A chemoenzymatic strategy for site-selective functionalization of native peptides and proteins.

The emergence of new therapeutic modalities requires complementary tools for their efficient syntheses. Availability of methodologies for site-selective modification of biomolecules remains a long-standing challenge, given the inherent complexity and the presence of repeating residues that bear functional groups with similar reactivity profiles. We describe a bioconjugation strategy for modification of native peptides relying on high site selectivity conveyed by enzymes. We engineered penicillin G acylases to distinguish among free amino moieties of insulin (two at amino termini and an internal lysine) and manipulate cleavable phenylacetamide groups in a programmable manner to form protected insulin derivatives. This enables selective and specific chemical ligation to synthesize homogeneous bioconjugates, improving yield and purity compared to the existing methods, and generally opens avenues in the functionalization of native proteins to access biological probes or drugs.

Genetic Encoding and Enzymatic Deprotection of a Latent Thiol Side Chain to Enable New Protein Bioconjugation Applications.

The thiol group of the cysteine side chain is arguably the most versatile chemical handle in proteins. To expand the scope of established and commercially available thiol bioconjugation reagents, we genetically encoded a second such functional moiety in form of a latent thiol group that can be unmasked under mild physiological conditions. Phenylacetamidomethyl (Phacm) protected homocysteine (HcP) was incorporated and its latent thiol group unmasked on purified proteins using penicillin G acylase (PGA). The enzymatic deprotection depends on steric accessibility, but can occur efficiently within minutes on exposed positions in flexible sequences. The freshly liberated thiol group does not require treatment with reducing agents. We demonstrate the potential of this approach for protein modification with conceptually new schemes for regioselective dual labeling, thiol bioconjugation in presence of a preserved disulfide bond and formation of a novel intramolecular thioether crosslink.

Cell-specific Profiling of Nascent Proteomes Using Orthogonal Enzyme-mediated Puromycin Incorporation.

Translation regulation is a fundamental component of gene expression, allowing cells to respond rapidly to a variety of stimuli in the absence of new transcription. The lack of methods for profiling nascent proteomes in distinct cell populations in heterogeneous tissues has precluded an understanding of translational regulation in physiologically relevant contexts. Here, we describe a chemical genetic method that involves orthogonal enzyme-mediated incorporation of a clickable puromycin analogue into nascent polypeptides. Using this method, we show that we can label newly synthesized proteins in a cell-specific manner in cells grown in culture and in heterogeneous tissues. We also show that we can identify the nascent proteome in genetically targeted cell populations using affinity enrichment and tandem mass spectrometry. Our method has the potential to provide unprecedented insights into cell-specific translational regulation in heterogeneous tissues.

An approach for the improved immobilization of penicillin G acylase onto macroporous poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) as a potential industrial biocatalyst.

The use of penicillin G acylase (PGA) covalently linked to insoluble carrier is expected to produce major advances in pharmaceutical processing industry and the enzyme stability enhancement is still a significant challenge. The objective of this study was to improve catalytic performance of the covalently immobilized PGA on a potential industrial carrier, macroporous poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) [poly(GMA-co-EGDMA)], by optimizing the copolymerization process and the enzyme attachment procedure. This synthetic copolymer could be a very promising alternative for the development of low-cost, easy-to-prepare, and stable biocatalyst compared to expensive commercially available epoxy carriers such as Eupergit or Sepabeads. The PGA immobilized on poly(GMA-co-EGDMA) in the shape of microbeads obtained by suspension copolymerization appeared to have higher activity yield compared to copolymerization in a cast. Optimal conditions for the immobilization of PGA on poly(GMA-co-EGDMA) microbeads were 1 mg/mL of PGA in 0.75 mol/L phosphate buffer pH 6.0 at 25°C for 24 h, leading to the active biocatalyst with the specific activity of 252.7 U/g dry beads. Chemical amination of the immobilized PGA could contribute to the enhanced stability of the biocatalyst by inducing secondary interactions between the enzyme and the carrier, ensuring multipoint attachment. The best balance between the activity yield (51.5%), enzyme loading (25.6 mg/g), and stability (stabilization factor 22.2) was achieved for the partially modified PGA.

