some variants, i.e., the OXA-48 V120L and OXA-23 V128L variants, catalyze increased βlactone formation compared to the wild-type enzymes. The results of kinetic and product studies reveal that variations of residues other than those directly involved in catalysis, including those arising from clinically observed mutations, can alter the reaction outcome of class D SBL catalysis. NMR studies show some Class D SBLs variants catalyze formation of β-lactones from all clinically relevant carbapenems regardless of the presence or not of a 1β-methyl substituent. Analysis of reported crystal structures for carbapenems derived acylenzyme complexes reveals preferred conformations for hydrolysis and β-lactone formation. The observation of increased β-lactone formation by class D SBLs variants, including the clinically observed carbapenemase OXA-48 V120L, supports the proposal that class D SBL-catalyzed rearrangement of β-lactams to β-lactones is important as a resistance mechanism.

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Section: Conformations Of the C-6 Hydroxyethyl Side Chain Observed Bymentioning

confidence: 97%

Analysis of β-lactone formation by clinically observed carbapenemases informs on a novel antibiotic resistance mechanism

Aertker

Chan

Lohans

et al. 2020

Journal of Biological Chemistry

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“… 24 , 25 It was validated in OXA-655 with V117L that alterations of the interactions with carbapenems were brought by substitutions at this key site. 26 In both crystal structures of OXA-655 and OXA-677 proteins, their position 117 residues are closer to Leu155 in the omega loop, and their interactions seem stronger. This conformational change probably alters the accessibility of the meropenem to the active site.…”

Section: Discussionmentioning

confidence: 97%

First Report of blaOXA-677 with Enhanced Meropenem-Hydrolyzing Ability in Pseudomonas aeruginosa in China

Sun¹,

Han²,

Yang³

et al. 2021

IDR

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Purpose OXA-10-type class D β-lactamases have shown their evolutionary potential of enhancing carbapenem resistance. This study aimed to elucidate the role of OXA-10 variants in clinical isolated multidrug resistant (MDR) Pseudomonas aeruginosa and characterize the first appearance of OXA-677 in China. Methods Six bla OXA-10-like -positive strains were screened by PCR from 41 P. aeruginosa strains, which were resistant to both carbapenems and ceftazidime-avibactam, collected across China in 2018. The minimum inhibitory concentrations (MIC) were determined with the broth microdilution method. The resistance-associated genes and genetic environment were investigated by whole-genome sequencing (WGS). The function and mechanism of OXA-677 β-lactamase were identified by molecular cloning and protein structure modeling. Results All the bla OXA-10-like -positive Pseudomonas aeruginosa were MDR strains. They also had outer membrane porin defects and produced β-lactam resistance gene bla PER-1, fluoroquinolone-resistant gene crpP , aminoglycoside-resistance gene aph(3ʹ)-IIb, aph(6)-Id, aacA and aadA , fosfomycin-resistance gene fosA , sulfamethoxazole-resistance gene sul1 , and chloramphenicol-resistance gene catB7 . All bla OXA-10 variants were located in a Tn1403-related transposon, containing aacA4-12-bla OXA-677 - aadA1, aacA4-12-bla OXA-101 - aadA5 , and bla OXA-246 - aacA3-aadA13 gene cassette arrays, respectively. Notably, the bla OXA-677 producer showed a high MIC level of meropenem (MIC>64 mg/L). Compared to bla OXA-10 , bla OXA-677 was found a G-to-T transversion at position 350, leading to a phenylalanine-for-valine substitution in position 117, which is closer to leucine155 in the omega loop of the active site. MIC of meropenem for E. coli DH5α with the recombinant plasmid pHSG398 carrying bla OXA-677 was elevated by 8 times. Conclusion We speculate that the OXA-10-like enzymes and the decrease of membrane permeability confer carbapenem resistance, and the V117 substitution in OXA-677 might lead to a higher resistance level of meropenem.

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“…Like other DBLs, OXA-48 harbors three highly conserved motifs: (I) S 70 -X-X-K 73 , where X represents a variable residue; (II) S 118 -X-V/I, which is equivalent to the S 130 -D-N motif in class A β-lactamases and Y-A/S/N in AmpC β-lactamases and (III) K 208 -T-G [4,12]. OXA-48 also has two other conserved motifs including Y/F 144 -G-N and W 221 -X-X-G that only exist in class D β-lactamases [13,14].…”

Section: Introductionmentioning

confidence: 99%

Structural and Functional Characterization of OXA-48: Insight into Mechanism and Structural Basis of Substrate Recognition and Specificity

Chiou

Cheng

Shum

et al. 2021

IJMS

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Class D β-lactamase OXA-48 is widely distributed among Gram-negative bacteria and is an important determinant of resistance to the last-resort carbapenems. Nevertheless, the detailed mechanism by which this β-lactamase hydrolyzes its substrates remains poorly understood. In this study, the complex structures of OXA-48 and various β-lactams were modeled and the potential active site residues that may interact with various β-lactams were identified and characterized to elucidate their roles in OXA-48 substrate recognition. Four residues, namely S70, K73, S118, and K208 were found to be essential for OXA-48 to undergo catalytic hydrolysis of various penicillins and carbapenems both in vivo and in vitro. T209 was found to be important for hydrolysis of imipenem, whereas R250 played a major role in hydrolyzing ampicillin, imipenem, and meropenem most likely by forming a H-bond or salt-bridge between the side chain of these two residues and the carboxylate oxygen ions of the substrates. Analysis of the effect of substitution of alanine in two residues, W105 and L158, revealed their roles in mediating the activity of OXA-48. Our data show that these residues most likely undergo hydrophobic interaction with the R groups and the core structure of the β-lactam ring in penicillins and the carbapenems, respectively. Unlike OXA-58, mass spectrometry suggested a loss of the C6-hydroxyethyl group during hydrolysis of meropenem by OXA-48, which has never been demonstrated in Class D carbapenemases. Findings in this study provide comprehensive knowledge of the mechanism of the substrate recognition and catalysis of OXA-type β-lactamases.

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Structural insights into the enhanced carbapenemase efficiency of OXA‐655 compared to OXA‐10

Cited by 11 publications

References 23 publications

Analysis of β-lactone formation by clinically observed carbapenemases informs on a novel antibiotic resistance mechanism

Analysis of β-lactone formation by clinically observed carbapenemases informs on a novel antibiotic resistance mechanism

First Report of blaOXA-677 with Enhanced Meropenem-Hydrolyzing Ability in Pseudomonas aeruginosa in China

Structural and Functional Characterization of OXA-48: Insight into Mechanism and Structural Basis of Substrate Recognition and Specificity

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