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

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.

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Crystal Structure of Carbapenemase OXA-58 from Acinetobacter baumannii

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01983-13 ◽

2014 ◽

Vol 58 (4) ◽

pp. 2135-2143 ◽

Cited By ~ 21

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.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01393-15 ◽

2015 ◽

Vol 60 (1) ◽

pp. 75-86 ◽

Cited By ~ 12

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.

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Characterization of the AmpC β-Lactamase fromBurkholderia multivorans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01140-18 ◽

2018 ◽

Vol 62 (10) ◽

Cited By ~ 5

Author(s):

Scott A. Becka ◽

Elise T. Zeiser ◽

Melissa D. Barnes ◽

Magdalena A. Taracila ◽

Kevin Nguyen ◽

...

Keyword(s):

Cystic Fibrosis ◽

Active Site ◽

Burkholderia Cepacia ◽

Single Amino Acid ◽

Amino Acid Substitutions ◽

Enzyme Complex ◽

Active Site Residues ◽

Content Type ◽

Burkholderia Multivorans

ABSTRACTBurkholderia multivoransis a member of theBurkholderia cepaciacomplex, a group of >20 related species of nosocomial pathogens that commonly infect individuals suffering from cystic fibrosis. β-Lactam antibiotics are recommended as therapy for infections due toB.multivorans, which possesses two β-lactamase genes,blapenAandblaAmpC. PenA is a carbapenemase with a substrate profile similar to that of theKlebsiella pneumoniaecarbapenemase (KPC); in addition, expression of PenA is inducible by β-lactams inB.multivorans. Here, we characterize AmpC fromB.multivoransATCC 17616. AmpC possesses only 38 to 46% protein identity with non-BurkholderiaAmpC proteins (e.g., PDC-1 and CMY-2). Among 49 clinical isolates ofB.multivorans, we identified 27 different AmpC variants. Some variants possessed single amino acid substitutions within critical active-site motifs (Ω loop and R2 loop). Purified AmpC1 demonstrated minimal measurable catalytic activity toward β-lactams (i.e., nitrocefin and cephalothin). Moreover, avibactam was a poor inhibitor of AmpC1 (Kiapp> 600 μM), and acyl-enzyme complex formation with AmpC1 was slow, likely due to lack of productive interactions with active-site residues. Interestingly, immunoblotting using a polyclonal anti-AmpC antibody revealed that protein expression of AmpC1 was inducible inB.multivoransATCC 17616 after growth in subinhibitory concentrations of imipenem (1 μg/ml). AmpC is a unique inducible class C cephalosporinase that may play an ancillary role inB.multivoranscompared to PenA, which is the dominant β-lactamase inB.multivoransATCC 17616.

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A general method to predict the effect of single amino acid substitutions on enzyme catalytic activity

10.1101/236265 ◽

2017 ◽

Author(s):

Yu-Hsiu T. Lin ◽

Cheng Lai Victor Huang ◽

Christina Ho ◽

Max Shatsky ◽

Jack F. Kirsch

Keyword(s):

Amino Acid ◽

Kinetic Parameters ◽

Active Site ◽

Enzyme Reaction ◽

Site Directed Mutagenesis ◽

Enzyme Function ◽

Single Amino Acid ◽

Kinetic Properties ◽

Amino Acid Substitutions ◽

Active Site Residues

ABSTRACTOver the past thirty years, site-directed mutagenesis has become established as one of the most powerful techniques to probe enzyme reaction mechanisms1-3. Substitutions of active site residues are most likely to yield significant perturbations in kinetic parameters, but there are many examples of profound changes in these values elicited by remote mutations4-6. Ortholog comparisons of extant sequences show that many mutations do not have profound influence on enzyme function. As the number of potential single natural amino acid substitutions that can be introduced in a protein of N amino acids in length by directed mutation is very large (19 * N), it would be useful to have a method to predict which amino acid substitutions are more likely to introduce significant changes in kinetic parameters in order to design meaningful probes into enzyme function. What is especially desirable is the identification of critical residues that do not contact the substrate directly, and may be remote from the active site.We collected literature data reflecting the effects of 2,804 mutations on kinetic properties for 12 enzymes. These data along with characteristic predictors were used in a machine-learning scheme to train a classifier to predict the effect of mutation. Use of this algorithm allows one to predict with a 2.5-fold increase in precision, if a given mutation, made anywhere in the enzyme, will cause a decrease in kcat/Km value of ≥ 95%. The improved precision allows the experimentalist to reduce the number of mutations necessary to probe the enzyme reaction mechanism.