Structural analysis of a penicillin V acylase from Pectobacterium atrosepticum confirms the importance of two Trp residues for activity and specificity.

Penicillin V acylases (PVA) catalyze the deacylation of the beta-lactam antibiotic phenoxymethylpenicillin (Pen V). They are members of the Ntn hydrolase family and possess an N-terminal cysteine as the main catalytic nucleophile residue. They form the evolutionarily related cholylglycine hydrolase (CGH) group which includes bile salt hydrolases (BSH) responsible for bile deconjugation. Even though a few PVA and BSH structures have been reported, no structure of a functional PVA from Gram-negative bacteria is available. Here, we report the crystal structure of a highly active PVA from Gram-negative Pectobacterium atrosepticum (PaPVA) at 2.5Å resolution. Structural comparison with PVAs from Gram-positive bacteria revealed that PaPVA had a distinctive tetrameric structure and active site organization. In addition, mutagenesis of key active site residues and biochemical characterization of the resultant variants elucidated the role of these residues in substrate binding and catalysis. The importance of residue Trp23 and Trp87 side chains in binding and correct positioning of Pen V by PVAs was confirmed using mutagenesis and substrate docking with a 15ns molecular dynamics simulation. These results establish the unique nature of Gram-negative CGHs and necessitate further research about their substrate spectrum.

Structure mediation in substrate binding and post-translational processing of penicillin acylases: Information from mutant structures of Kluyvera citrophila penicillin G acylase.

Penicillin acylases are industrially important enzymes for the production of 6-APA, which is used extensively in the synthesis of secondary antibiotics. The enzyme translates into an inactive single chain precursor that subsequently gets processed by the removal of a spacer peptide connecting the chains of the mature active heterodimer. We have cloned the penicillin G acylase from Kluyvera citrophila (KcPGA) and prepared two mutants by site-directed mutagenesis. Replacement of N-terminal serine of the ß-subunit with cysteine (Serß1Cys) resulted in a fully processed but inactive enzyme. The second mutant in which this serine is replaced by glycine (Serß1Gly) remained in the unprocessed and inactive form. The crystals of both mutants belonged to space group P1 with four molecules in the asymmetric unit. The three-dimensional structures of these mutants were refined at resolutions 2.8 and 2.5 Å, respectively. Comparison of these structures with similar structures of Escherichia coli PGA (EcPGA) revealed various conformational changes that lead to autocatalytic processing and consequent removal of the spacer peptide. The large displacements of residues such as Arg168 and Arg477 toward the N-terminal cleavage site of the spacer peptide or the conformational changes of Arg145 and Phe146 near the active site in these structures suggested probable steps in the processing dynamics. A comparison between the structures of the processed Serß1Cys mutant and that of the processed form of EcPGA showed conformational differences in residues Argα145, Pheα146, and Pheß24 at the substrate binding pocket. Three conformational transitions of Argα145 and Pheα146 residues were seen when processed and unprocessed forms of KcPGA were compared with the substrate bound structure of EcPGA. Structure mediation in activity difference between KcPGA and EcPGA toward acyl homoserine lactone (AHL) is elucidated.

Penicillin V acylase from Pectobacterium atrosepticum exhibits high specific activity and unique kinetics.

