Cathepsin D primes caspase-8 activation by multiple intra-chain proteolysis

During the resolution of inflammatory responses, neutrophils rapidly undergo apoptosis. A direct and fast activation of caspase-8 by cathepsin D was shown to be crucial in the initial steps of neutrophil apoptosis. Nevertheless, the activation mechanism of caspase-8 remains unclear. Here, by using site-specific mutants of caspase-8, we show that both cathepsin D-mediated proteolysis and homodimerization of caspase-8 are necessary to generate an active caspase-8. At acidic pH, cathepsin D specifically cleaved caspase-8 but not the initiator caspase-9 or -10 and significantly increased caspase-8 activity in dimerizing conditions. These events were completely abolished by pepstatin A, a pharmacological inhibitor of cathepsin D. The cathepsin D intra-chain proteolysis greatly stabilized the active site of caspase-8. Moreover, the main caspase-8 fragment generated by cathepsin D cleavage could be affinity-labeled with the active site probe biotin-VAD-fluoromethyl ketone, suggesting that this fragment is enzymatically active. Importantly, in an in vitro cell-free assay, the addition of recombinant human caspase-8 protein, pre-cleaved by cathepsin D, was followed by caspase-3 activation. Our data therefore indicate that cathepsin D is able to initiate the caspase cascade by direct activation of caspase-8. As cathepsin D is ubiquitously expressed, this may represent a general mechanism to induce apoptosis in a variety of immune and nonimmune cells.

Crosslinking Studies To Probe Substrate Binding By Soybean Lipoxygenase-1

Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of polyunsaturated fatty acids into conjugated diene hydroperoxides. The three dimensional structure of SBLO-1 is known, but it is not certain how substrates bind. One hypothesis involves the transient separation of helix-2 and helix-11 located on the exterior of the molecule in front of the active site iron. A second hypothesis involves a conformational change in the side chains of residues leucine 541 and threonine 259. To test these hypotheses, site directed mutagenesis was used to create a cysteine mutation on each helix, which could allow for the formation of a disulfide linkage. Disulfide formation between the two cysteines in the T259C,S545C mutant was found to be unfavorable, but later shown to be present at higher pH values using SDS-PAGE. Treatment of the T259C,S545C with the crosslinker 2,3-dibromomaleimide (DBM) resulted in a 50% reduction in catalytic activity. No loss of activity was observed when the single mutant, S545C, or the wild type was treated with DBM. Single mutants T259C and L541C both showed approximately 20% reduction in the rate after addition of DBM. Double mutants T259C,L541C and S263C,S545C showed approximately 30% reduction in the rate after addition of DBM. Single mutants T259C and L541C showed an increase in activity after incubation with NEM. Double mutants T259C,S545C and T259C,L541C showed an increase in activity after incubation with NEM. The S263C,S545C double mutant showed a slight decrease in activity in the presence of NEM. It is unclear how the NEM and DBM are interacting with the molecule, but this can easily be determined through mass spectrometry experiments.

Purification and Preliminary Characterization of the TM0727 Protein from Thermotoga maritima