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Structures of the Class D Carbapenemases OXA-23 and OXA-146: Mechanistic Basis of Activity against Carbapenems, Extended-Spectrum Cephalosporins, and Aztreonam

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00762-13 ◽

2013 ◽

Vol 57 (10) ◽

pp. 4848-4855 ◽

Cited By ~ 44

Author(s):

Kip-Chumba J. Kaitany ◽

Neil V. Klinger ◽

Cynthia M. June ◽

Maddison E. Ramey ◽

Robert A. Bonomo ◽

...

Keyword(s):

Active Site ◽

Single Amino Acid ◽

Side Chain ◽

Content Type ◽

X Ray ◽

Amino Acid Insertion ◽

Extended Spectrum ◽

Class D ◽

Mechanistic Basis ◽

Over The Top

ABSTRACTClass D β-lactamases that hydrolyze carbapenems such as imipenem and doripenem are a recognized danger to the efficacy of these “last-resort” β-lactam antibiotics. Like all known class D carbapenemases, OXA-23 cannot hydrolyze the expanded-spectrum cephalosporin ceftazidime. OXA-146 is an OXA-23 subfamily clinical variant that differs from the parent enzyme by a single alanine (A220) inserted in the loop connecting β-strands β5 and β6. We discovered that this insertion enables OXA-146 to bind and hydrolyze ceftazidime with an efficiency comparable to those of other extended-spectrum class D β-lactamases. OXA-146 also binds and hydrolyzes aztreonam, cefotaxime, ceftriaxone, and ampicillin with higher efficiency than OXA-23 and preserves activity against doripenem. In this study, we report the X-ray crystal structures of both the OXA-23 and OXA-146 enzymes at 1.6-Å and 1.2-Å resolution. A comparison of the two structures shows that the extra alanine moves a methionine (M221) out of its normal position, where it forms a bridge over the top of the active site. This single amino acid insertion also lengthens the β5-β6 loop, moving the entire backbone of this region further away from the active site. A model of ceftazidime bound in the active site reveals that these two structural alterations are both likely to relieve steric clashes between the bulky R1 side chain of ceftazidime and OXA-23. With activity against all four classes of β-lactam antibiotics, OXA-146 represents an alarming new threat to the treatment of infections caused byAcinetobacterspp.

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Structural Analysis of the Role of Pseudomonas aeruginosa Penicillin-Binding Protein 5 in β-Lactam Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00505-13 ◽

2013 ◽

Vol 57 (7) ◽

pp. 3137-3146 ◽

Cited By ~ 25

Author(s):

Jeffrey D. Smith ◽

Malika Kumarasiri ◽

Weilie Zhang ◽

Dusan Hesek ◽

Mijoon Lee ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Active Site ◽

Binding Protein ◽

Main Function ◽

Penicillin Binding Protein ◽

Active Site Residues ◽

Content Type ◽

A Cell ◽

Dynamics Simulations

ABSTRACTPenicillin-binding protein 5 (PBP5) is one of the most abundant PBPs inPseudomonas aeruginosa. Although its main function is that of a cell walldd-carboxypeptidase, it possesses sufficient β-lactamase activity to contribute to the ability ofP. aeruginosato resist the antibiotic activity of the β-lactams. The study of these dual activities is important for understanding the mechanisms of antibiotic resistance byP. aeruginosa, an important human pathogen, and to the understanding of the evolution of β-lactamase activity from the PBP enzymes. We purified a soluble version ofP. aeruginosaPBP5 (designated Pa sPBP5) by deletion of its C-terminal membrane anchor. Underin vitroconditions, Pa sPBP5 demonstrates bothdd-carboxypeptidase and expanded-spectrum β-lactamase activities. Its crystal structure at a 2.05-Å resolution shows features closely resembling those of the class A β-lactamases, including a shortened loop spanning residues 74 to 78 near the active site and with respect to the conformations adopted by two active-site residues, Ser101 and Lys203. These features are absent in the related PBP5 ofEscherichia coli. A comparison of the two Pa sPBP5 monomers in the asymmetric unit, together with molecular dynamics simulations, revealed an active-site flexibility that may explain its carbapenemase activity, a function that is absent in theE. coliPBP5 enzyme. Our functional and structural characterizations underscore the versatility of this PBP5 in contributing to the β-lactam resistance ofP. aeruginosawhile highlighting how broader β-lactamase activity may be encoded in the structural folds shared by the PBP and serine β-lactamase classes.