Penicillin V acylases (PVAs, E.C.3.5.11) belong to the Ntn hydrolase super family of enzymes that catalyze the deacylation of the side chain from phenoxymethyl penicillin (penicillin V). Penicillin acylases find use in the pharmaceutical industry for the production of semi-synthetic antibiotics. PVAs employ the N-terminal cysteine residue as catalytic nucleophile and are structurally and evolutionarily related to bile salt hydrolases (BSHs). Here, we report the cloning and characterization of a PVA enzyme from the Gram-negative plant pathogen, Pectobacterium atrosepticum (PaPVA). The enzyme was cloned and expressed in Escherichia coli attaining a very high yield (250 mg/l) and a comparatively high specific activity (430 IU/mg). The enzyme showed marginally better pH and thermo-stability over PVAs characterized from Gram-positive bacteria. The enzyme also showed enhanced activity in presence of organic solvents and detergents. The enzyme kinetics turned out to be significantly different from that of previously reported PVAs, displaying positive cooperativity and substrate inhibition. The presence of bile salts had a modulating effect on PaPVA activity. Sequence analysis and characterization reveal the distinctive nature of these enzymes and underscore the need to study PVAs from Gram-negative bacteria.

Riesgo potencial de parásitos zoonóticos presentes en heces caninas en Puerto Escondido, Oaxaca / Potential hazard of zoonotic parasites present in canine feces in Puerto Escondido, Oaxaca

Objetivo. Estimar la prevalencia de parásitos potencialmente zoonóticos en heces caninas de Puerto Escondido. Material y métodos. La ciudad se dividió en diez zonas de estudio y éstas se categorizaron en hábitats natural, urbano y suburbano. Se colectaron muestras fecales caninas del piso. Se recuperaron los parásitos por medio de técnicas coproparasitológicas de flotación y frotis directo para su observación microscópica y posterior identificación. Se estimó la prevalencia parasitaria en las heces caninas. Resultados. Todas las zonas presentaron fecalismo canino. La prevalencia parasitaria fue de 73.33%. Los parásitos con mayor prevalencia fueron Toxocara canis (47.78%), Ancylostoma caninum (17.88%) y Dipylidium caninum (13.89%). Conclusión. El fecalismo canino proviene de perros errantes y con dueño. Del total de parásitos encontrados, 66.66% son zoonóticos. Los factores que favorecen la problemática son el hábitat suburbano, el manejo indeseable de la basura y la tenencia irresponsable de los cánidos.

Objective. To estimate the zoonotic parasites prevalence in feral dog feces in Puerto Escondido. Material and methods. The fecalism frecuency was estimated in ten zones. To identify the parasites parasitological flotation and direct smear methods were used. The parasitic prevalence was estimated in the canine feces. Results. All the zones presented canine fecalism. The parasitic prevalence in the feces was 73.33%. The parasites with the highest prevalence were Toxocara canis (47.78%), Ancylostoma caninum (17.88%), and Dipylidium caninum (13.89%). Conclusion. Canine fecalism comes from strayed and owned dogs. 66.66% of the parasites found in the dog feces are zoonotics. The factors associated to this problem are the suburban habitat, waste mishandling and nil tenure of stray dogs.

An iodine-free and directed-disulfide-bond-forming route to insulin analogues.

An iodine-free synthetic route to insulin analogues has been established via a directed disulfide bond formation strategy. This method is completely compatible with oxidation-sensitive residues. The key step is constructing the third disulfide bond via a novel procedure involving phenylacetylaminomethyl group (Phacm), immobilized Penicillin G Acylase, and Ellman's reagent. We expect that this method could be broadly utilized for synthesizing insulin-like and other cysteine-rich peptides, in particular, where oxidation-sensitive residues are present in the sequence.

Modulation of the microenvironment surrounding the active site of penicillin G acylase immobilized on acrylic carriers improves the enzymatic synthesis of cephalosporins.

