Simultaneous Irradiation with UV-A, -B, and -C Lights Promotes Effective Decontamination of Planktonic and Sessile Bacteria: A Pilot Study.

Surfaces in highly anthropized environments are frequently contaminated by both harmless and pathogenic bacteria. Accidental contact between these contaminated surfaces and people could contribute to uncontrolled or even dangerous microbial diffusion. Among all possible solutions useful to achieve effective disinfection, ultraviolet irradiations (UV) emerge as one of the most "Green" technologies since they can inactivate microorganisms via the formation of DNA/RNA dimers, avoiding the environmental pollution associated with the use of chemical sanitizers. To date, mainly UV-C irradiation has been used for decontamination purposes, but in this study, we investigated the cytotoxic potential on contaminated surfaces of combined UV radiations spanning the UV-A, UV-B, and UV-C spectrums, obtained with an innovative UV lamp never conceived so far by analyzing its effect on a large panel of collection and environmental strains, further examining any possible adverse effects on eukaryotic cells. We found that this novel device shows a significant efficacy on different planktonic and sessile bacteria, and, in addition, it is compatible with eukaryotic skin cells for short exposure times. The collected data strongly suggest this new lamp as a useful device for fast and routine decontamination of different environments to ensure appropriate sterilization procedures.

Antimicrobial D-amino acid oxidase-derived peptides specify gut microbiota.

The flavoenzyme D-amino acid oxidase (DAAO) is deputed to the degradation of D-enantiomers of amino acids. DAAO plays various relevant physiological roles in different organisms and tissues. Thus, it has been recently suggested that the goblet cells of the mucosal epithelia secrete into the lumen of intestine, a processed and active form of DAAO that uses the intestinal D-amino acids to generate hydrogen peroxide (H2O2), an immune messenger that helps fighting gut pathogens, and by doing so controls the homeostasis of gut microbiota. Here, we show that the DAAO form lacking the 1-16 amino acid residues (the putative secretion signal) is unstable and inactive, and that DAAO is present in the epithelial layer and the mucosa of mouse gut, where it is largely proteolyzed. In silico predicted DAAO-derived antimicrobial peptides show activity against various Gram-positive and Gram-negative bacteria but not on Lactobacilli species, which represent the commensal microbiota. Peptidomic analysis reveals the presence of such peptides in the mucosal fraction. Collectively, we identify a novel mechanism for gut microbiota selection implying DAAO-derived antimicrobial peptides which are generated by intestinal proteases and that are secreted in the gut lumen. In conclusion, we herein report an additional, ancillary role for mammalian DAAO, unrelated to its enzymatic activity.

Protective Effects of Recombinant Human Angiogenin in Keratinocytes: New Insights on Oxidative Stress Response Mediated by RNases.

Human angiogenin (ANG) is a 14-kDa ribonuclease involved in different pathophysiological processes including tumorigenesis, neuroprotection, inflammation, innate immunity, reproduction, the regeneration of damaged tissues and stress cell response, depending on its intracellular localization. Under physiological conditions, ANG moves to the cell nucleus where it enhances rRNA transcription; conversely, recent reports indicate that under stress conditions, ANG accumulates in the cytoplasmic compartment and modulates the production of tiRNAs, a novel class of small RNAs that contribute to the translational inhibition and recruitment of stress granules (SGs). To date, there is still limited and controversial experimental evidence relating to a hypothetical role of ANG in the epidermis, the outermost layer of human skin, which is continually exposed to external stressors. The present study collects compelling evidence that endogenous ANG is able to modify its subcellular localization on HaCaT cells, depending on different cellular stresses. Furthermore, the use of recombinant ANG allowed to determine as this special enzyme is effectively able to counter at various levels the alterations of cellular homeostasis in HaCaT cells, actually opening a new vision on the possible functions that this special enzyme can support also in the stress response of human skin.

Host defence peptides identified in human apolipoprotein B as promising antifungal agents.

