Opening of a cryptic pocket in ß-lactamase increases penicillinase activity.

Understanding the functional role of protein-excited states has important implications in protein design and drug discovery. However, because these states are difficult to find and study, it is still unclear if excited states simply result from thermal fluctuations and generally detract from function or if these states can actually enhance protein function. To investigate this question, we consider excited states in ß-lactamases and particularly a subset of states containing a cryptic pocket which forms under the Ω-loop. Given the known importance of the Ω-loop and the presence of this pocket in at least two homologs, we hypothesized that these excited states enhance enzyme activity. Using thiol-labeling assays to probe Ω-loop pocket dynamics and kinetic assays to probe activity, we find that while this pocket is not completely conserved across ß-lactamase homologs, those with the Ω-loop pocket have a higher activity against the substrate benzylpenicillin. We also find that this is true for TEM ß-lactamase variants with greater open Ω-loop pocket populations. We further investigate the open population using a combination of NMR chemical exchange saturation transfer experiments and molecular dynamics simulations. To test our understanding of the Ω-loop pocket's functional role, we designed mutations to enhance/suppress pocket opening and observed that benzylpenicillin activity is proportional to the probability of pocket opening in our designed variants. The work described here suggests that excited states containing cryptic pockets can be advantageous for function and may be favored by natural selection, increasing the potential utility of such cryptic pockets as drug targets.

Whole-Cell Biotransformation of Penicillin G by a Three-Enzyme Co-expression System with Engineered Deacetoxycephalosporin†C Synthase.

Deacetoxycephalosporin C synthase (DAOCS) catalyzes the transformation of penicillin G to phenylacetyl-7-aminodeacetoxycephalosporanic acid (G-7-ADCA) for which it depends on 2-oxoglutarate (2OG) as co-substrate. However, the low activity of DAOCS and the expense of 2OG restricts its practical applications in the production of G-7-ADCA. Herein, a rational design campaign was performed on a DAOCS from Streptomyces clavuligerus (scDAOCS) in the quest to construct novel expandases. The resulting mutants showed 25∼58 % increase in activity compared to the template. The dominant DAOCS variants were then embedded into a three-enzyme co-expression system, consisting of a catalase and an L-glutamic oxidase for the generation of 2OG, to convert penicillin G to G-7-ADCA in E. coli. The engineered whole-cell enzyme cascade was applied to an up-scaled reaction, exhibiting a yield of G-7-ADCA up to 39.21 mM (14.6 g ⋅ L-1 ) with a conversion of 78.42 mol %. This work highlights the potential of the integrated whole-cell system that may inspire further research on green and efficient production of 7-ADCA.

Vesicular transport and secretion of penicillin G in Penicillium rubens P2-32-T.

The compartmentalization of penicillin G biosynthesis in Penicillium rubens has been extensively studied. However, how this compound is secreted has not been completely elucidated, although its transport could be of the vesicular type. This work was aimed at observing vesicles and penicillin secretion and proposing a hypothetical model for their compartmentalization and secretion. For this purpose, a high-penicillin-producing strain (P. rubens P2-32-T) was compared by transmission electron microscopy (TEM) and atomic force microscopy (AFM) with a null-producing strain (P. rubens npe10) in 24- and 48-h cultures. The results showed multivesicular bodies and secretory vesicles, suggesting that P. rubens transports and secretes penicillin G through vesicular excretion.

Detection of Penicillin G Produced by Penicillium chrysogenum with Raman Microspectroscopy and Multivariate Curve Resolution-Alternating Least-Squares Methods.

Raman microspectroscopy is a minimally invasive technique that can identify molecules without labeling. In this study, we demonstrate the detection of penicillin G inside Penicillium chrysogenum KF425 fungal cells. Raman spectra acquired from the fungal cells had highly overlapped spectroscopic signatures and hence were analyzed with multivariate curve resolution by alternating least-squares (MCR-ALS) to extract the spectra of individual molecular constituents. In addition to detecting spatial distribution of multiple constituents such as proteins and lipids inside the fungal body, we could also observe the subcellular localization of penicillin G. This methodology has the potential to be employed in screening the production of bioactive compounds by microorganisms.

Fiber Mach-Zehnder-interferometer-based liquid crystal biosensor for detecting enzymatic reactions of penicillinase.

