scholarly journals Crystal Structure of Carbapenemase OXA-58 from Acinetobacter baumannii

2014 ◽  
Vol 58 (4) ◽  
pp. 2135-2143 ◽  
Author(s):  
Clyde A. Smith ◽  
Nuno Tiago Antunes ◽  
Marta Toth ◽  
Sergei B. Vakulenko
Keyword(s):  
Water Molecule ◽  
Acinetobacter Baumannii ◽  
Active Site ◽  
Bacterial Infections ◽  
Surrounding Medium ◽  
Active Site Residues ◽  
Content Type ◽  
Class D ◽  
Enzyme Complexes ◽  
Wide Dissemination

ABSTRACTClass D β-lactamases capable of hydrolyzing last-resort carbapenem antibiotics represent a major challenge for treatment of bacterial infections. Wide dissemination of these enzymes inAcinetobacter baumanniielevated this pathogen to the category of most deadly and difficult to treat. We present here the structure of the OXA-58 β-lactamase, a major class D carbapenemase ofA. baumannii, determined to 1.30-Å resolution. Unlike two otherAcinetobactercarbapenemases, OXA23 and OXA-24, the OXA-58 enzyme lacks the characteristic hydrophobic bridge over the active site, despite conservation of the residues which participate in its formation. The active-site residues in OXA-58 are spatially conserved in comparison to those in other class D β-lactamases. Lys86, which activates water molecules during the acylation and deacylation steps, is fully carboxylated in the OXA-58 structure. In the absence of a substrate, a water molecule is observed in the active site of the enzyme and is positioned in the pocket that is usually occupied by the 6α-hydroxyethyl moiety of carbapenems. A water molecule in this location would efficiently deacylate good substrates, such as the penicillins, but in the case of carbapenems, it would be expelled by the 6α-hydroxyethyl moiety of the antibiotics and a water from the surrounding medium would find its way to the vicinity of the carboxylated Lys86 to perform deacylation. Subtle differences in the position of this water in the acyl-enzyme complexes of class D β-lactamases could ultimately be responsible for differences in the catalytic efficiencies of these enzymes against last-resort carbapenem antibiotics.

scholarly journals Clinical Variants of the Native Class D β-Lactamase of Acinetobacter baumannii Pose an Emerging Threat through Increased Hydrolytic Activity against Carbapenems

2016 ◽  
Vol 60 (10) ◽  
pp. 6155-6164 ◽  
Author(s):  
Emma C. Schroder ◽  
Zachary L. Klamer ◽  
Aysegul Saral ◽  
Kyle A. Sugg ◽  
Cynthia M. June ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Active Site ◽  
Hydrolytic Activity ◽  
Single Amino Acid ◽  
Kinetic Properties ◽  
Active Site Residues ◽  
Content Type ◽  
Class D ◽  
Clinical Variants ◽  
Dynamics Simulations

ABSTRACTThe threat posed by the chromosomally encoded class D β-lactamase ofAcinetobacter baumannii(OXA-51/66) has been unclear, in part because of its relatively low affinity and turnover rate for carbapenems. Several hundred clinical variants of OXA-51/66 have been reported, many with substitutions of active-site residues. We determined the kinetic properties of OXA-66 and five clinical variants with respect to a wide variety of β-lactam substrates. The five variants displayed enhanced activity against carbapenems and in some cases against penicillins, late-generation cephalosporins, and the monobactam aztreonam. Molecular dynamics simulations show that in OXA-66, P130 inhibits the side-chain rotation of I129 and thereby prevents doripenem binding because of steric clash. A single amino acid substitution at this position (P130Q) in the variant OXA-109 greatly enhances the mobility of both I129 and a key active-site tryptophan (W222), thereby facilitating carbapenem binding. This expansion of substrate specificity represents a very worrisome development for the efficacy of β-lactams against this troublesome pathogen.

scholarly journals Active-Site Plasticity Is Essential to Carbapenem Hydrolysis by OXA-58 Class D β-Lactamase of Acinetobacter baumannii

2015 ◽  
Vol 60 (1) ◽  
pp. 75-86 ◽  
Author(s):  
Shivendra Pratap ◽  
Madhusudhanarao Katiki ◽  
Preet Gill ◽  
Pravindra Kumar ◽  
Dasantila Golemi-Kotra
Keyword(s):  
Crystal Structure ◽  
Acinetobacter Baumannii ◽  
Active Site ◽  
Active Sites ◽  
Multidrug Resistant ◽  
Crystal Structure Analysis ◽  
Content Type ◽  
Class D ◽  
Enzyme Species ◽  
Hydroxyethyl Group