Therapeutic options to treat invasive fungal infections are still limited. This makes the development of novel antifungal agents highly desirable. Naturally occurring antifungal peptides represent valid candidates, since they are not harmful for human cells and are endowed with a wide range of activities and their mechanism of action is different from that of conventional antifungal drugs. Here, we characterized for the first time the antifungal properties of novel peptides identified in human apolipoprotein B. ApoB-derived peptides, here named r(P)ApoBLPro, r(P)ApoBLAla and r(P)ApoBSPro, were found to have significant fungicidal activity towards Candida albicans (C. albicans) cells. Peptides were also found to be able to slow down metabolic activity of Aspergillus niger (A. niger) spores. In addition, experiments were carried out to clarify the mechanism of fungicidal activity of ApoB-derived peptides. Peptides immediately interacted with C. albicans cell surfaces, as indicated by fluorescence live cell imaging analyses, and induced severe membrane damage, as indicated by propidium iodide uptake induced upon treatment of C. albicans cells with ApoB-derived peptides. ApoB-derived peptides were also tested on A. niger swollen spores, initial hyphae and branched mycelium. The effects of peptides were found to be more severe on swollen spores and initial hyphae compared to mycelium. Fluorescence live cell imaging analyses confirmed peptide internalization into swollen spores with a consequent accumulation into hyphae. Altogether, these findings open interesting perspectives to the application of ApoB-derived peptides as effective antifungal agents. KEY POINTS: Human cryptides identified in ApoB are effective antifungal agents. ApoB-derived cryptides exert fungicidal effects towards C. albicans cells. ApoB-derived cryptides affect different stages of growth of A. niger. Graphical abstract.

Environment-Sensitive Fluorescent Labelling of Peptides by Luciferin Analogues.

Environment-sensitive fluorophores are very valuable tools in the study of molecular and cellular processes. When used to label proteins and peptides, they allow for the monitoring of even small variations in the local microenvironment, thus acting as reporters of conformational variations and binding events. Luciferin and aminoluciferin, well known substrates of firefly luciferase, are environment-sensitive fluorophores with unusual and still-unexploited properties. Both fluorophores show strong solvatochromism. Moreover, luciferin fluorescence is influenced by pH and water abundance. These features allow to detect local variations of pH, solvent polarity and local water concentration, even when they occur simultaneously, by analyzing excitation and emission spectra. Here, we describe the characterization of (amino)luciferin-labeled derivatives of four bioactive peptides: the antimicrobial peptides GKY20 and ApoBL, the antitumor peptide p53pAnt and the integrin-binding peptide RGD. The two probes allowed for the study of the interaction of the peptides with model membranes, SDS micelles, lipopolysaccharide micelles and Escherichia coli cells. Kd values and binding stoichiometries for lipopolysaccharide were also determined. Aminoluciferin also proved to be very well-suited to confocal laser scanning microscopy. Overall, the characterization of the labeled peptides demonstrates that luciferin and aminoluciferin are previously neglected environment-sensitive labels with widespread potential applications in the study of proteins and peptides.

Membrane disintegration by the antimicrobial peptide (P)GKY20: lipid segregation and domain formation.

Antimicrobial peptides (AMPs) are membrane-active peptides with a broad spectrum of activity against different pathogenic organisms and they represent promising new drugs to overcome the emergence of resistance to antibiotics in bacteria. (P)GKY20 is an antimicrobial peptide with a low hemolytic effect on eukaryotic cells and a strong antimicrobial activity especially against Gram-negative bacteria. However, its mechanism of action is still unknown. Here, we use fluorescence spectroscopy and differential scanning calorimetry combined with atomic force microscopy to characterise the binding of (P)GKY20 with model biomembranes and its effect on the membrane's microstructure and thermotropic properties. We found that (P)GKY20 selectively perturbs the bacterial-like membrane via a carpet-like mechanism employing peptide conformational changes, lipid segregation and domain formation as key steps in promoting membrane disruption. These results shed a first light on the action mechanism of (P)GKY20 and could represent an important contribution to the development of new peptides serving as antimicrobial agents.

Chemical Cleavage of an Asp-Cys Sequence Allows Efficient Production of Recombinant Peptides with an N-Terminal Cysteine Residue.