A novel, to the best of our knowledge, liquid crystal (LC) biosensor, based on an optical fiber Mach-Zehnder interferometer (MZI), is proposed. The proposed optical fiber MZI consists of two single-mode fibers and a tapered photonic crystal fiber (PCF). The PCF is coated with 4'-pentyl-biphenyl-4-carboxylic acid (PBA)-doped 4-cyano-4'-pentylbiphenyl (5CB). Being a pH-sensitive material, PBA can manipulate LC molecules to different orientations according to their pH values. When the orientation of LC molecules changes with varying pH, the effective refractive index of the cladding modes also is accordingly affected. Enzymatic reactions of penicillinase can release H+, which causes the decrease of the pH. Therefore, the enzymatic reactions of penicillinase can be sensed by monitoring the peak shift in the interference spectrum. The effects of the tapered diameter on the sensitivity of the sensor were experimentally investigated as well.

Crystal Structures of Penicillin-Binding Protein D2 from Listeria monocytogenes and Structural Basis for Antibiotic Specificity.

ß-Lactam antibiotics that inhibit penicillin-binding proteins (PBPs) have been widely used in the treatment of bacterial infections. However, the molecular basis underlying the different inhibitory potencies of ß-lactams against specific PBPs is not fully understood. Here, we present the crystal structures of penicillin-binding protein D2 (PBPD2) from Listeria monocytogenes, a Gram-positive foodborne bacterial pathogen that causes listeriosis in humans. The acylated structures in complex with four antibiotics (penicillin G, ampicillin, cefotaxime, and cefuroxime) revealed that the ß-lactam core structures were recognized by a common set of residues; however, the R1 side chains of each antibiotic participate in different interactions with PBPD2. In addition, the structural complementarities between the side chains of ß-lactams and the enzyme were found to be highly correlated with the relative reactivities of penam or cephem antibiotics against PBPD2. Our study provides the structural basis for the inhibition of PBPD2 by clinically important ß-lactam antibiotics that are commonly used in listeriosis treatment. Our findings imply that the modification of ß-lactam side chains based on structural complementarity could be useful for the development of potent inhibitors against ß-lactam-resistant PBPs.

Whole-Genome Analysis of an Extensively Drug-Resistance Empedobacter falsenii Strain Reveals Distinct Features and the Presence of a Novel Metallo-ß-Lactamase (EBR-2).

The spread of antibiotic resistance is rapidly threatening the effectiveness of antibiotics in the clinical setting. Many infections are being caused by known and unknown pathogenic bacteria that are resistant to many or all antibiotics currently available. Empedobacter falsenii is a nosocomial pathogen that can cause human infections. E. falsenii Wf282 strain was found to be resistant to many antibiotics, including carbapenems and colistin. Whole-genome shotgun sequencing of the strain was performed, and distinct features were identified. A novel metallo-ß-lactamase, named EBR-2, was found, suggesting a potential role of E. falsenii as a reservoir of ß-lactamases and other resistance determinants also found in its genome. The EBR-2 protein showed the highest catalytic efficiency for penicillin G as compared to meropenem and ampicillin and was unable to hydrolyze cefepime. The results described in this work broaden the current understanding of the role of ß-lactamases in the Flavobacteriaceae family and suggest that E. falsenii Wf282 may be a reservoir of these novel resistance determinants.

Reconstitution of TCA cycle with DAOCS to engineer Escherichia coli into an efficient whole cell catalyst of penicillin G.

Many medically useful semisynthetic cephalosporins are derived from 7-aminodeacetoxycephalosporanic acid (7-ADCA), which has been traditionally made by the polluting chemical method. Here, a whole-cell biocatalytic process based on an engineered Escherichia coli strain expressing 2-oxoglutarate-dependent deacetoxycephalosporin C synthase (DAOCS) for converting penicillin G to G-7-ADCA is developed. The major engineering strategy is to reconstitute the tricarboxylic acid (TCA) cycle of E. coli to force the metabolic flux to go through DAOCS catalyzed reaction for 2-oxoglutarate to succinate conversion. Then the glyoxylate bypass was disrupted to eliminate metabolic flux that may circumvent the reconstituted TCA cycle. Additional engineering steps were taken to reduce the degradation of penicillin G and G-7-ADCA in the bioconversion process. These steps include engineering strategies to reduce acetate accumulation in the biocatalytic process and to knock out a host ß-lactamase involved in the degradation of penicillin G and G-7-ADCA. By combining these manipulations in an engineered strain, the yield of G-7-ADCA was increased from 2.50 ± 0.79 mM (0.89 ± 0.28 g/L, 0.07 ± 0.02 g/gDCW) to 29.01 ± 1.27 mM (10.31 ± 0.46 g/L, 0.77 ± 0.03 g/gDCW) with a conversion rate of 29.01 mol%, representing an 11-fold increase compared with the starting strain (2.50 mol%).

Engineering deacetoxycephalosporin C synthase as a catalyst for the bioconversion of penicillins.