ABSTRACTCarbapenem-hydrolyzing class D β-lactamases (CHDLs) are a subgroup of class D β-lactamases, which are enzymes that hydrolyze β-lactams. They have attracted interest due to the emergence of multidrug-resistantAcinetobacter baumannii, which is not responsive to treatment with carbapenems, the usual antibiotics of choice for this bacterium. Unlike other class D β-lactamases, these enzymes efficiently hydrolyze carbapenem antibiotics. To explore the structural requirements for the catalysis of carbapenems by these enzymes, we determined the crystal structure of the OXA-58 CHDL ofA. baumanniifollowing acylation of its active-site serine by a 6α-hydroxymethyl penicillin derivative that is a structural mimetic for a carbapenem. In addition, several point mutation variants of the active site of OXA-58, as identified by the crystal structure analysis, were characterized kinetically. These combined studies confirm the mechanistic relevance of a hydrophobic bridge formed over the active site. This structural feature is suggested to stabilize the hydrolysis-productive acyl-enzyme species formed from the carbapenem substrates of this enzyme. Furthermore, our structural studies provide strong evidence that the hydroxyethyl group of carbapenems samples different orientations in the active sites of CHDLs, and the optimum orientation for catalysis depends on the topology of the active site allowing proper closure of the active site. We propose that CHDLs use the plasticity of the active site to drive the mechanism of carbapenem hydrolysis toward efficiency.

scholarly journals Cyclic Boronates Inhibit All Classes of β-Lactamases

2017 ◽  
Vol 61 (4) ◽  
Author(s):  
Samuel T. Cahill ◽  
Ricky Cain ◽  
David Y. Wang ◽  
Christopher T. Lohans ◽  
David W. Wareham ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Bacterial Infections ◽  
Binding Mode ◽  
Gram Negative ◽  
Content Type ◽  
Class A ◽  
Class D ◽  
Dual Action ◽  
The Common ◽  
Continued Use

ABSTRACT β-Lactamase-mediated resistance is a growing threat to the continued use of β-lactam antibiotics. The use of the β-lactam-based serine-β-lactamase (SBL) inhibitors clavulanic acid, sulbactam, and tazobactam and, more recently, the non-β-lactam inhibitor avibactam has extended the utility of β-lactams against bacterial infections demonstrating resistance via these enzymes. These molecules are, however, ineffective against the metallo-β-lactamases (MBLs), which catalyze their hydrolysis. To date, there are no clinically available metallo-β-lactamase inhibitors. Coproduction of MBLs and SBLs in resistant infections is thus of major clinical concern. The development of “dual-action” inhibitors, targeting both SBLs and MBLs, is of interest, but this is considered difficult to achieve due to the structural and mechanistic differences between the two enzyme classes. We recently reported evidence that cyclic boronates can inhibit both serine- and metallo-β-lactamases. Here we report that cyclic boronates are able to inhibit all four classes of β-lactamase, including the class A extended spectrum β-lactamase CTX-M-15, the class C enzyme AmpC from Pseudomonas aeruginosa, and class D OXA enzymes with carbapenem-hydrolyzing capabilities. We demonstrate that cyclic boronates can potentiate the use of β-lactams against Gram-negative clinical isolates expressing a variety of β-lactamases. Comparison of a crystal structure of a CTX-M-15:cyclic boronate complex with structures of cyclic boronates complexed with other β-lactamases reveals remarkable conservation of the small-molecule binding mode, supporting our proposal that these molecules work by mimicking the common tetrahedral anionic intermediate present in both serine- and metallo-β-lactamase catalysis.

scholarly journals Cryo-electron Microscopy Structure of the Acinetobacter baumannii 70S Ribosome and Implications for New Antibiotic Development

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Christopher E. Morgan ◽  
Wei Huang ◽  
Susan D. Rudin ◽  
Derek J. Taylor ◽  
James E. Kirby ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Acinetobacter Baumannii ◽  
Bacterial Infections ◽  
Bound States ◽  
Antimicrobial Agents ◽  
Gram Negative ◽  
Content Type ◽  
Antibiotic Development ◽  
Cryo Electron Microscopy ◽  
70S Ribosome