Peptides with an N-terminal cysteine residue allow site-specific modification of proteins and peptides and chemical synthesis of proteins. They have been widely used to develop new strategies for imaging, drug discovery, diagnostics, and chip technologies. Here we present a method to produce recombinant peptides with an N-terminal cysteine residue as a convenient alternative to chemical synthesis. The method is based on the release of the desired peptide from a recombinant fusion protein by mild acid hydrolysis of an Asp-Cys sequence. To test the general validity of the method we prepared four fusion proteins bearing three different peptides (20-37 amino acid long) at the C-terminus of a ketosteroid isomerase-derived and two Onconase-derived carriers for the production of toxic peptides in E. coli. The chosen peptides were (C)GKY20, an antimicrobial peptide from the C-terminus of human thrombin, (C)ApoBL, an antimicrobial peptide from an inner region of human Apolipoprotein B, and (C)p53pAnt, an anticancer peptide containing the C-terminal region of the p53 protein fused to the cell penetrating peptide Penetratin. Cleavage efficiency of Asp-Cys bonds in the four fusion proteins was studied as a function of pH, temperature, and incubation time. In spite of the differences in the amino acid sequence (GTGDCGKY, GTGDCHVA, GSGTDCGSR, SQGSDCGSR) we obtained for all the proteins a cleavage efficiency of about 70-80% after 24 h incubation at 60 °C and pH 2. All the peptides were produced with very good yield (5-16 mg/L of LB cultures), high purity (>96%), and the expected content of free thiol groups (1 mol per mole of peptide). Furthermore, (C)GKY20 was modified with PyMPO-maleimide, a commercially available fluorophore bearing a thiol reactive group, and with 6-hydroxy-2-cyanobenzothiazole, a reagent specific for N-terminal cysteines, with yields of 100% thus demonstrating that our method is very well suited for the production of fully reactive peptides with an N-terminal cysteine residue.

Structural and functional insights into RHA-P, a bacterial GH106 α-L-rhamnosidase from Novosphingobium sp. PP1Y.

α-L-Rhamnosidases (α-RHAs, EC 3.2.1.40) are glycosyl hydrolases (GHs) hydrolyzing terminal α-l-rhamnose residues from different substrates such as heteropolysaccharides, glycosylated proteins and natural flavonoids. Although the possibility to hydrolyze rhamnose from natural flavonoids has boosted the use of these enzymes in several biotechnological applications over the past decades, to date only few bacterial rhamnosidases have been fully characterized and only one crystal structure of a rhamnosidase of the GH106 family has been described. In our previous work, an α-l-rhamnosidase belonging to this family, named RHA-P, was isolated from the marine microorganism Novosphingobium sp. PP1Y. The initial biochemical characterization highlighted the biotechnological potential of RHA-P for bioconversion applications. In this work, further functional and structural characterization of the enzyme is provided. The recombinant protein was obtained fused to a C-terminal His-tag and, starting from the periplasmic fractions of induced recombinant cells of E. coli strain BL21(DE3), was purified through a single step purification protocol. Homology modeling of RHA-P in combination with a site directed mutagenesis analysis confirmed the function of residues D503, E506, E644, likely located at the catalytic site of RHA-P. In addition, a kinetic characterization of the enzyme on natural flavonoids such as naringin, rutin, hesperidin and quercitrin was performed. RHA-P showed activity on all flavonoids tested, with a catalytic efficiency comparable or even higher than other bacterial α-RHAs described in literature. The results confirm that RHA-P is able to hydrolyze both α-1,2 and α-1,6 glycosidic linkages, and suggest that the enzyme may locate different polyphenolic aromatic moities in the active site.

Antimicrobial potency of cationic antimicrobial peptides can be predicted from their amino acid composition: Application to the detection of "cryptic" antimicrobial peptides.