The TM0727 gene of Thermotoga maritima is responsible for encoding what has been reported to be a modulator of DNA gyrase (pmbA). Although the function of pmbA is still unknown, it is believedto be involved in cell division, carbon storage regulation, and the synthesis of the antibiotic peptide microcin B17. It is suggested that it serves together with tldD, a known zinc dependent protease, tomodulate DNA gyrase. TM0727 is believed to be a zinc dependent protease that binds zinc in the central active site of the molecule, located between two equivalent monomeric units. However, thecrystal structure determined by Wilson et al. (2005) did not contain zinc. It therefore remains to be seen if TM0727 requires zinc for activity, or regulation, and if the protein is indeed a protease. To begin studying this protein, the gene was expressed in BL21(DE3) pLysS cells and the induction time was optimized. Using affinity and ion exchange chromatography, the protein has been successfully purified. The purification procedure can be replicated to obtain sufficient protein for characterization. Purification results show that the protein loses stability after 24 hours and remains stable under an imidazole-free lysis workup. Preliminary characterization of TM0727 has focused on understanding the protein’s structuralproperties through tryptophan fluorescence anisotropy measurements. The four tryptophan residues located within the TM0727 dimer fluoresce at different maximum wavelengths and with differentintensities upon excitation with 295nm light. These emission properties are highly sensitive to the environment (solvent, surrounding residues) of each tryptophan residue. The low number oftryptophans allows for a specific monitoring of the protein’s structure as it denatures. As more denaturant is added to the protein, its tryptophan environments have clearly altered. This is indicative of unfolding and increased solvent exposure of the protein. This unfolding has been confirmed with the addition of a fluorescent quencher. Additionally, fluorescence anisotropy measurements have been carried out on the protein to gain a preliminary understanding of the rotational dynamics of the tryptophan residues. These experiments excite the tryptophan residues within the sample using a polarized light source. Polarized emission is then detected, the degree of which depends on the rotational dynamics and local environment of the tryptophan residues. The protein was denatured and the changes in emission were recorded to detect these structural changes. Results have shown a large change in quaternary structure, consistent with a dimer to monomer transition, occurs at 1.5M Guandidine HCl. There has also been an examination of the crystal structure for the location of a potential active site. The inner cavity of the protein was inspected visually to locate a potential location for a catalytic triad, specifically the amino acids found in the active sites of serine, cyteine, and aspartateproteases. It was found that a potential aspartic protease active site may be located between the Asparate286 and Aspartate287 residues. Further investigation is warranted to test this remotepossibility.

Enzymatic Activity of Soybean Lipoxygenase-1 on Linoleoyl-Derivatized Agarose

Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of linoleic acid to form 13(S) and 9(R) hydroperoxides. The manner in which substrates bind to the lipoxygenase family of enzymes is not known. It is believed fatty acid substrates may bind either with the aliphatic end first or with the carboxylate group facing the interior of the protein. This thesis tested a potential methyl-end first substrate binding mechanism by studying the activity of SBLO-1 to oxygenate immobilized linoleoyl residues attached to an insoluble polymer. Linoleic acid was attached to aminohexyl agarose in the presence of N-(3- dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) and Nhydroxysuccinimide (NHS). The concentration of the covalently attached residues was facilitated by enriching linoleic acid with a small amount of the radioactive 14C-isotope. Functionalization yields of 3% available primary amines on the resin were obtained. Enzymatic oxygenation of the linoleoyl-residues was verified using the ferrous oxidation in xylenol orange (FOX) assay. Approximately 30% of the attached linoleoyl moieties were converted to hydroperoxides in the presence of SBLO-1. A disulfide-containing cleavable linker, cystamine, was used as part of an improved method to isolate the product in a facile manner. Cystamine was attached to NHS-activated agarose with approximately 5% overall functionalization yield of available functional groups. 14C-linoleic acid was successfully covalently linked to the cystamine moieties in the presence of EDC and NHS. The FOX assay verified the enzymatic oxygenation of the linoleoyl residues attached to cystamine-derivatized agarose. The isolation of the peroxide product was attempted in a series of extractions in organic solvents. The product was analyzed using GC/MS which did not show a new peak indicative of product. Further work is needed to successfully analyze the stereoand regiochemistry of the oxygenated product. The presence of the peroxides in this study indicated the linoleoyl residues behave as substrates of SBLO-1. It is unknown how bulky substrates bind to the active site; however, it is difficult to rationalize a carboxylate group-first binding mode. Discovery of the 13(S)-hydroperoxide product on the linoleoyl-agarose would support the claim of a potential methyl-end first binding mechanism.