ABSTRACT Antimicrobial resistance is a major health threat as it limits treatment options for infection. At the forefront of this serious issue is Acinetobacter baumannii, a Gram-negative opportunistic pathogen that exhibits the remarkable ability to resist antibiotics through multiple mechanisms. As bacterial ribosomes represent a target for multiple distinct classes of existing antimicrobial agents, we here use single-particle cryo-electron microscopy (cryo-EM) to elucidate five different structural states of the A. baumannii ribosome, including the 70S, 50S, and 30S forms. We also determined interparticle motions of the 70S ribosome in different tRNA bound states using three-dimensional (3D) variability analysis. Together, our structural data further our understanding of the ribosome from A. baumannii and other Gram-negative pathogens and will enable structure-based drug discovery to combat antibiotic-resistant bacterial infections. IMPORTANCE Acinetobacter baumannii is a severe nosocomial threat largely due to its intrinsic antibiotic resistance and remarkable ability to acquire new resistance determinants. The bacterial ribosome serves as a major target for modern antibiotics and the design of new therapeutics. Here, we present cryo-EM structures of the A. baumannii 70S ribosome, revealing several unique species-specific structural features that may facilitate future drug development to combat this recalcitrant bacterial pathogen.

scholarly journals Horizontal Transfer of the OXA-24 Carbapenemase Gene via Outer Membrane Vesicles: a New Mechanism of Dissemination of Carbapenem Resistance Genes in Acinetobacter baumannii

2011 ◽  
Vol 55 (7) ◽  
pp. 3084-3090 ◽  
Author(s):  
Carlos Rumbo ◽  
Esteban Fernández-Moreira ◽  
María Merino ◽  
Margarita Poza ◽  
Jose Antonio Mendez ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Resistance Genes ◽  
Carbapenem Resistance ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Content Type ◽  
Class D ◽  
Carbapenemase Gene

ABSTRACTThe resistance ofAcinetobacter baumanniistrains to carbapenems is a worrying problem in hospital settings. The main mechanism of carbapenem resistance is the expression of β-lactamases (metalloenzymes or class D enzymes). The mechanisms of the dissemination of these genes amongA. baumanniistrains are not fully understood. In this study we used two carbapenem-resistant clinical strains ofA. baumannii(AbH12O-A2 and AbH12O-CU3) expressing the plasmid-borneblaOXA-24gene (plasmids pMMA2 and pMMCU3, respectively) to demonstrate thatA. baumanniireleases outer membrane vesicles (OMVs) duringin vitrogrowth. The use of hybridization studies enabled us to show that these OMVs harbored theblaOXA-24gene. The incubation of these OMVs with the carbapenem-susceptibleA. baumanniiATCC 17978 host strain yielded full resistance to carbapenems. The presence of the original plasmids harboring theblaOXA-24gene was detected in strain ATCC 17978 after the transformation of OMVs. New OMVs harboringblaOXA-24were released byA. baumanniiATCC 17978 after it was transformed with the original OMV-mediated plasmids, indicating the universality of the process. We present the first experimental evidence that clinical isolates ofA. baumanniimay release OMVs as a mechanism of horizontal gene transfer whereby carbapenem resistance genes are delivered to surroundingA. baumanniibacterial isolates.

scholarly journals Mutagenesis and crystallographic studies of the catalytic residues of the papain family protease bleomycin hydrolase: new insights into active-site structure

2006 ◽  
Vol 401 (2) ◽  
pp. 421-428 ◽  
Author(s):  
Paul A. O'Farrell ◽  
Leemor Joshua-Tor
Keyword(s):  
Water Molecule ◽  
Active Site ◽  
Bound Water ◽  
Active Site Residues ◽  
Site Structure ◽  
Active Site Cysteine ◽  
C Terminus ◽  
Bleomycin Hydrolase ◽  
Active Site Mutants

Bleomycin hydrolase (BH) is a hexameric papain family cysteine protease which is involved in preparing peptides for antigen presentation and has been implicated in tumour cell resistance to bleomycin chemotherapy. Structures of active-site mutants of yeast BH yielded unexpected results. Replacement of the active-site asparagine with alanine, valine or leucine results in the destabilization of the histidine side chain, demonstrating unambiguously the role of the asparagine residue in correctly positioning the histidine for catalysis. Replacement of the histidine with alanine or leucine destabilizes the asparagine position, indicating a delicate arrangement of the active-site residues. In all of the mutants, the C-terminus of the protein, which lies in the active site, protrudes further into the active site. All mutants were compromised in their catalytic activity. The structures also revealed the importance of a tightly bound water molecule which stabilizes a loop near the active site and which is conserved throughout the papain family. It is displaced in a number of the mutants, causing destabilization of this loop and a nearby loop, resulting in a large movement of the active-site cysteine. The results imply that this water molecule plays a key structural role in this family of enzymes.