Cationic antimicrobial peptides (CAMPs) are essential components of innate immunity. Here we show that antimicrobial potency of CAMPs is linearly correlated to the product CmHnL where C is the net charge of the peptide, H is a measure of its hydrophobicity and L its length. Exponents m and n define the relative contribution of charge and hydrophobicity to the antimicrobial potency. Very interestingly the values of m and n are strain specific. The ratio n/(m+n) can vary between ca. 0.5 and 1, thus indicating that some strains are sensitive to highly charged peptides, whereas others are particularly susceptible to more hydrophobic peptides. The slope of the regression line describing the correlation "antimicrobial potency"/"CmHnL product" changes from strain to strain indicating that some strains acquired a higher resistance to CAMPs than others. Our analysis provides also an effective computational strategy to identify CAMPs included inside the structure of larger proteins or precursors, which can be defined as "cryptic" CAMPs. We demonstrate that it is not only possible to identify and locate with very good precision the position of cryptic peptides, but also to analyze the internal structure of long CAMPs, thus allowing to draw an accurate map of the molecular determinants of their antimicrobial activity. A spreadsheet, provided in the Supplementary material, allows performing the analysis of protein sequences. Our strategy is also well suited to analyze large pools of sequences, thus significantly improving the identification of new CAMPs and the study of innate immunity.

Complete sequencing of Novosphingobium sp. PP1Y reveals a biotechnologically meaningful metabolic pattern.

BACKGROUND: Novosphingobium sp. strain PP1Y is a marine α-proteobacterium adapted to grow at the water/fuel oil interface. It exploits the aromatic fraction of fuel oils as a carbon and energy source. PP1Y is able to grow on a wide range of mono-, poly- and heterocyclic aromatic hydrocarbons. Here, we report the complete functional annotation of the whole Novosphingobium genome. RESULTS: PP1Y genome analysis and its comparison with other Sphingomonadal genomes has yielded novel insights into the molecular basis of PP1Y's phenotypic traits, such as its peculiar ability to encapsulate and degrade the aromatic fraction of fuel oils. In particular, we have identified and dissected several highly specialized metabolic pathways involved in: (i) aromatic hydrocarbon degradation; (ii) resistance to toxic compounds; and (iii) the quorum sensing mechanism. CONCLUSIONS: In summary, the unraveling of the entire PP1Y genome sequence has provided important insight into PP1Y metabolism and, most importantly, has opened new perspectives about the possibility of its manipulation for bioremediation purposes.

Methylobacterium populi VP2: plant growth-promoting bacterium isolated from a highly polluted environment for polycyclic aromatic hydrocarbon (PAH) biodegradation.

The use of microorganisms to accelerate the natural detoxification processes of toxic substances in the soil represents an alternative ecofriendly and low-cost method of environmental remediation compared to harmful incineration and chemical treatments. Fourteen strains able to grow on minimal selective medium with a complex mixture of different classes of xenobiotic compounds as the sole carbon source were isolated from the soil of the ex-industrial site ACNA (Aziende Chimiche Nazionali Associate) in Cengio (Savona, Italy). The best putative degrading isolate, Methylobacterium populi VP2, was identified using a polyphasic approach on the basis of its phenotypic, biochemical, and molecular characterisation. Moreover, this strain also showed multiple plant growth promotion activities: it was able to produce indole-3-acetic acid (IAA) and siderophores, solubilise phosphate, and produce a biofilm in the presence of phenanthrene and alleviate phenanthrene stress in tomato seeds. This is the first report on the simultaneous occurrence of the PAH-degrading ability by Methylobacterium populi and its multiple plant growth-promoting activities. Therefore, the selected indigenous strain, which is naturally present in highly contaminated soils, is good candidate for plant growth promotion and is capable of biodegrading xenobiotic organic compounds to remediate contaminated soil alone and/or soil associated with plants.

Bioconversion of 4-hydroxyestradiol by extradiol ring-cleavage dioxygenases from Novosphingobium sp. PP1Y.