The catalytic properties of penicillin G acylase (PGA) from Escherichia coli in kinetically controlled synthesis of ß-lactam antibiotics are negatively affected upon immobilization on hydrophobic acrylic carriers. Two strategies have been here pursued to improve the synthetic performance of PGA immobilized on epoxy-activated acrylic carriers. First, an aldehyde-based spacer was inserted on the carrier surface by glutaraldehyde activation (immobilization yield = 50%). The resulting 3-fold higher synthesis/hydrolysis ratio (vs/vh1 = 9.7 ± 0.7 and 10.9 ± 0.7 for Eupergit® C and Sepabeads® EC-EP, respectively) with respect to the unmodified support (vs/vh1 = 3.3 ± 0.4) was ascribed to a facilitated diffusion of substrates and products as a result of the increased distance between the enzyme and the carrier surface. A second series of catalysts was prepared by direct immobilization of PGA on epoxy-activated acrylic carriers (Eupergit® C), followed by quenching of oxiranes not involved in the binding with the protein with different nucleophiles (amino acids, amines, amino alcohols, thiols and amino thiols). In most cases, this derivatization increased the synthesis/hydrolysis ratio with respect to the non derivatized carrier. Particularly, post-immobilization treatment with cysteine resulted in about 2.5-fold higher vs/vh1 compared to the untreated biocatalyst, although the immobilization yield decreased from 70% (untreated Eupergit® C) to 20%. Glutaraldehyde- and cysteine-treated Eupergit® C catalyzed the synthesis of cefazolin in 88% (±0.9) and 87% (±1.6) conversion, respectively, whereas untreated Eupergit® C afforded this antibiotic in 79% (±1.2) conversion.

Eco-friendly combination of the immobilized PGA enzyme and the S-Phacm protecting group for the synthesis of Cys-containing peptides.

Enzyme-labile protecting groups have emerged as a green alternative to conventional protecting groups. These groups introduce a further orthogonal dimension and eco-friendliness into protection schemes for the synthesis of complex polyfunctional organic molecules. S-Phacm, a Cys-protecting group, can be easily removed by the action of a covalently immobilized PGA enzyme under very mild conditions. Herein, the versatility and reliability of an eco-friendly combination of the immobilized PGA enzyme and the S-Phacm protecting group has been evaluated for the synthesis of diverse Cys-containing peptides.

Modular theranostic prodrug based on a FRET-activated self-immolative linker.

New chemotherapeutic prodrugs that can report on the localization and activation of the drug upon internalization into select cells are being widely investigated for cancer treatment. Here, we introduce a new molecular design for a theranostic prodrug based on a self-immolative linker attached to a pair of FRET dyes that produces a fluorescent signal upon disassembly. The prodrug evaluated here was designed to release the chemotherapeutic drug camptothecin upon activation by the model enzyme penicillin-G-amidase. Similar patterns of the disassembly of the prodrug were observed in HPLC and fluorescence assays. The obtained results demonstrate that upon specific activation of the prodrug, the increase of the emitted fluorescent signal is linearly correlated with the observed drug release. Such a design could potentially be used to monitor prodrug activation in real-time and provide information regarding the location and the amount of active drug molecules.

Purification and characterization of YxeI, a penicillin acylase from Bacillus subtilis.

The paper reports the purification and characterization of the first penicillin acylase from Bacillus subtilis. YxeI, the protein annotated as hypothetical, coded by the gene yxeI in the open reading frame between iol and hut operons in B. subtilis was cloned and expressed in Eshcherichia coli, purified and characterized. The purified protein showed measurable penicillin acylase activity with penicillin V. The enzyme was a homotetramer of 148 kDa. The apparent K(m) of the enzyme for penicillin V and the synthetic substrate 2-nitro-5-(phenoxyacetamido)-benzoic acid was 40 mM and 0.63 mM, respectively, and the association constants were 8.93×10(2) M(-1) and 2.51×10(5) M(-1), respectively. It was inhibited by cephalosporins and conjugated bile salts, substrates of the closely related bile acid hydrolases. It had good sequence homology with other penicillin V acylases and conjugated bile acid hydrolases, members of the Ntn hydrolase family. The N-terminal nucleophile was a cysteine which is revealed by a simple removal of N-formyl-methionine. The activity of the protein was affected by high temperature, acidic pH and the presence of the denaturant guanidine hydrochloride.

The N-terminal nucleophile serine of cephalosporin acylase executes the second autoproteolytic cleavage and acylpeptide hydrolysis.