scholarly journals Early Insights into the Interactions of Different β-Lactam Antibiotics and β-Lactamase Inhibitors against Soluble Forms of Acinetobacter baumannii PBP1a and Acinetobacter sp. PBP3

2012 ◽  
Vol 56 (11) ◽  
pp. 5687-5692 ◽  
Author(s):  
Krisztina M. Papp-Wallace ◽  
Baui Senkfor ◽  
Julian Gatta ◽  
Weirui Chai ◽  
Magdalena A. Taracila ◽  
...  
Keyword(s):  
United States ◽  
Cell Wall ◽  
Acinetobacter Baumannii ◽  
Active Site ◽  
Molecular Model ◽  
Content Type ◽  
Penicillin Binding Proteins ◽  
One Step ◽  
Susceptibility And Resistance

ABSTRACTAcinetobacter baumanniiis an increasingly problematic pathogen in United States hospitals. Antibiotics that can treatA. baumanniiare becoming more limited. Little is known about the contributions of penicillin binding proteins (PBPs), the target of β-lactam antibiotics, to β-lactam–sulbactam susceptibility and β-lactam resistance inA. baumannii. Decreased expression of PBPs as well as loss of binding of β-lactams to PBPs was previously shown to promote β-lactam resistance inA. baumannii. Using anin vitroassay with a reporter β-lactam, Bocillin, we determined that the 50% inhibitory concentrations (IC50s) for PBP1a fromA. baumanniiand PBP3 fromAcinetobactersp. ranged from 1 to 5 μM for a series of β-lactams. In contrast, PBP3 demonstrated a narrower range of IC50s against β-lactamase inhibitors than PBP1a (ranges, 4 to 5 versus 8 to 144 μM, respectively). A molecular model with ampicillin and sulbactam positioned in the active site of PBP3 reveals that both compounds interact similarly with residues Thr526, Thr528, and Ser390. Accepting that many interactions with cell wall targets are possible with the ampicillin-sulbactam combination, the low IC50s of ampicillin and sulbactam for PBP3 may contribute to understanding why this combination is effective againstA. baumannii. Unraveling the contribution of PBPs to β-lactam susceptibility and resistance brings us one step closer to identifying which PBPs are the best targets for novel β-lactams.

scholarly journals Multiple substitutions lead to increased loop flexibility and expanded specificity in Acinetobacter baumannii carbapenemase OXA-239

2018 ◽  
Vol 475 (1) ◽  
pp. 273-288 ◽  
Author(s):  
Thomas M. Harper ◽  
Cynthia M. June ◽  
Magdalena A. Taracila ◽  
Robert A. Bonomo ◽  
Rachel A. Powers ◽  
...  
Keyword(s):  
Amino Acid ◽  
Acinetobacter Baumannii ◽  
Active Site ◽  
Active Sites ◽  
Protein Structures ◽  
Amino Acid Substitutions ◽  
The Third ◽  
Class D ◽  
Late Generation ◽  
Loop Flexibility

OXA-239 is a class D carbapenemase isolated from an Acinetobacter baumannii strain found in Mexico. This enzyme is a variant of OXA-23 with three amino acid substitutions in or near the active site. These substitutions cause OXA-239 to hydrolyze late-generation cephalosporins and the monobactam aztreonam with greater efficiency than OXA-23. OXA-239 activity against the carbapenems doripenem and imipenem is reduced ∼3-fold and 20-fold, respectively. Further analysis demonstrated that two of the substitutions (P225S and D222N) are largely responsible for the observed alteration of kinetic parameters, while the third (S109L) may serve to stabilize the protein. Structures of OXA-239 with cefotaxime, doripenem and imipenem bound as acyl-intermediates were determined. These structures reveal that OXA-239 has increased flexibility in a loop that contains P225S and D222N. When carbapenems are bound, the conformation of this loop is essentially identical with that observed previously for OXA-23, with a narrow active site that makes extensive contacts to the ligand. When cefotaxime is bound, the loop can adopt a different conformation that widens the active site to allow binding of that bulky drug. This alternate conformation is made possible by P225S and further stabilized by D222N. Taken together, these results suggest that the three substitutions were selected to expand the substrate specificity profile of OXA-23 to cephalosporins and monobactams. The loss of activity against imipenem, however, suggests that there may be limits to the plasticity of class D enzymes with regard to evolving active sites that can effectively bind multiple classes of β-lactam drugs.