Livestock breeding activities and pharmaceutical wastes lead to considerable accumulation of steroid hormones and estrogens in wastewaters. Here estrogens act as pro-cancerogenic agents and endocrine disruptors interfering with the sexual development of aquatic animals and having toxic effects in humans. Environmental bacteria play a vital role in estrogens degradation. Their wide reservoir of enzymes, such as ring cleavage dioxygenases (RCDs), can degrade the steroid nucleus, catalyzing the meta-cleavage of A, B or D steroid rings. In this work, 4 extra-diol ring cleavage dioxygenases (ERCDs), PP28735, PP26077, PP00124 and PP00193, were isolated from the marine sphingomonad Novosphingobium sp. PP1Y and characterized. Enzymes kinetic parameters were determined on different synthetic catecholic substrates. Then, the bioconversion of catechol estrogens was evaluated. PP00124 showed to be an efficient catalyst for the degradation of 4-hydroxyestradiol (4-OHE2), a carcinogenic hydroxylated derivate of E2. 4-OHE2 complete cleavage was obtained using PP00124 both in soluble form and in whole recombinant E. coli cells. LC-MS/MS analyses confirmed the generation of a semialdehyde product, through A-ring meta cleavage. To the best of our knowledge, PP00124 is the first characterized enzyme able to directly degrade 4-OHE2 via meta cleavage. Moreover, the complete 4-OHE2 biodegradation using recombinant whole cells highlighted advantages for bioremediation purposes.

The Antimicrobial, Antibiofilm and Anti-Inflammatory Activities of P13#1, a Cathelicidin-like Achiral Peptoid.

Cationic antimicrobial peptides (CAMPs) are powerful molecules with antimicrobial, antibiofilm and endotoxin-scavenging activities. These properties make CAMPs very attractive drugs in the face of the rapid increase in multidrug-resistant (MDR) pathogens, but they are limited by their susceptibility to proteolytic degradation. An intriguing solution to this issue could be the development of functional mimics of CAMPs with structures that enable the evasion of proteases. Peptoids (N-substituted glycine oligomers) are an important class of peptidomimetics with interesting benefits: easy synthetic access, intrinsic proteolytic stability and promising bioactivities. Here, we report the characterization of P13#1, a 13-residue peptoid specifically designed to mimic cathelicidins, the best-known and most widespread family of CAMPs. P13#1 showed all the biological activities typically associated with cathelicidins: bactericidal activity over a wide spectrum of strains, including several ESKAPE pathogens; the ability to act in combination with different classes of conventional antibiotics; antibiofilm activity against preformed biofilms of Pseudomonas aeruginosa, comparable to that of human cathelicidin LL-37; limited toxicity; and an ability to inhibit LPS-induced proinflammatory effects which is comparable to that of "the last resource" antibiotic colistin. We further studied the interaction of P13#1 with SDS, LPSs and bacterial cells by using a fluorescent version of P13#1. Finally, in a subcutaneous infection mouse model, it showed antimicrobial and anti-inflammatory activities comparable to ampicillin and gentamicin without apparent toxicity. The collected data indicate that P13#1 is an excellent candidate for the formulation of new antimicrobial therapies.

Human Cryptic Host Defence Peptide GVF27 Exhibits Anti-Infective Properties against Biofilm Forming Members of the Burkholderia cepacia Complex.

Therapeutic solutions to counter Burkholderia cepacia complex (Bcc) bacteria are challenging due to their intrinsically high level of antibiotic resistance. Bcc organisms display a variety of potential virulence factors, have a distinct lipopolysaccharide naturally implicated in antimicrobial resistance. and are able to form biofilms, which may further protect them from both host defence peptides (HDPs) and antibiotics. Here, we report the promising anti-biofilm and immunomodulatory activities of human HDP GVF27 on two of the most clinically relevant Bcc members, Burkholderia multivorans and Burkholderia cenocepacia. The effects of synthetic and labelled GVF27 were tested on B. cenocepacia and B. multivorans biofilms, at three different stages of formation, by confocal laser scanning microscopy (CLSM). Assays on bacterial cultures and on human monocytes challenged with B. cenocepacia LPS were also performed. GVF27 exerts, at different stages of formation, anti-biofilm effects towards both Bcc strains, a significant propensity to function in combination with ciprofloxacin, a relevant affinity for LPSs isolated from B. cenocepacia as well as a good propensity to mitigate the release of pro-inflammatory cytokines in human cells pre-treated with the same endotoxin. Overall, all these findings contribute to the elucidation of the main features that a good therapeutic agent directed against these extremely leathery biofilm-forming bacteria should possess.