Synthesis Of Linoleate Analogs With Longer Alkyl Termini To Test For Proposed "Tail-First" Binding In SBLO-1

Lipoxygenases are nonheme-iron proteins that catalyze the oxygenation of polyunsaturated fatty acids to give conjugated diene hydroperoxides. For example, soybean lipoxygenase-1 (SBLO-1) converts linoleate into 13-(S)-hydroperoxy-9(Z),11(E)-octadecadienoate (13(S)-HPOD). Although the crystal structure of SBLO-1 has been determined, it is still unclear how the substrate binds at the active site. This absence of knowledge makes it difficult to understand the role of the enzyme during catalysis of the reaction. We hypothesize that SBLO-1 binds linoleate ¿tail-first¿, so that the methyl terminus is within a hydrophobic pocket deep within the enzyme. It is believed that the hydrophobic residue phenylalanine-557 at this site has stabilizing interactions with the terminal methyl group on linoleate. To test this hypothesis, we have developed a synthetic pathway that will yield linoleate analogs with longer fatty acid chains by 1 and 2 more carbons at the alkyl terminus. These substrates will be analyzed through kinetic assays done in combination with wild type SBLO-1 and mutants in which we have replaced phenylalanine-557 with valine.

Structure-Function Relations in the PI-PLC/GDPD Enzyme Superfamily

Phosphatidylinositol-specific phospholipases C (PI-PLC) are known to participate in many eukaryotic signal transduction pathways and act as virulence factors in lower organisms. Glycerophosphoryl diester phosphodiesterase (GDPD) enzymes are involved in phosphate homeostasis and phospholipid catabolism for energy production. Streptomyces antibioticus phosphatidylinositol-specific phospholipase C (SaPLC1) is a 38 kDa enzyme that displays characteristics of both enzyme superfamilies, representing an evolutionary link between these divergent enzyme classes. SaPLC1 also boasts a unique catalytic mechanism that involves a trans 1,6-cyclic inositol phosphate intermediate instead of the typical cis 1,2-cyclic inositol phosphate. The mechanism by which this occurs is still unclear. To attack this problem, we established a wide mutagenesis scan of the active site and measured activities of alanine mutants. A chemical rescue assay was developed to verify that the activity loss was due to the removal of the functional role of the mutated residue. 31P-NMR was employed in characterizing and quantifying intermediates in mutants that slowed the reaction sufficiently. We found that the H37A and H76A mutations support the hypothesis that these structurally conserved residues are also conserved in terms of their catalytic roles. H37 was found to be the general base (GB), while H76 plays the role of general acid (GA). K131 was identified as a semi-conserved key positive charge donor found at the entrance of the active site. By elucidating the SaPLC1 mechanism in relation to its active site architecture, we have increased our understanding of the structure-function relations that support catalysis in the PI-PLC/GDPD superfamily. These findings provide groundwork for in vivo studies of SaPLC1 function and its possible role in novel signaling or metabolism in Streptomyces.

Luminal starch substrate "brake" on maltase-glucoamylase activity is located within the glucoamylase subunit

The detailed mechanistic aspects for the final starch digestion process leading to effective alpha-glucogenesis by the 2 mucosal alpha-glucosidases, human sucrase-isomaltase complex (SI) and human maltase-glucoamylase (MGAM), are poorly understood. This is due to the structural complexity and vast variety of starches and their intermediate digestion products, the poorly understood enzyme-substrate interactions occurring during the digestive process, and the limited knowledge of the structure-function properties of SI and MGAM. Here we analyzed the basic catalytic properties of the N-terminal subunit of MGAM (ntMGAM) on the hydrolysis of glucan substrates and compared it with those of human native MGAM isolated by immunochemical methods. In relation to native MGAM, ntMGAM displayed slower activity against maltose to maltopentose (G5) series glucose oligomers, as well as maltodextrins and alpha-limit dextrins, and failed to show the strong substrate inhibitory "brake" effect caused by maltotriose, maltotetrose, and G5 on the native enzyme. In addition, the inhibitory constant for acarbose was 2 orders of magnitude higher for ntMGAM than for native MGAM, suggesting lower affinity and/or fewer binding configurations of the active site in the recombinant enzyme. The results strongly suggested that the C-terminal subunit of MGAM has a greater catalytic efficiency due to a higher affinity for glucan substrates and larger number of binding configurations to its active site. Our results show for the first time, to our knowledge, that the C-terminal subunit of MGAM is responsible for the MGAM peptide's "glucoamylase" activity and is the location of the substrate inhibitory brake. In contrast, the membrane-bound ntMGAM subunit contains the poorly inhibitable "maltase" activity of the internally duplicated enzyme.