7-aminodeacetoxycephalosporanic acid (7-ADCA) is a key intermediate of many clinically useful semisynthetic cephalosporins that were traditionally prepared by processes involving chemical ring expansion of penicillin G. Bioconversion of penicillins to cephalosporins using deacetoxycephalosporin C synthase (DAOCS) is an alternative and environmentally friendly process for 7-ADCA production. Arnold Demain and co-workers pioneered such a process. Later, protein engineering efforts to improve the substrate specificity and catalytic efficiency of DAOCS for penicillins have been made by many groups, and a whole cell process using Escherichia coli for bioconversion of penicillins has been developed.

Penicillin G increases the synthesis of a suicidal marker (CidC) and virulence (HlgBC) proteins in Staphylococcus aureus biofilm cells.

The present study reports the effect of Penicillin G (PenG) on the proteome dynamics of the Staphylococcus aureus strain Newman during biofilm mode of growth. The viability of the 18-h-old biofilm cells challenged with PenG at the concentration of 1mgmL(-1) was first assessed by plate counting, resazurin and LIVE/DEAD fluorescence staining, which indicated that the viability was reduced by ∼35% and ∼90% at 2h and 24h, respectively, after the addition of PenG. Subsequent two-dimensional difference gel electrophoresis (2D DIGE) assay of the treated and non-treated biofilm cells at the indicated time points revealed 45 proteins showing time- and treatment-specific change (1.5-fold, p<0.01). The 2D DIGE results suggested that the PenG-induced decrease in viability was accompanied by an increased synthesis of pyruvate oxidase (CidC), a suicidal marker known to potentiate acetate-dependent cell death in S. aureus. Increased abundance was also found for the TCA cycle associated malate-quinone oxidoreductase (Mqo), the ClpC ATPase, the HlgBC toxin and phage-associated proteins, which suggests that surviving cells have induced these activities as a last effort to overcome lethal doses of PenG. Proteomic results also revealed that the surviving cells were likely to strengthen their peptidoglycan due to the increased abundance of cell-wall biogenesis associated proteins, FemA and Pbp2; a phenomenon associated with dormancy in S. aureus.

Investigation of Endogenous Compounds Applicable to Drug-Drug Interaction Studies Involving the Renal Organic Anion Transporters, OAT1 and OAT3, in Humans.

This study was a comprehensive analysis of metabolites in plasma and urine specimens from subjects who received probenecid, a potent inhibitor of renal organic anion transporters (OATs). Taurine and glycochenodeoxycholate sulfate (GCDCA-S) could be identified using authentic standards. Probenecid had no effect on the area under the plasma-concentration time curves of taurine and GCDCA-S, whereas it significantly inhibited their urinary excretion in a dose-dependent manner. Probenecid at 500, 750, and 1500 mg orally decreased the renal clearance (CLR) values of taurine and GCDCA-S by 45% and 60%, 59% and 79%, and 70% and 88%, respectively. The CLR values correlated strongly (r > 0.96) between the test compounds (benzylpenicillin, 6ß-hydroxycortisol, taurine, and GCDCA-S). Taurine and GCDCA-S were substrates of OAT1 and OAT3, with Km values of 379 ± 58 and 64.3 ± 3.9 µM, respectively. The Ki values of probenecid for the OAT1- and OAT3-mediated uptake of taurine and GCDCA-S (9.49 ± 1.27 and 7.40 ± 0.70 µM, respectively) were similar to those of their typical substrate drugs. The magnitude of the reduction in the CLR of taurine and GCDCA-S by probenecid could be reasonably explained using the geometric mean values of unbound probenecid concentration and Ki values. These results suggest that taurine and GCDCA-S can be used as probes for evaluating pharmacokinetic drug-drug interactions involving OAT1 and OAT3, respectively, in humans.

Identification of peptide inhibitors of penicillinase using a phage display library.

There is a constant need to identify novel inhibitors to combat ß-lactamase-mediated antibiotic resistance. In this study, we identify three penicillinase-binding peptides, P1 (DHIHRSYRGEFD), P2 (NIYTTPWGSNWS), and P3 (SHSLPASADLRR), using a phage display library. Surface plasmon resonance (SPR) is utilized for quantitative determination and comparison of the binding specificity of selected peptides to penicillinase. An SPR biosensor functionalized with P3-GGGC (SHSLPASADLRRGGGC) is developed for detection of penicillinase with excellent sensitivity (15.8 RU nM(-1)) and binding affinity (KD = 0.56 nM). To determine if peptides can be good inhibitors for penicillinase, these peptides are mixed with penicillinase and their inhibition efficiency is determined by measuring the hydrolysis of substrate penicillin G using UV-vis spectrophotometry. Peptide P2 (NIYTTPWGSNWS) is found to be a promising penicillinase inhibitor with a Ki of 9.22 µM and a Ki' of 33.12 µM, suggesting that the inhibition mechanism is a mixed pattern. This peptide inhibitor (P2) can be used as a lead compound to identify more potent small molecule inhibitors for penicillinase. This study offers a potential approach to both detection of ß-lactamases and development of novel inhibitors of ß-lactamases.