The marine isolate Novosphingobium sp. PP1Y shows specific adaptation to use the aromatic fraction of fuels as the sole carbon and energy source.

Novosphingobium sp. PP1Y, isolated from a surface seawater sample collected from a closed bay in the harbour of Pozzuoli (Naples, Italy), uses fuels as its sole carbon and energy source. Like some other Sphingomonads, this strain can grow as either planktonic free cells or sessile-aggregated flocks. In addition, this strain was found to grow as biofilm on several types of solid and liquid hydrophobic surfaces including polystyrene, polypropylene and diesel oil. Strain PP1Y is not able to grow on pure alkanes or alkane mixtures but is able to grow on a surprisingly wide range of aromatic compounds including mono, bi, tri and tetracyclic aromatic hydrocarbons and heterocyclic compounds. During growth on diesel oil, the organic layer is emulsified resulting in the formation of small biofilm-coated drops, whereas during growth on aromatic hydrocarbons dissolved in paraffin the oil layer is emulsified but the drops are coated only if the mixtures contain selected aromatic compounds, like pyrene, propylbenzene, tetrahydronaphthalene and heterocyclic compounds. These peculiar characteristics suggest strain PP1Y has adapted to efficiently grow at the water/fuel interface using the aromatic fraction of fuels as the sole carbon and energy source.

Transglutaminase-mediated crosslinking of a host defence peptide derived from human apolipoprotein B and its effect on the peptide antimicrobial activity.

Background Microbial transglutaminase (mTG) has been successfully used to produce site-specific protein conjugates derivatized at the level of Gln and/or Lys residues for different biotechnological applications. Here, a recombinant peptide identified in human apolipoprotein B sequence, named r(P)ApoBL and endowed with antimicrobial activity, was studied as a possible acyl acceptor substrate of mTG with at least one of the six Lys residues present in its sequence. Methods The enzymatic crosslinking reaction was performed in vitro using N,N-dimethylcasein, substance P and bitter vetch (Vicia ervilia) seed proteins, well known acyl donor substrates in mTG-catalyzed reactions. Mass spectrometry analyses were performed for identifying the Lys residue(s) involved in the crosslinking reaction. Finally, bitter vetch protein-based antimicrobial films grafted with r(P)ApoBL were prepared and, their biological activity evaluated. Results r(P)ApoBL was able to be enzymatically modified by mTG. In particular, it was demonstrated the highly selective crosslinking of the peptide under study by mTG at level of Lys-18. Interestingly, the biological activity of the peptide when grafted into protein-based films was found to be lost following mTG-catalyzed crosslinking. Conclusions r(P)ApoBL was shown to be an effective acyl acceptor substrate of mTG. The involvement of Lys-18 in the enzymatic reaction was demonstrated. In addition, films grafted with r(P)ApoBL in the presence of mTG lost antimicrobial property. General significance A possible role of mTG as biotechnological tool to modulate the r(P)ApoBL antimicrobial activity was hypothesized, and a potential use in food packaging of protein-based films grafted with r(P)ApoBL was suggested.

Enzymes as a Reservoir of Host Defence Peptides.

Host defence peptides (HDPs) are powerful modulators of cellular responses to various types of insults caused by pathogen agents. To date, a wide range of HDPs, from species of different kingdoms including bacteria, plant and animal with extreme diversity in structure and biological activity, have been described. Apart from a limited number of peptides ribosomally synthesized, a large number of promising and multifunctional HDPs have been identified within protein precursors, with properties not necessarily related to innate immunity, consolidating the fascinating hypothesis that proteins have a second or even multiple biological mission in the form of one or more bio-active peptides. Among these precursors, enzymes constitute certainly an interesting group, because most of them are mainly globular and characterized by a fine specific internal structure closely related to their catalytic properties and also because they are yet little considered as potential HDP releasing proteins. In this regard, the main aim of the present review is to describe a panel of HDPs, identified in all canonical classes of enzymes, and to provide a detailed description on hydrolases and their corresponding HDPs, as there seems to exist a striking link between these structurally sophisticated catalysts and their high content in cationic and amphipathic cryptic peptides.