scholarly journals The role of conserved surface hydrophobic residues in the carbapenemase activity of the class D β-lactamases

2017 ◽  
Vol 73 (8) ◽  
pp. 692-701 ◽  
Author(s):  
Marta Toth ◽  
Clyde A. Smith ◽  
Nuno T. Antunes ◽  
Nichole K. Stewart ◽  
Lauren Maltz ◽  
...  
Keyword(s):  
Water Molecule ◽  
Active Site ◽  
Clinical Importance ◽  
Catalytic Efficiency ◽  
Structural Data ◽  
Molecular Surface ◽  
Water Molecules ◽  
Hydrophobic Residues ◽  
Class D ◽  
Life Threatening

Carbapenem-hydrolyzing class D β-lactamases (CHDLs) produce resistance to the last-resort carbapenem antibiotics and render these drugs ineffective for the treatment of life-threatening infections. Here, it is shown that among the clinically important CHDLs, OXA-143 produces the highest levels of resistance to carbapenems and has the highest catalytic efficiency against these substrates. Structural data demonstrate that acylated carbapenems entirely fill the active site of CHDLs, leaving no space for water molecules, including the deacylating water. Since the entrance to the active site is obstructed by the acylated antibiotic, the deacylating water molecule must take a different route for entry. It is shown that in OXA-143 the movement of a conserved hydrophobic valine residue on the surface opens a channel to the active site of the enzyme, which would not only allow the exchange of water molecules between the active site and the milieu, but would also create extra space for a water molecule to position itself in the vicinity of the scissile bond of the acyl-enzyme intermediate to perform deacylation. Structural analysis of the OXA-23 carbapenemase shows that in this enzyme movement of the conserved leucine residue, juxtaposed to the valine on the molecular surface, creates a similar channel to the active site. These data strongly suggest that all CHDLs may employ a mechanism whereupon the movement of highly conserved valine or leucine residues would allow a water molecule to access the active site to promote deacylation. It is further demonstrated that the 6α-hydroxyethyl group of the bound carbapenem plays an important role in the stabilization of this channel. The recognition of a universal deacylation mechanism for CHDLs suggests a direction for the future development of inhibitors and novel antibiotics for these enzymes of utmost clinical importance.

scholarly journals Activity of the β-Lactamase Inhibitor LN-1-255 against Carbapenem-Hydrolyzing Class D β-Lactamases from Acinetobacter baumannii

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
Juan Carlos Vázquez-Ucha ◽  
María Maneiro ◽  
Marta Martínez-Guitián ◽  
John Buynak ◽  
Christopher R. Bethel ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Multidrug Resistant ◽  
Kinetics Parameters ◽  
Clinical Issue ◽  
Content Type ◽  
Carbapenem Resistant ◽  
Class D ◽  
Continued Use ◽  
Global Threat ◽  
Lactamase Inhibitor

ABSTRACT The number of infections caused by Gram-negative pathogens carrying carbapenemases is increasing, and the group of carbapenem-hydrolyzing class D β-lactamases (CHDLs) is especially problematic. Several clinically important CHDLs have been identified in Acinetobacter baumannii, including OXA-23, OXA-24/40, OXA-58, OXA-143, OXA-235, and the chromosomally encoded OXA-51. The selection and dissemination of carbapenem-resistant A. baumannii strains constitutes a serious global threat. Carbapenems have been successfully utilized as last-resort antibiotics for the treatment of multidrug-resistant A. baumannii infections. However, the spread of OXA carbapenemases is compromising the continued use of these antimicrobials. In response to this clinical issue, it is necessary and urgent to design and develop new specific inhibitors with efficacy against these enzymes. The aim of this work was to characterize the inhibitory activity of LN-1-255 (a 6-alkylidene-2-substituted penicillin sulfone) and compare it to that of two established inhibitors (avibactam and tazobactam) against the most relevant enzymes of each group of class D carbapenemases in A. baumannii. The β-lactamase inhibitor LN-1-255 demonstrated excellent microbiological synergy and inhibition kinetics parameters against all tested CHDLs and a significantly higher activity than tazobactam and avibactam. A combination of carbapenems and LN-1-255 was effective against A. baumannii class D carbapenemases. Docking assays confirmed the affinity of LN-1-255 for the active site of these enzymes. LN-1-255 represents a potential new β-lactamase inhibitor that may have a significant role in eradicating infections caused by A. baumannii isolates carrying CHDLs.

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