Removal of Penicillin G and Erythromycin with Ionizing Radiation Followed by Biological Treatment.

The decomposition of penicillin G and erythromycin antibiotics at concentration of 0.2 mg ml(-1) by gamma irradiation at 50 kGy followed by biological treatment with Cupriavidus metallidurans CH34 was evaluated. Degradation of penicillin G and erythromycin was analyzed using nuclear magnetic resonance analysis (NMR), fourier transform infrared spectroscopy (FTIR), and chemical oxygen demand (COD). The exposure to the absorbed dose of 50 kGy caused degradation of penicillin G and erythromycin in the aqueous solution. The complete disappearance of NMR and FTIR peaks following irradiation confirmed the breakage of the ß-lactam ring in penicillin G, and the decarboxylation and cleavage of the thiazolidine ring and for erythromycin, the complete destruction of the three aromatic rings. Irradiation alone removed 52.8 and 65.5 % of penicillin G and erythromycin, respectively. Further reduction to 12.6 and 14 % of the original penicillin G and erythromycin COD, respectively, was achieved using treatment of the irradiation products with C. metallidurans.

Pexophagy in Penicillin G Secretion by Penicillium chrysogenum PQ-96.

Penicillin G oversecretion by Penicillium chrysogenum PQ-96 is associated with a strictly adjusted cellular organization of the mature and senescent mycelial cells. Abundant vacuolar phagy and extended cellular vacuolization combined with vacuolar budding resulting in the formation of vacuolar vesicles that fuse with the cell membrane are the most important characteristic features of those cells. We suggest as follows: if the peroxisomes are integrated into vacuoles, the penicillin G formed in peroxisomes might be transferred to vacuoles and later secreted out of the cells by an exocytosis process. The peroxisomal cells of the mycelium are privileged in penicillin G secretion.

Design, synthesis, and functional evaluation of CO-releasing molecules triggered by Penicillin†G amidase as a model protease.

Protease-triggered CO-releasing molecules (CORMs) were developed. The viability of the approach was demonstrated through the synthesis of compounds consisting of an η(4) -oxydiene-Fe(CO)3 moiety connected to a penicillin G amidase (PGA)-cleavable unit through a self-immolative linker. The rate of PGA-induced hydrolysis was investigated by HPLC analysis and the subsequent CO release was quantitatively assessed through headspace gas chromatography. In an in vitro assay with human endothelial cells, typical biological effects of CO, that is, inhibition of the inflammatory response and the induction of heme oxygenase-1 expression, were observed only upon co-administration of the CORM and PGA. This work forms a promising basis for the future development of protease-specific CORMs for potential medicinal applications.

Research on degradation of penicillins in milk by ß-lactamase using ultra-performance liquid chromatography coupled with time-of-flight mass spectrometry.

The degradation of penicillin G, penicillin V, and ampicillin in milk in the presence of ß-lactamase was investigated by ultra-performance liquid chromatography coupled with electrospray ionization-time-of-flight mass spectrometry. Degradation products of the 3 penicillins in milk were identified based on the fact that the metabolites or degradation products contain a substructure of penicillin, and their degradation pathways in acidic milk in presence of ß-lactamase were developed. The influence of factors on the degradation was investigated, including ß-lactamase dosage, temperature, time, and acidity. The ratio of the 2 degradation products (penicilloic acid and penilloic acid) is different at different temperatures and pH. Penicilloic acid was the dominant species obtained at pH 6 under 40°C, but, being unstable, it could not be used as a standard for accurate analysis of penicilloic acid, and also could not be used as target for detection of penicillins in milk. Penilloic acid was the dominant species obtained at pH 2 above 40°C; it was stable and could be used as a standard for quantitative analysis and as target for detecting whether penicillins were used in milk.

Compartmentalization in penicillin G biosynthesis by Penicillium chrysogenum PQ-96.