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Exploiting correlated molecular-dynamics networks to counteract enzyme activity–stability trade-off

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1812204115 ◽

2018 ◽

Vol 115 (52) ◽

pp. E12192-E12200 ◽

Cited By ~ 13

Author(s):

Haoran Yu ◽

Paul A. Dalby

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulations ◽

Active Site ◽

Dynamic Networks ◽

Aromatic Aldehydes ◽

Active Site Residues ◽

Trade Off ◽

Highly Correlated ◽

Dynamics Simulations ◽

The Impact

The directed evolution of enzymes for improved activity or substrate specificity commonly leads to a trade-off in stability. We have identified an activity–stability trade-off and a loss in unfolding cooperativity for a variant (3M) of Escherichia coli transketolase (TK) engineered to accept aromatic substrates. Molecular dynamics simulations of 3M revealed increased flexibility in several interconnected active-site regions that also form part of the dimer interface. Mutating the newly flexible active-site residues to regain stability risked losing the new activity. We hypothesized that stabilizing mutations could be targeted to residues outside of the active site, whose dynamics were correlated with the newly flexible active-site residues. We previously stabilized WT TK by targeting mutations to highly flexible regions. These regions were much less flexible in 3M and would not have been selected a priori as targets using the same strategy based on flexibility alone. However, their dynamics were highly correlated with the newly flexible active-site regions of 3M. Introducing the previous mutations into 3M reestablished the WT level of stability and unfolding cooperativity, giving a 10.8-fold improved half-life at 55 °C, and increased midpoint and aggregation onset temperatures by 3 °C and 4.3 °C, respectively. Even the activity toward aromatic aldehydes increased up to threefold. Molecular dynamics simulations confirmed that the mutations rigidified the active-site via the correlated network. This work provides insights into the impact of rigidifying mutations within highly correlated dynamic networks that could also be useful for developing improved computational protein engineering strategies.

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Growth Retardation, Reduced Invasiveness, and Impaired Colistin-Mediated Cell Death Associated with Colistin Resistance Development in Acinetobacter baumannii

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01439-13 ◽

2013 ◽

Vol 58 (2) ◽

pp. 828-832 ◽

Cited By ~ 54

Author(s):

Spyros Pournaras ◽

Aggeliki Poulou ◽

Konstantina Dafopoulou ◽

Yassine Nait Chabane ◽

Ioulia Kristo ◽

...

Keyword(s):

Cell Death ◽

Acinetobacter Baumannii ◽

Biofilm Formation ◽

Bloodstream Infections ◽

Single Amino Acid ◽

Colistin Resistance ◽

Resistance Development ◽

Content Type ◽

Alkyl Hydroperoxide ◽

Severe Soft Tissue

ABSTRACTTwo colistin-susceptible/colistin-resistant (Cols/Colr) pairs ofAcinetobacter baumanniistrains assigned to international clone 2, which is prevalent worldwide, were sequentially recovered from two patients after prolonged colistin administration. Compared with the respective Colsisolates (Ab248 and Ab299, both having a colistin MIC of 0.5 μg/ml), both Colrisolates (Ab249 and Ab347, with colistin MICs of 128 and 32 μg/ml, respectively) significantly overexpressedpmrCABgenes, had single-amino-acid shifts in the PmrB protein, and exhibited significantly slower growth. The Colrisolate Ab347, tested by proteomic analysis in comparison with its Colscounterpart Ab299, underexpressed the proteins CsuA/B and C from thecsuoperon (which is necessary for biofilm formation). This isolate also underexpressed aconitase B and different enzymes involved in the oxidative stress response (KatE catalase, superoxide dismutase, and alkyl hydroperoxide reductase), suggesting a reduced response to reactive oxygen species (ROS) and, consequently, impaired colistin-mediated cell death through hydroxyl radical production. Colsisolates that were indistinguishable by macrorestriction analysis from Ab299 caused six sequential bloodstream infections, and isolates indistinguishable from Ab248 caused severe soft tissue infection, while Colrisolates indistinguishable from Ab347 and Ab249 were mainly colonizers. In particular, a Colsisolate identical to Ab299 was still invading the bloodstream 90 days after the colonization of this patient by Colrisolates. These observations indicate considerably lower invasiveness ofA. baumanniiclinical isolates following the development of colistin resistance.

