Nonribosomal peptide synthetases and their biotechnological potential in Penicillium rubens

Nonribosomal peptide synthetases (NRPSs) are large multimodular enzymes that synthesize a diverse variety of peptides. Many of these are currently used as pharmaceuticals, thanks to their activity as antimicrobials (penicillin, vancomycin, daptomycin, echinocandin), immunosuppressant (cyclosporin) and anticancer compounds (bleomycin). Because of their biotechnological potential, NRPSs have been extensively studied in the past decades. In this review, we provide an overview of the main structural and functional features of these enzymes, and we consider the challenges and prospects of engineering NRPSs for the synthesis of novel compounds. Furthermore, we discuss secondary metabolism and NRP synthesis in the filamentous fungus Penicillium rubens and examine its potential for the production of novel and modified β-lactam antibiotics

Genome sequencing and molecular networking analysis of the wild fungusAnthostomella pineareveal its ability to produce a diverse range of secondary metabolites

Background Filamentous fungi are prolific producers of bioactive molecules and enzymes with important applications in industry. Yet, the vast majority of fungal species remain undiscovered or uncharacterized. Here we focus our attention to a wild fungal isolate that we identified as Anthostomella pinea. The fungus belongs to a complex polyphyletic genus in the family of Xylariaceae, which is known to comprise endophytic and pathogenic fungi that produce a plethora of interesting secondary metabolites. Despite that, Anthostomella is largely understudied and only two species have been fully sequenced and characterized at a genomic level.Results In this work, we used long-read sequencing to obtain the complete 53.7 Mb genome sequence including the full mitochondrial DNA. We performed extensive structural and functional annotation of coding sequences, including genes encoding enzymes with potential applications in biotechnology. Among others, we found that the genome of A. pinea encodes 91 biosynthetic gene clusters, more than 600 CAZymes, and 164 P450s. Furthermore, untargeted metabolomics and molecular networking analysis of the cultivation extracts revealed a rich secondary metabolism, and in particular an abundance of sesquiterpenoids and sesquiterpene lactones. We also identified the polyketide antibiotic xanthoepocin, to which we attribute the anti–Gram-positive effect of the extracts that we observed in antibacterial plate assays.Conclusions Taken together, our results provide a first glimpse into the potential of Anthstomella pinea to provide new bioactive molecules and biocatalysts and will facilitate future research into these valuable metabolites

Biochemical characterization of the Nocardia lactamdurans ACV synthetase

The L-δ-(α-aminoadipoyl)-L-cysteinyl-D-valine synthetase (ACVS) is a nonribosomal peptide synthetase (NRPS) that fulfills a crucial role in the synthesis of β-lactams. Although some of the enzymological aspects of this enzyme have been elucidated, its large size, at over 400 kDa, has hampered heterologous expression and stable purification attempts. Here we have successfully overexpressed the Nocardia lactamdurans ACVS in E. coli HM0079. The protein was purified to homogeneity and characterized for tripeptide formation with a focus on the substrate specificity of the three modules. The first L-α-aminoadipic acid-activating module is highly specific, whereas the modules for L-cysteine and L-valine are more promiscuous. Engineering of the first module of ACVS confirmed the strict specificity observed towards its substrate, which can be understood in terms of the non-canonical peptide bond position

Dimethylmyricacene: An In Vitro and In Silico Study of a Semisynthetic Non-Camptothecin Derivative Compound, Targeting Human DNA Topoisomerase 1B

Human topoisomerase 1B regulates the topological state of supercoiled DNA enabling all fundamental cell processes. This enzyme, which is the unique molecular target of the natural anticancer compound camptothecin, acts by nicking one DNA strand and forming a transient protein–DNA covalent complex. The interaction of human topoisomerase 1B and dimethylmyricacene, a compound prepared semisynthetically from myricanol extracted from Myrica cerifera root bark, was investigated using enzymatic activity assays and molecular docking procedures. Dimethylmyricacene was shown to inhibit both the cleavage and the religation steps of the enzymatic reaction, and cell viability of A-253, FaDu, MCF-7, HeLa and HCT-116 tumor cell lines