Cephalosporin acylase (CA) precursor is translated as a single polypeptide chain and folds into a self-activating pre-protein. Activation requires two peptide bond cleavages that excise an internal spacer to form the mature αß heterodimer. Using Q-TOF LC-MS, we located the second cleavage site between Glu(159) and Gly(160), and detected the corresponding 10-aa spacer (160)GDPPDLADQG(169) of CA mutants. The site of the second cleavage depended on Glu(159): moving Glu into the spacer or removing 5-10 residues from the spacer sequence resulted in shorter spacers with the cleavage at the carboxylic side of Glu. The mutant E159D was cleaved more slowly than the wild-type, as were mutants G160A and G160L. This allowed kinetic measurements showing that the second cleavage reaction was a first-order, intra-molecular process. Glutaryl-7-aminocephalosporanic acid is the classic substrate of CA, in which the N-terminal Ser(170) of the ß-subunit, is the nucleophile. Glu and Asp resemble glutaryl, suggesting that CA might also remove N-terminal Glu or Asp from peptides. This was indeed the case, suggesting that the N-terminal nucleophile also performed the second proteolytic cleavage. We also found that CA is an acylpeptide hydrolase rather than a previously expected acylamino acid acylase. It only exhibited exopeptidase activity for the hydrolysis of an externally added peptide, supporting the intra-molecular interaction. We propose that the final CA activation is an intra-molecular process performed by an N-terminal nucleophile, during which large conformational changes in the α-subunit C-terminal region are required to bridge the gap between Glu(159) and Ser(170).

Glyoxyl-disulfide agarose: a tailor-made support for site-directed rigidification of proteins.

A new strategy has been developed for site-directed immobilization/rigidification of genetically modified enzymes through multipoint covalent attachment on bifunctional disulfide-glyoxyl supports. Here the mechanism is described as a two-step immobilization/rigidification protocol where the enzyme is directly immobilized by thiol-disulfide exchange between the ß-thiol of the single genetically introduced cysteine and the few disulfide groups presented on the support surface (3 µmol/g). Afterward, the enzyme is uniquely rigidified by multipoint covalent attachment (MCA) between the lysine residues in the vicinity of the introduced cysteine and the many glyoxyl groups (220 µmol/g) on the support surface. Both site-directed immobilization and rigidification have been possible only on these novel bifunctional supports. In fact, this technology has made possible to elucidate the protein regions where rigidification by MCA promoted higher protein stabilizations. Hence, rigidification of vicinity of position 333 from lipase 2 from Geobacillus thermocatenulatus (BTL2) promoted a stabilization factor of 33 regarding the unipunctual site-directed immobilized derivative. In the same context, rigidification of penicillin G acylase from E. coli (PGA) through position ß201 resulted in a stabilization factor of 1069. Remarkably, when PGA was site-directed rigidified through that position, it presented a half-life time of 140 h under 60% (v/v) of dioxane and 4 °C, meaning a derivative eight times more stable than the PGA randomly immobilized on glyoxyl-disulfide agarose. Herein we have opened a new scenario to optimize the stabilization of proteins via multipoint covalent immobilization, which may represent a breakthrough in tailor-made tridimensional rigidification of proteins.

Initial insight into the function of the lysosomal 66.3 kDa protein from mouse by means of X-ray crystallography.