Antimicrobial peptide Temporin-L complexed with anionic cyclodextrins results in a potent and safe agent against sessile bacteria.

Concern over antibiotic resistance is growing, and new classes of antibiotics, particularly against Gram-negative bacteria, are needed. Antimicrobial peptides (AMPs) have been proposed as a new class of clinically useful antimicrobials. Special attention has been devoted to frog-skin temporins. In particular, temporin L (TL) is strongly active against Gram-positive, Gram-negative bacteria and yeast strains. With the aim of overcoming some of the main drawbacks preventing the widespread clinical use of this peptide, i.e. toxicity and unfavorable pharmacokinetics profile, we designed new formulations combining TL with different types of cyclodextrins (CDs). TL was associated to a panel of neutral or negatively charged, monomeric and polymeric CDs. The impact of CDs association on TL solubility, as well as the transport through bacterial alginates was assessed. The biocompatibility on human cells together with the antimicrobial and antibiofilm properties of TL/CD systems was explored.

Molecular determinants of the regioselectivity of toluene/o-xylene monooxygenase from Pseudomonas sp. strain OX1.

Bacterial multicomponent monooxygenases (BMMs) are a heterogeneous family of di-iron monooxygenases which share the very interesting ability to hydroxylate aliphatic and/or aromatic hydrocarbons. Each BMM possesses defined substrate specificity and regioselectivity which match the metabolic requirements of the strain from which it has been isolated. Pseudomonas sp. strain OX1, a strain able to metabolize o-, m-, and p-cresols, produces the BMM toluene/o-xylene monooxygenase (ToMO), which converts toluene to a mixture of o-, m-, and p-cresol isomers. In order to investigate the molecular determinants of ToMO regioselectivity, we prepared and characterized 15 single-mutant and 3 double-mutant forms of the ToMO active site pocket. Using the Monte Carlo approach, we prepared models of ToMO-substrate and ToMO-reaction intermediate complexes which allowed us to provide a molecular explanation for the regioselectivities of wild-type and mutant ToMO enzymes. Furthermore, using binding energy values calculated by energy analyses of the complexes and a simple mathematical model of the hydroxylation reaction, we were able to predict quantitatively the regioselectivities of the majority of the variant proteins with good accuracy. The results show not only that the fine-tuning of ToMO regioselectivity can be achieved through a careful alteration of the shape of the active site but also that the effects of the mutations on regioselectivity can be quantitatively predicted a priori.

Cost-effective production of recombinant peptides in Escherichia coli.

Among bioactive peptides, cationic antimicrobial peptides (AMPs), also referred to as host defence peptides (HDPs), are valuable tools to treat infections, being able to kill a wide variety of microbes directly and/or modulate host immunity. HDPs have great therapeutic potential against antibiotic-resistant bacteria, viruses and even parasites. However, high manufacturing costs have greatly limited their development as drugs, thus highlighting the need to develop novel and competitive production strategies. Here, a cost-effective procedure was established to produce the high amounts of peptides required for basic and clinical research. Firstly, a novel culture medium was designed, which was found to support significantly higher cell densities and recombinant expression levels of peptides under test compared to conventional media. The procedure has been also efficiently scaled up by using a 5 L fermenter, while the costs have been lowered significantly by developing a successful auto-induction strategy, which has been found to support higher yields of target constructs and cell biomass compared to conventional strategies based on expression induction by IPTG. Interestingly, it was estimated that by increasing production scale from 100 to 1000 mg/batch, unit costs decreased strongly from 253 to 42 €/mg. These costs appear highly competitive when compared to chemical synthesis strategies. Altogether, the data indicate that the strategy represents an important starting point for the future development of large-scale manufacture of HDPs.