Dataset Information

Class D ?-lactamases: are they all carbapenemases?

ABSTRACT: Carbapenem-hydrolyzing class D ?-lactamases (CHDLs) are enzymes of the utmost clinical importance due to their ability to produce resistance to carbapenems, the antibiotics of last resort for the treatment of various life-threatening infections. The vast majority of these enzymes have been identified in Acinetobacter spp., notably in Acinetobacter baumannii. The OXA-2 and OXA-10 enzymes predominantly occur in Pseudomonas aeruginosa and are currently classified as narrow-spectrum class D ?-lactamases. Here we demonstrate that when OXA-2 and OXA-10 are expressed in Escherichia coli strain JM83, they produce a narrow-spectrum antibiotic resistance pattern. When the enzymes are expressed in A. baumannii ATCC 17978, however, they behave as extended-spectrum ?-lactamases and confer resistance to carbapenem antibiotics. Kinetic studies of OXA-2 and OXA-10 with four carbapenems have demonstrated that their catalytic efficiencies with these antibiotics are in the same range as those of some recognized class D carbapenemases. These results are in disagreement with the classification of the OXA-2 and OXA-10 enzymes as narrow-spectrum ?-lactamases, and they suggest that other class D enzymes that are currently regarded as noncarbapenemases may in fact be CHDLs.

SUBMITTER: Antunes NT

PROVIDER: S-EPMC4023754 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Class D β-lactamases: are they all carbapenemases?

Antunes Nuno T NT Lamoureaux Toni L TL Toth Marta M Stewart Nichole K NK Frase Hilary H Vakulenko Sergei B SB

Antimicrobial agents and chemotherapy 20140127 4

Carbapenem-hydrolyzing class D β-lactamases (CHDLs) are enzymes of the utmost clinical importance due to their ability to produce resistance to carbapenems, the antibiotics of last resort for the treatment of various life-threatening infections. The vast majority of these enzymes have been identified in Acinetobacter spp., notably in Acinetobacter baumannii. The OXA-2 and OXA-10 enzymes predominantly occur in Pseudomonas aeruginosa and are currently classified as narrow-spectrum class D β-lactam ...[more]

PMID: 24468778

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Project description:Despite 70 years of clinical use, ?-lactam antibiotics still remain at the forefront of antimicrobial chemotherapy. The major challenge to these life-saving therapeutics is the presence of bacterial enzymes (i.e., ?-lactamases) that can hydrolyze the ?-lactam bond and inactivate the antibiotic. These enzymes can be grouped into four classes (A-D). Among the most genetically diverse are the class D ?-lactamases. In this class are ?-lactamases that can inactivate the entire spectrum of ?-lactam antibiotics (penicillins, cephalosporins, and carbapenems). Class D ?-lactamases are mostly found in Gram-negative bacteria such as Pseudomonas aeruginosa , Escherichia coli , Proteus mirabilis , and Acinetobacter baumannii . The active-sites of class D ?-lactamases contain an unusual N-carboxylated lysine post-translational modification. A strongly hydrophobic active-site helps create the conditions that allow the lysine to combine with CO2, and the resulting carbamate is stabilized by a number of hydrogen bonds. The carboxy-lysine plays a symmetric role in the reaction, serving as a general base to activate the serine nucleophile in the acylation reaction, and the deacylating water in the second step. There are more than 250 class D ?-lactamases described, and the full set of variants shows remarkable diversity with regard to substrate binding and turnover. Narrow-spectrum variants are most effective against the earliest generation penicillins and cephalosporins such as ampicillin and cephalothin. Extended-spectrum variants (also known as extended-spectrum ?-lactamases, ESBLs) pose a more dangerous clinical threat as they possess a small number of substitutions that allow them to bind and hydrolyze later generation cephalosporins that contain bulkier side-chain constituents (e.g., cefotaxime, ceftazidime, and cefepime). Mutations that permit this versatility seem to cluster in the area surrounding an active-site tryptophan resulting in a widened active-site to accommodate the oxyimino side-chains of these cephalosporins. More concerning are the class D ?-lactamases that hydrolyze clinically important carbapenem ?-lactam drugs (e.g., imipenem). Whereas carbapenems irreversibly acylate and inhibit narrow-spectrum ?-lactamases, class D carbapenemases are able to recruit and activate a deacylating water. The rotational orientation of the C6 hydroxyethyl group found on all carbapenem antibiotics likely plays a role in whether the deacylating water is effective or not. Inhibition of class D ?-lactamases is a current challenge. Commercially available inhibitors that are active against other classes of ?-lactamases are ineffective against class D enzymes. On the horizon are several compounds, consisting of both ?-lactam derivatives and non-?-lactams, that have the potential of providing novel leads to design new mechanism-based inactivators that are effective against the class D enzymes. Several act synergistically when given in combination with a ?-lactam antibiotic, and others show a unique mechanism of inhibition that is distinct from the traditional ?-lactamase inhibitors. These studies will bolster structure-based inhibitor design efforts to facilitate the optimization and development of these compounds as class D inactivators.

Project description:OXA beta-lactamases are largely responsible for beta-lactam resistance in Acinetobacter spp. and Pseudomonas aeruginosa, two of the most difficult-to-treat nosocomial pathogens. In general, the beta-lactamase inhibitors used in clinical practice (clavulanic acid, sulbactam, and tazobactam) demonstrate poor activity against class D beta-lactamases. To overcome this challenge, we explored the abilities of beta-lactamase inhibitors of the C-2- and C-3-substituted penicillin and cephalosporin sulfone families against OXA-1, extended-spectrum (OXA-10, OXA-14, and OXA-17), and carbapenemase-type (OXA-24/40) class D beta-lactamases. Three C-2-substituted penicillin sulfone compounds (JDB/LN-1-255, JDB/LN-III-26, and JDB/ASR-II-292) showed low K(i) values for the OXA-1 beta-lactamase (0.70 +/- 0.14 --> 1.60 +/- 0.30 microM) and demonstrated significant K(i) improvements compared to the C-3-substituted cephalosporin sulfone (JDB/DVR-II-214), tazobactam, and clavulanic acid. The C-2-substituted penicillin sulfones JDB/ASR-II-292 and JDB/LN-1-255 also demonstrated low K(i)s for the OXA-10, -14, -17, and -24/40 beta-lactamases (0.20 +/- 0.04 --> 17 +/- 4 microM). Furthermore, JDB/LN-1-255 displayed stoichiometric inactivation of OXA-1 (the turnover number, i.e., the partitioning of the initial enzyme inhibitor complex between hydrolysis and enzyme inactivation [t(n)] = 0) and t(n)s ranging from 5 to 8 for the other OXA enzymes. Using mass spectroscopy to study the intermediates in the inactivation pathway, we determined that JDB/LN-1-255 inhibited OXA beta-lactamases by forming covalent adducts that do not fragment. On the basis of the substrate and inhibitor kinetics of OXA-1, we constructed a model showing that the C-3 carboxylate of JDB/LN-1-255 interacts with Ser115 and Thr213, the R-2 group at C-2 fits between the space created by the long B9 and B10 beta strands, and stabilizing hydrophobic interactions are formed between the pyridyl ring of JDB/LN-1-255 and Val116 and Leu161. By exploiting conserved structural and mechanistic features, JDB/LN-1-255 is a promising lead compound in the quest for effective inhibitors of OXA-type beta-lactamases.

Dataset Information

Class D ?-lactamases: are they all carbapenemases?

Publications

Class D β-lactamases: are they all carbapenemases?

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