A framework for deriving semantic web services

Web service-based development represents an emerging approach for the development of distributed information systems. Web services have been mainly applied by software practitioners as a means to modularize system functionality that can be offered across a network (e.g., intranet and/or the Internet). Although web services have been predominantly developed as a technical solution for integrating software systems, there is a more business-oriented aspect that developers and enterprises need to deal with in order to benefit from the full potential of web services in an electronic market. This ‘ignored’ aspect is the representation of the semantics underlying the services themselves as well as the ‘things’ that the services manage. Currently languages like the Web Services Description Language (WSDL) provide the syntactic means to describe web services, but lack in providing a semantic underpinning. In order to harvest all the benefits of web services technology, a framework has been developed for deriving business semantics from syntactic descriptions of web services. The benefits of such a framework are two-fold. Firstly, the framework provides a way to gradually construct domain ontologies from previously defined technical services. Secondly, the framework enables the migration of syntactically defined web services toward semantic web services. The study follows a design research approach which (1) identifies the problem area and its relevance from an industrial case study and previous research, (2) develops the framework as a design artifact and (3) evaluates the application of the framework through a relevant scenario

In vitro and in silico characterization of an antimalarial compound with antitumor activity targeting human DNA topoisomerase IB

Human DNA topoisomerase IB controls the topological state of supercoiled DNA through a complex catalytic cycle that consists of cleavage and religation reactions, allowing the progression of fundamental DNA metabolism. The catalytic steps of human DNA topoisomerase IB were analyzed in the presence of a drug, obtained by the open-access drug bank Medicines for Malaria Venture. The experiments indicate that the compound strongly and irreversibly inhibits the cleavage step of the enzyme reaction and reduces the cell viability of three different cancer cell lines. Molecular docking and molecular dynamics simulations suggest that the drug binds to the human DNA topoisomerase IB-DNA complex sitting inside the catalytic site of the enzyme, providing a molecular explanation for the cleavage-inhibition effect. For all these reasons, the aforementioned drug could be a possible lead compound for the development of an efficient anti-tumor molecule targeting human DNA topoisomerase IB

The influence of gravimetric moisture content on studded shoe–surface interactions in soccer

It is desirable for the studs of a soccer shoe to penetrate the sport surface and provide the player with sufficient traction when accelerating. Mechanical tests are often used to measure the traction of shoe–surface combinations. Mechanical testing offers a repeatable measure of shoe–surface traction, eliminating the inherent uncertainties that exist when human participant testing is employed, and are hence used to directly compare the performance of shoe–surface combinations. However, the influence specific surface characteristics has on traction is often overlooked. Examining the influence of surface characteristics on mechanical test results improves the understanding of the traction mechanisms at the shoe–surface interface. This allows footwear developers to make informed decisions on the design of studded outsoles. The aim of this paper is to understand the effect gravimetric moisture content has on the tribological mechanisms at play during stud–surface interaction. This study investigates the relationships between: the gravimetric moisture content of a natural sand-based soccer surface; surface stiffness measured via a bespoke impact test device; and surface traction measured via a bespoke mechanical test device. Regression analysis revealed that surface stiffness decreases linearly with increased gravimetric moisture content (p = 0.04). Traction was found to initially increase and then decrease with gravimetric moisture content. It was observed that: a surface of low moisture content provides low stud penetration and therefore reduced traction; a surface of high moisture content provides high stud penetration but also reduced traction due to a lubricating effect; and surfaces with moisture content in between the two extremes provide increased traction. In this study a standard commercially available stud was used and other studs may provide slightly different results. The results provide insight into the traction mechanisms at the stud–surface interface which are described in the paper. The variation between traction measurements shows the influence gravimetric moisture content will have on player performance. This highlights the requirement to understand surface conditions prior to making comparative shoe–surface traction studies and the importance of using a studded outsole that is appropriate to the surface condition during play

Regulation of mGlu4 metabotropic glutamate receptor signaling by type-2 G-protein coupled receptor kinase (GRK2