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Single Amino Acid Substitutions in HXT2.4 from Scheffersomyces stipitis Lead to Improved Cellobiose Fermentation by Engineered Saccharomyces cerevisiae

Applied and Environmental Microbiology ◽

10.1128/aem.03253-12 ◽

2012 ◽

Vol 79 (5) ◽

pp. 1500-1507 ◽

Cited By ~ 25

Author(s):

Suk-Jin Ha ◽

Heejin Kim ◽

Yuping Lin ◽

Myoung-Uoon Jang ◽

Jonathan M. Galazka ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Single Amino Acid ◽

Kinetic Properties ◽

Wild Type ◽

Scheffersomyces Stipitis ◽

Content Type ◽

Charged Amino Acids ◽

Cellodextrin Transporter ◽

Engineered Strain

ABSTRACTSaccharomyces cerevisiaecannot utilize cellobiose, but this yeast can be engineered to ferment cellobiose by introducing both cellodextrin transporter (cdt-1) and intracellular β-glucosidase (gh1-1) genes fromNeurospora crassa. Here, we report that an engineeredS. cerevisiaestrain expressing the putative hexose transporter geneHXT2.4fromScheffersomyces stipitisandgh1-1can also ferment cellobiose. This result suggests that HXT2.4p may function as a cellobiose transporter whenHXT2.4is overexpressed inS. cerevisiae. However, cellobiose fermentation by the engineered strain expressingHXT2.4andgh1-1was much slower and less efficient than that by an engineered strain that initially expressedcdt-1andgh1-1. The rate of cellobiose fermentation by theHXT2.4-expressing strain increased drastically after serial subcultures on cellobiose. Sequencing and retransformation of the isolated plasmids from a single colony of the fast cellobiose-fermenting culture led to the identification of a mutation (A291D) in HXT2.4 that is responsible for improved cellobiose fermentation by the evolvedS. cerevisiaestrain. Substitutions for alanine (A291) of negatively charged amino acids (A291E and A291D) or positively charged amino acids (A291K and A291R) significantly improved cellobiose fermentation. The mutant HXT2.4(A291D) exhibited 1.5-fold higherKmand 4-fold higherVmaxvalues than those from wild-type HXT2.4, whereas the expression levels were the same. These results suggest that the kinetic properties of wild-type HXT2.4 expressed inS. cerevisiaeare suboptimal, and mutations of A291 into bulky charged amino acids might transform HXT2.4p into an efficient transporter, enabling rapid cellobiose fermentation by engineeredS. cerevisiaestrains.

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In Vitro Activity of Sulbactam-Durlobactam against Acinetobacter baumannii-calcoaceticus Complex Isolates Collected Globally in 2016 and 2017

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02534-19 ◽

2020 ◽

Vol 64 (4) ◽

Cited By ~ 4

Author(s):

Sarah M. McLeod ◽

Samir H. Moussa ◽

Meredith A. Hackel ◽

Alita A. Miller

Keyword(s):

Acinetobacter Baumannii ◽

Antibacterial Activities ◽

Multidrug Resistant ◽

Clinical Isolates ◽

Single Amino Acid ◽

Content Type ◽

Level Of Activity ◽

Severe Infections ◽

Sources Of Infection

ABSTRACT Acinetobacter baumannii-calcoaceticus complex (ABC) organisms cause severe infections that are difficult to treat due to preexisting antibiotic resistance. Sulbactam-durlobactam (formerly sulbactam-ETX2514) (SUL-DUR) is a β-lactam–β-lactamase inhibitor combination antibiotic designed to treat serious infections caused by ABC organisms, including multidrug-resistant (MDR) strains. The in vitro antibacterial activities of SUL-DUR and comparator agents were determined by broth microdilution against 1,722 clinical isolates of ABC organisms collected in 2016 and 2017 from 31 countries across Asia/South Pacific, Europe, Latin America, the Middle East, and North America. Over 50% of these isolates were resistant to carbapenems. Against this collection of global isolates, SUL-DUR had a MIC50/MIC90 of 1/2 μg/ml compared to a MIC50/MIC90 of 8/64 μg/ml for sulbactam alone. This level of activity was found to be consistent across organisms, regions, sources of infection, and subsets of resistance phenotypes, including MDR and extensively drug-resistant isolates. The SUL-DUR activity was superior to those of the tested comparators, with only colistin having similar potency. Whole-genome sequencing of the 39 isolates (2.3%) with a SUL-DUR MIC of >4 μg/ml revealed that these strains encoded either the metallo-β-lactamase NDM-1, which durlobactam does not inhibit, or single amino acid substitutions near the active site of penicillin binding protein 3 (PBP3), the primary target of sulbactam. In summary, SUL-DUR demonstrated potent antibacterial activity against recent, geographically diverse clinical isolates of ABC organisms, including MDR isolates.

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