The arrangement of organelles in the sub-apical productive non-growing vacuolated hyphal cells of the high- and the low-penicillin-pro- ducing strains Penicillium chrysogenum was compared using transmission electron microscopy. In the productive cells of the high-yielding strain the endoplasmic reticulum and the polyribosomes with associated peroxisomes are frequently arranged at the periphery of the cytoplasm and around the vacuoles. At the high activity of penicillin G biosynthesis the immuno-label of the cytosolic isopenicillin N synthase is concentrated at the polyribosomes arranged in the peripheral cytoplasm and along the tonoplast as well as around the peroxisomes. On the basis of the obtained results the compartmentalization of the pathway of penicillin G biosymthesis is discussed. The obtained results support the phenylacetic acid detoxification hypothesis of penicillin G biosynthesis.

R164H and V240H replacements by site-directed mutagenesis of TEM-149 extended-spectrum ß-lactamase: kinetic analysis of TEM-149H240 and TEM-149H164-H240 laboratory mutants.

Two laboratory mutant forms, TEM-149(H240) and TEM-149(H164-H240), of the TEM-149 extended-spectrum ß-lactamase enzyme were constructed by site-directed mutagenesis. TEM-149(H240) and TEM-149(H164-H240) were similar in kinetic behavior, except with respect to benzylpenicillin and ceftazidime. Molecular modeling of the two mutant enzymes demonstrated the role of histidine at position 240 in the reduction of the affinity of the enzyme for ceftazidime.

Spectrophotometric-dual-enzyme-simultaneous assay in one reaction solution: chemometrics and experimental models.

Spectrophotometric-dual-enzyme-simultaneous assay in one reaction solution (SDESA) is proposed. SDESA requires the following: (a) Enzyme A acts on Substrate A to release Product A bearing the longest difference absorbance peak (λ(A)) much larger than that of Product B (λ(B)) formed by Enzyme B action on Substrate B; λ(B) is close to the longest isoabsorbance wavelength of Product A and Substrate A (λ(0)); (b) absorbance at λ(A) and λ(0) is quantified via swift alternation of detection wavelengths and corrected on the basis of absorbance additivity; (c) inhibition/activation on either enzyme by any substance is eliminated; (d) Enzyme A is quantified via an integration strategy if levels of Substrate A are lower than the Michaelis constant. Chemometrics of SDESA was tested with γ-glutamyltransferase and lactate-dehydrogenase of complicated kinetics. γ-Glutamyltransferase releases p-nitroaniline from γ-glutamyl-p-nitroaniline with λ(0) at 344 nm and λ(A) close to 405 nm, lactate-dehydrogenase consumes reduced nicotinamide dinucleotide bearing λ(B) at 340 nm. Kinetic analysis of reaction curve yielded lactate-dehydrogenase activity free from inhibition by p-nitroaniline; the linear range of initial rates of γ-glutamyltransferase via the integration strategy, and that of lactate-dehydrogenase after interference elimination, was comparable to those by separate assays, respectively; the quantification limit of either enzyme by SDESA at 25-fold higher activity of the other enzyme remained comparable to that by a separate assay. To test potential application, SDESA of alkaline phosphatase (ALP) and ß-D-galactosidase as enzyme-linked-immunoabsorbent assay (ELISA) labels were examined. ALP releases 4-nitro-1-naphthol from 4-nitronaphthyl-1-phosphate with λ(0) at 405 nm and λ(A) at 458 nm, ß-D-galactosidase releases 4-nitrophenol from ß-D-(4-nitrophenyl)-galactoside with λ(B) at 405 nm. No interference from substrates/products made SDESA of ß-galactosidase and ALP simple for ELISA of penicillin G and clenbuterol in one well, and the quantification limit of either hapten was comparable to that via a separate assay. Hence, SDESA is promising.

Engineering the substrate specificity of a thermophilic penicillin acylase from thermus thermophilus.

A homologue of the Escherichia coli penicillin acylase is encoded in the genomes of several thermophiles, including in different Thermus thermophilus strains. Although the natural substrate of this enzyme is not known, this acylase shows a marked preference for penicillin K over penicillin G. Three-dimensional models were created in which the catalytic residues and the substrate binding pocket were identified. Through rational redesign, residues were replaced to mimic the aromatic binding site of the E. coli penicillin G acylase. A set of enzyme variants containing between one and four amino acid replacements was generated, with altered catalytic properties in the hydrolyses of penicillins K and G. The introduction of a single phenylalanine residue in position α188, α189, or ß24 improved the K(m) for penicillin G between 9- and 12-fold, and the catalytic efficiency of these variants for penicillin G was improved up to 6.6-fold. Structural models, as well as docking analyses, can predict the positioning of penicillins G and K for catalysis and can demonstrate how binding in a productive pose is compromised when more than one bulky phenylalanine residue is introduced into the active site.