Human intestinal maltase-glucoamylase: crystal structure of the N-terminal catalytic subunit and basis of inhibition and substrate specificity

Human maltase-glucoamylase (MGAM) is one of the two enzymes responsible for catalyzing the last glucose-releasing step in starch digestion. MGAM is anchored to the small-intestinal brush-border epithelial cells and contains two homologous glycosyl hydrolase family 31 catalytic subunits: an N-terminal subunit (NtMGAM) found near the membrane-bound end and a C-terminal luminal subunit (CtMGAM). In this study, we report the crystal structure of the human NtMGAM subunit in its apo form (to 2.0 A) and in complex with acarbose (to 1.9 A). Structural analysis of the NtMGAM-acarbose complex reveals that acarbose is bound to the NtMGAM active site primarily through side-chain interactions with its acarvosine unit, and almost no interactions are made with its glycone rings. These observations, along with results from kinetic studies, suggest that the NtMGAM active site contains two primary sugar subsites and that NtMGAM and CtMGAM differ in their substrate specificities despite their structural relationship. Additional sequence analysis of the CtMGAM subunit suggests several features that could explain the higher affinity of the CtMGAM subunit for longer maltose oligosaccharides. The results provide a structural basis for the complementary roles of these glycosyl hydrolase family 31 subunits in the bioprocessing of complex starch structures into glucose.

Characterizations of partially reduced polyoxometalate catalysts using ammonia adsorption microcalorimetry and methanol oxidation studies / by Suresh Babu Bommineni.

Phosphomolybdic acid (H3PMo12O40) along with niobium,pyridine and niobium exchanged phosphomolybdic acid catalysts were prepared. Ammonia adsorption microcalorimetry and methanol oxidation studies were carried out to investigate the acid sites strength acid/base/redox properties of each catalyst. The addition of niobium, pyridine or both increased the ammonia heat of adsorption and the total uptake. The catalyst with both niobium and pyridine demonstrated the largest number of strong sites. For the parent H3PMo12O40 catalyst, methanol oxidation favors the redox product. Incorporation of niobium results in similar selectivity to redox products but also results in no catalyst deactivation. Incorporation of pyridine instead changes to the selectivity to favor the acidic product. Finally, the inclusion of both niobium and pyridine results in strong selectivity to the acidic product while also showing no catalyst deactivation. Thus the presence of pyridine appears to enhance the acid property of the catalyst while niobium appears to stabilize the active site.

Equine cytochrome P450 2B6--genomic identification, expression and functional characterization with ketamine