ABSTRACT We examined the role of G-protein coupled receptor kinase-2 (GRK2) in the homologous desensitization of mGlu4 metabotropic glutamate receptors transiently expressed in human embryonic kidney (HEK) 293 cells. Receptor activation with the agonist L-2-amino-4-phosphonobutanoate (L-AP4) stimulated at least two distinct signaling pathways: inhibition of cAMP formation and activation of the mitogen-activated protein kinase (MAPK) pathway [assessed by Western blot analysis of phosphorylated extracellular signal-regulated kinase (ERK) 1 and 2]. Activation of both pathways was attenuated by pertussis toxin. Overexpression of GRK2 (but not GRK4) largely attenuated the stimulation of the MAPK pathway by L-AP4, whereas it slightly potentiated the inhibition of FSK-stimulated cAMP formation. Transfection with a kinase-dead mutant of GRK2 (GRK2-K220R) or with the C-terminal fragment of GRK2 also reduced the mGlu4-mediated stimulation of MAPK, suggesting that GRK2 binds to the G␤␥ subunits to inhibit signal propagation toward the MAPK pathway. This was confirmed by the evidence that GRK2 coimmunoprecipitated with G␤␥ subunits in an agonist-dependent manner. Finally, neither GRK2 nor its kinase-dead mutant had any effect on agonist-induced mGlu4 receptor internalization in HEK293 cells transiently transfected with GFP-tagged receptors. Agonist-dependent internalization was instead abolished by a negative-dominant mutant of dynamin, which also reduced the stimulation of MAPK pathway by L-AP4. We speculate that GRK2 acts as a "switch molecule" by inhibiting the mGlu4 receptor-mediated stimulation of MAPK and therefore directing the signal propagation toward the inhibition of adenylyl cyclase. Metabotropic glutamate (mGlu) receptors, which belong to the third class of the G protein-coupled receptor (GPCR) superfamily, modulate excitatory synaptic transmission and are implicated in different aspects of central nervous system physiology, including motor control, motor coordination, sensory perception and pain transmission, learning and memory processes, and developmental plasticity Homologous desensitization of GCPRs is mediated by a family of enzymes called G-protein coupled receptor kinases (GRKs). This family includes GRK1, which corresponds to rhodopsin kinase, GRK2, and -3, which are ubiquitous and are activated by G-protein ␤␥ subunits, and GRK4, -5, and -6

The comparative effects of mGlu5 receptor positive allosteric modulators VU0409551 and VU0360172 on cognitive deficits and signalling in the sub-chronic PCP rat model for schizophrenia

The metabotropic glutamate receptor mGlu5 has been implicated in the neuropathology of several debilitating disorders. In schizophrenia, mGlu5 receptor hypofunction has been linked with neuropathology and cognitive deficits, making it an attractive therapeutic target. The cognitive impairment associated with schizophrenia remains an unmet clinical need, with existing antipsychotics primarily targeting positive symptoms and failing to induce consistent, substantial improvements in cognition, thus providing incomplete treatment. Using the sub-chronic phencyclidine (scPCP) rat model, widely shown to mimic the cognitive impairment and neuropathology of schizophrenia, we have investigated two mGlu5 receptor positive allosteric modulators (PAMs), VU0409551 and VU0360172. We compared the efficacy of these compounds in restoring cognitive deficits and, since these two PAMs have reportedly distinct signalling mechanisms, changes in mGlu5 receptor signalling molecules AKT and MAPK in the prefrontal cortex. Although not effective at 0.05 and 1mg/kg, cognitive deficits were significantly alleviated by both PAMs at 10 and 20 mg/kg. The compounds appeared to have differential effects on the scPCP-induced increases in AKT and MAPK phosphorylation: VU0409551 induced a significant decrease in expression of p-AKT, whereas VU0360172 had this effect on p-MAPK levels. Thus, the beneficial effects of PAMs on scPCP-induced cognitive impairment are accompanied by at least partial reversal of scPCP-induced elevated levels of p-MAPK and p-AKT, dysfunction of which is strongly implicated in schizophrenia pathology. These promising data imply an important role for mGlu5 receptor signalling pathways in improving cognition in the scPCP model and provide support for mGlu5 receptor PAMs as a possible therapeutic intervention for schizophrenia