BACKGROUND: The lysosomal 66.3 kDa protein from mouse is a soluble, mannose 6-phosphate containing protein of so far unknown function. It is synthesized as a glycosylated 75 kDa precursor that undergoes limited proteolysis leading to a 28 kDa N- and a 40 kDa C-terminal fragment. RESULTS: In order to gain insight into the function and the post-translational maturation process of the glycosylated 66.3 kDa protein, three crystal structures were determined that represent different maturation states. These structures demonstrate that the 28 kDa and 40 kDa fragment which have been derived by a proteolytic cleavage remain associated. Mass spectrometric analysis confirmed the subsequent trimming of the C-terminus of the 28 kDa fragment making a large pocket accessible, at the bottom of which the putative active site is located. The crystal structures reveal a significant similarity of the 66.3 kDa protein to several bacterial hydrolases. The core alphabetabetaalpha sandwich fold and a cysteine residue at the N-terminus of the 40 kDa fragment (C249) classify the 66.3 kDa protein as a member of the structurally defined N-terminal nucleophile (Ntn) hydrolase superfamily. CONCLUSION: Due to the close resemblance of the 66.3 kDa protein to members of the Ntn hydrolase superfamily a hydrolytic activity on substrates containing a non-peptide amide bond seems reasonable. The structural homology which comprises both the overall fold and essential active site residues also implies an autocatalytic maturation process of the lysosomal 66.3 kDa protein. Upon the proteolytic cleavage between S248 and C249, a deep pocket becomes solvent accessible, which harbors the putative active site of the 66.3 kDa protein.

Increase of doxorubicin sensitivity for folate receptor positive cells when given as the prodrug N-(phenylacetyl) doxorubicin in combination with folate-conjugated PGA.

Folate receptor (FR) has been proposed as a promising target for tumor drug targeting. The aim of this study was to increase the chemo-sensitivity of FR-positive cells to doxorubicin by folate-directed enzyme prodrug therapy (FDEPT). Folate conjugated penicillin-G amidase was prepared and its ability to hydrolyze N-(phenylacetyl) doxorubicin was measured by HPLC. Fluorescence and confocal image analysis revealed that Folate-PGA can be specifically delivered into FR-positive HeLa and SKOV3 tumor cells. In vitro cytotoxity assays, IC50 was reduced with N-(phenylacetyl) doxorubicin versus doxorubicin for HeLa (3.1-fold reduction; p<0.001) and SKOV3 (3.3-fold reduction; p<0.001) when Folate-PGA was specifically bound to the cells. Complete activation was confirmed in HeLa and SKOV3 cells pretreated with free folic acid (1 mM), where the combination of N-(phenylacetyl) doxorubicin with Folate-PGA did not show any significant cell toxicity to the IC50 of doxorubicin. Pharmacokinetic clearance and biodistribution studies in vivo showed that 125I-Folate-PGA was cleared from blood within 24 h and had significantly higher tumor uptake compared to 125I-PGA (p<0.05). These results demonstrate that the FDEPT approach may be a potential promising strategy to improve chemotherapy-resistant cancers therapeutic ratio and warranted future studies.

Insight into autoproteolytic activation from the structure of cephalosporin acylase: a protein with two proteolytic chemistries.

Cephalosporin acylase (CA), a member of the N-terminal nucleophile hydrolase family, is activated through sequential primary and secondary autoproteolytic reactions with the release of a pro segment. We have determined crystal structures of four CA mutants. Two mutants are trapped after the primary cleavage, and the other two undergo secondary cleavage slowly. These structures provide a look at pro-segment conformation during activation in N-terminal nucleophile hydrolases. The highly strained helical pro segment of precursor is transformed into a relaxed loop in the intermediates, suggesting that the relaxation of structural constraints drives the primary cleavage reaction. The secondary autoproteolytic step has been proposed to be intermolecular. However, our analysis provides evidence that CA is processed in two sequential steps of intramolecular autoproteolysis involving two distinct residues in the active site, the first a serine and the second a glutamate.

Structure-based stabilization of an enzyme: the case of penicillin acylase from Alcaligenes faecalis.

The modeled structure of penicillin acylase from Alcaligenes faecali (AFPGA) was constructed by comparative modeling with the Modeller program. Candidate positions that could be replaced with cysteine were estimated by scanning the modeled structure of AFPGA with the program MODIP (modeling disulfide bond in protein). The mutant Q3C/P751C had a higher optimum temperature by three degrees than that of the wild type AFPGA. The half life of the double mutant Q3C/P751C at 55 degrees C was increased by 50%. To our knowledge, this was the first structure-based genetic modification of AFPGA.