Ketamine is an anesthetic and analgesic regularly used in veterinary patients. As ketamine is almost always administered in combination with other drugs, interactions between ketamine and other drugs bear the risk of either adverse effects or diminished efficacy. Since cytochrome P450 enzymes (CYPs) play a pivotal role in the phase I metabolism of the majority of all marketed drugs, drug-drug interactions often occur at the active site of these enzymes. CYPs have been thoroughly examined in humans and laboratory animals, but little is known about equine CYPs. The characterization of equine CYPs is essential for a better understanding of drug metabolism in horses. We report annotation, cloning and heterologous expression of the equine CYP2B6 in V79 Chinese hamster fibroblasts. After computational annotation of all CYP2B genes, the coding sequence (CDS) of equine CYP2B6 was amplified by RT-PCR from horse liver total RNA and revealed an amino acid sequence identity of 77% and a similarity of 93.7% to its human ortholog. A non-synonymous variant c.226G>A in exon 2 of the equine CYP2B6 was detected in 97 horses. The mutant A-allele showed an allele frequency of 82%. Two further variants in exon 3 were detected in one and two horses of this group, respectively. Transfected V79 cells were incubated with racemic ketamine and norketamine as probe substrates to determine metabolic activity. The recombinant equine CYP2B6 N-demethylated ketamine to norketamine and produced metabolites of norketamine, such as hydroxylated norketamines and 5,6-dehydronorketamine. V(max) for S-/and R-norketamine formation was 0.49 and 0.45nmol/h/mg cellular protein and K(m) was 3.41 and 2.66μM, respectively. The N-demethylation of S-/R-ketamine was inhibited concentration-dependently with clopidogrel showing an IC(50) of 5.63 and 6.26μM, respectively. The functional importance of the recorded genetic variants remains to be explored. Equine CYP2B6 was determined to be a CYP enzyme involved in ketamine and norketamine metabolism, thus confirming results from inhibition studies with horse liver microsomes. Clopidogrel seems to be a feasible inhibitor for equine CYP2B6. The specificity still needs to be established with other single equine CYPs. Heterologous expression of single equine CYP enzymes opens new possibilities to substantially improve the understanding of drug metabolism and drug interactions in horses.

THE DOMAINS OF THE CATALYTIC SUBUNIT OF THE EUKARYOTIC RNA DEGRADING EXOSOME, RRP44P, HAVE DISTINCT FUNCTIONS

The exosome is a 3’ to 5’ exoribonuclease complex that consists of ten essential subunits. In the cytoplasm, the exosome degrades mRNA in a general mRNA turnover pathway and in several mRNA surveillance pathways. In the nucleus, the exosome processes RNA precursors to form small, stable, mature RNA species, including rRNA, snRNA, and snoRNA. In addition to processing these RNAs, the nuclear exosome is also involved in degrading aberrantly processed forms of these RNAs, and others, including mRNA. The 3’ to 5’ exoribonuclease activity of the exosome is contributed by the RNB domain of the only catalytically active subunit, Rrp44p, a member of the RNase II family of enzymes. In addition to the RNB domain, Rrp44p consists of three putative RNA binding domains and has an uncharacterized N-terminus, which includes a CR3 region and PIN domain. In an effort to characterize the cellular functions of the domains of Rrp44p, this study identified a second nuclease active site in the PIN domain. Specifically, the PIN domain exhibits endoribonuclease activity in vitro and is essential for exosome function. Further analysis of the nuclease activities of Rrp44p indicate a role for the exoribonuclease activity of Rrp44p in the cytoplasmic and nuclear exosome. This work has also characterized the CR3 region of Rrp44p, a region that has not yet been characterized in any other protein. This region is needed for the majority, if not all, of the cytoplasmic exosome functions as well as for interaction with the exosome. The CR3 region, along with a histidine residue in the N-terminus of Rrp44p, may coordinate a zinc atom. Preliminary evidence supports a role for this coordination in exosome function. Further investigation, however, is needed to determine the molecular dependence of the exosome on the CR3 region of Rrp44p. Despite its initial discovery thirteen years ago, the essential function of Rrp44p, and the exosome, is not yet known. The studies presented here, however, indicate that the essential function of Rrp44p and the exosome is in the nucleus and depends on the nuclease activities of Rrp44p.

Crystal structure of the alpha(6)beta(6) holoenzyme of propionyl-coenzyme A carboxylase.

Propionyl-coenzyme A carboxylase (PCC), a mitochondrial biotin-dependent enzyme, is essential for the catabolism of the amino acids Thr, Val, Ile and Met, cholesterol and fatty acids with an odd number of carbon atoms. Deficiencies in PCC activity in humans are linked to the disease propionic acidaemia, an autosomal recessive disorder that can be fatal in infants. The holoenzyme of PCC is an alpha(6)beta(6) dodecamer, with a molecular mass of 750 kDa. The alpha-subunit contains the biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) domains, whereas the beta-subunit supplies the carboxyltransferase (CT) activity. Here we report the crystal structure at 3.2-A resolution of a bacterial PCC alpha(6)beta(6) holoenzyme as well as cryo-electron microscopy (cryo-EM) reconstruction at 15-A resolution demonstrating a similar structure for human PCC. The structure defines the overall architecture of PCC and reveals unexpectedly that the alpha-subunits are arranged as monomers in the holoenzyme, decorating a central beta(6) hexamer. A hitherto unrecognized domain in the alpha-subunit, formed by residues between the BC and BCCP domains, is crucial for interactions with the beta-subunit. We have named it the BT domain. The structure reveals for the first time the relative positions of the BC and CT active sites in the holoenzyme. They are separated by approximately 55 A, indicating that the entire BCCP domain must translocate during catalysis. The BCCP domain is located in the active site of the beta-subunit in the current structure, providing insight for its involvement in the CT reaction. The structural information establishes a molecular basis for understanding the large collection of disease-causing mutations in PCC and is relevant for the holoenzymes of other biotin-dependent carboxylases, including 3-methylcrotonyl-CoA carboxylase (MCC) and eukaryotic acetyl-CoA carboxylase (ACC).

Aminoacyl-tRNA synthetases: Probing the molecular basis of catalysis and discrimination

Aminoacyl-tRNA synthetases (RSs) are responsible for the essential connection of amino acids with trinucleotide sequences of tRNA's. The RS family constitutes two structurally dissimilar groups of proteins, class I and class II. Methionyl-tRNA synthetase (MetRS) and isoleucyl-tRNA synthetase (IleRS), both members of class I, were the focus of this work. Both enzymes are zinc-containing proteins; show a high degree of amino acid specificity; and edit activated noncognate amino acids, thereby ensuring the fidelity of the genetic code. The goals of this work were to further delineate the molecular basis of catalysis and discrimination in these enzymes by mapping active site geometries using high-resolution nuclear magnetic resonance spectroscopy (NMR).^ Internuclear distances obtained from transferred nuclear Overhauser effects were used to define the conformations of Mg($\alpha$,$\beta$-methylene)ATP bound to E. coli MetRS and E. coli IleRS in multiple complexes. Identical conformations were found for the bound ATP. Thus, the predicted structural homology between IleRS and MetRS is supported by consensus enzyme-bound nucleotide conformations. The conformation of the bound nucleotide is not sensitive to occupation of the amino acid site of MetRS or IleRS. Therefore, conformational changes known to occur in the synthetases upon ligand binding appear not to alter the bound conformation of the adenosine portion of the nucleotide. Nuclear Overhauser effects on the substrate amino acid L-selenomethionine were also used to evaluate the enzyme-bound conformation of the cognate amino acid. The amino acid assumes a conformation which is consistent with a proposed editing mechanism.^ The E. coli MetRS was shown to catalyze amino acid $\alpha$-proton exchange in the presence of deuterium oxide of all cognate amino acids. It is proposed that the enzyme-bound zinc coordinates the $\alpha$-carboxylate of the amino acid, rendering the $\alpha$-proton more acidic. An enzymic base is responsible for exchange of the $\alpha$-proton. This proposal suggests that the enzyme-bound zinc may have a role in amino acid discrimination in MetRS. However, the role of this exchange reaction in catalysis remains unknown. ^

Experimental dissection and characterization of the functional domains in cardiac troponin C

Contraction of vertebrate cardiac muscle is regulated by the binding of Ca$\sp{2+}$ to the troponin C (cTnC) subunit of the troponin complex. In this study, we have used site-directed mutagenesis and a variety of assay techniques to explore the functional roles of regions in cTnC, including Ca$\sp{2+}$/Mg$\sp{2+}$-binding sites III and IV, the functionally inactive site I, the N-terminal helix, the N-terminal hydrophobic pocket and the two cysteine residues with regard to their ability to form disulfide bonds. Conversion of the first Ca$\sp{2+}$ ligand from Asp to Ala inactivated sites III and IV and decreased the apparent affinity of cTnC for the thin filament. Conversion of the second ligand from Asn to Ala also inactivated these sites in the free protein but Ca$\sp{2+}$-binding was recovered upon association with troponin I and troponin T. The Ca$\sp{2+}$-concentrations required for tight thin filament-binding by proteins containing second-ligand mutations were significantly greater than that required for the wild-type protein. Mutation of site I such that the primary sequence was that of an active site with the first Ca$\sp{2+}$ ligand changed from Asp to Ala resulted in a 70% decrease in maximal Ca$\sp{2\sp+}$ dependent ATPase activity in both cardiac and fast skeletal myofibrils. Thus, the primary sequence of the inactive site I in cTnC is functionally important. Major changes in the sequence of the N-terminus had little effect on the ability of cTnC to recover maximal activity but deletion of the first nine residues resulted in a 60 to 80% decrease in maximal activity with only a minor decrease in the pCa$\sb{50}$ of activation, suggesting that the N-terminal helix must be present but that a specific sequence is not required. The formation of an inter- or intramolecular disulfide bonds caused the exposure of hydrophobic surfaces on cTnC and rendered the protein Ca$\sp{2+}$ independent. Finally, elution patterns from a hydrophobic interactions column suggest that cTnC undergoes a significant change in hydrophobicity upon Ca$\sp{2+}$ binding, the majority of which is caused by site II. These latter data show an interesting correlation between exposure of hydrophobic surfaces on and activation of cTnC. Overall, these results represent significant progress toward the elucidation of the functional roles of a variety of structural regions in cTnC. ^

In vivo Evolution of CMY-2 to CMY-33 β-Lactamase in Escherichia coli ST131: Characterization of an Acquired Extended-Spectrum AmpC (ESAC) Conferring Resistance to Cefepime.

Cefepime is frequently prescribed to treat infections caused by AmpC-producing Gram-negative bacteria. CMY-2 is the most common plasmid-mediated AmpC (pAmpC) β-lactamase. Unfortunately, CMY variants conferring enhanced cefepime resistance are reported. Here, we describe the evolution of CMY-2 to an extended-spectrum AmpC (ESAC) in clonally identical E. coli isolates obtained from a patient. The CMY-2-producing E. coli (CMY-2-Ec) was isolated from a wound. Thirty days later, one CMY-33-producing E. coli (CMY-33-Ec) was detected in bronchoalveolar lavage. Two weeks before the isolation of CMY-33-Ec, the patient received cefepime.CMY-33-Ec and CMY-2-Ec were identical by rep-PCR, being of hyperepidemic ST131, but showed different β-lactam MICs (e.g., cefepime 16 vs. ≤0.5 μg/ml). Identical CMY-2-Ec isolates were also found in a rectal swab. CMY-33 differs from CMY-2 by a Leu293-Ala294 deletion. Expressed in E. coli DH10B, both CMYs conferred resistance to ceftazidime (≥256 μg/ml), but cefepime MICs were higher for CMY-33 than CMY-2 (8 vs. 0.25 μg/ml). The kcat/Km or kinact/KI (μM(-1) s(-1)) indicated that CMY-33 possesses an ESBL-like spectrum compared to CMY-2 (cefoxitin: 0.2 vs. 0.4; ceftazidime: 0.2 vs. not measurable; cefepime: 0.2 vs. not measurable; tazobactam 0.0018 vs. 0.0009). Using molecular modeling, we show that a widened active site (∼4 Å shift) may play a significant role in enhancing cefepime hydrolysis. This is the first in vivo demonstration of a pAmpC that under cephalosporin treatment expands its substrate spectrum resembling an ESBL. The prevalence of CMY-2-Ec isolates is rapidly increasing worldwide, therefore awareness that cefepime treatment may select for resistant isolates is critical.