Giuseppe Forlani - Academia.edu (original) (raw)

Papers by Giuseppe Forlani

Journal of Agricultural and Food Chemistry, Jul 3, 2013

Analogues of previously studied phenyl-substituted aminomethylene-bisphosphonic acids were synthe... more Analogues of previously studied phenyl-substituted aminomethylene-bisphosphonic acids were synthesized and evaluated as inhibitors of Arabidopsis thaliana δ 1-pyrroline-5-carboxylate reductase. With the aim of improving their effectiveness, two main modifications were introduced into the inhibitory scaffold: the aminomethylenebisphosphonic moiety was replaced with a hydroxymethylenebisphosphonic group, and the length of the molecule was increased by replacing the methylene linker with an ethylidene chain. In addition, chlorine atoms in the phenyl ring were replaced with various other substituents. Most of the studied derivatives showed activity in the micromolar to millimolar range, with two of them being more effective than the lead compound, with concentrations inhibiting 50% of enzyme activity as low as 50 μM. Experimental evidence supporting the ability of these inhibitors to interfere with proline synthesis in vivo is also shown.

Journal of Agricultural and Food Chemistry, Apr 10, 2008

Using the structure of (3,5-dichlorophenyl)aminomethylenebisphosphonic acid as a lead compound, 2... more Using the structure of (3,5-dichlorophenyl)aminomethylenebisphosphonic acid as a lead compound, 25 new phosphonates were synthesized and evaluated as possible inhibitors of Arabidopsis thaliana δ 1-pyrroline-5-carboxylate (P5C) reductase. Derivatives substituted in the phenyl ring retained the inhibitory potential, though to a different extent. On the contrary any variation in the scaffold, i.e., the replacement of the second phosphonate moiety with a hydroxyl or an amino residue, resulted in a significant loss of biological activity. The availability of several structures capable of interfering with the catalytic mechanism in the micromolar to millimolar range allowed a proper structure-activity relationship analysis, leading us to hypothesize about the steric and electronic requirements for maintenance or enhancement of the inhibitory properties. Reversal experiments with suspension cultured cells provided evidence for the occurrence of enzyme inhibition in vivo. Because in higher plants the step catalyzed by P5C reductase is shared by all pathways leading to proline synthesis, these compounds may be exploited for the design of new substances endowed with herbicidal activity.

Journal of Agricultural and Food Chemistry, May 1, 2007

A series of N-substituted derivatives of aminomethylenebisphosphonic acid were evaluated as poten... more A series of N-substituted derivatives of aminomethylenebisphosphonic acid were evaluated as potential inhibitors of δ 1-pyrroline-5-carboxylate reductase (EC 1.5.1.2), the enzyme that catalyzes the last step in proline biosynthesis, partially purified from Arabidopsis thaliana suspension cultured cells. At millimolar concentrations, three compounds out of 26 were found to interfere with the catalytic mechanism. One of them, namely, 3,5-dichloropyridyl-aminomethylenebisphosphonic acid, retained such inhibitory activity in the micromolar range. Kinetic analyses ruled out the possibility that the inhibition could simply rely upon the chelating properties of bisphosphonates and showed mechanisms of a noncompetitive type against NADH and an uncompetitive type against δ 1-pyrroline-5-carboxylic acid, with K I values of 199 (6 and 10.3 (1.5 µM, respectively. A computer-aided docking analysis, performed on the basis of the crystal structure of the enzyme from Streptococcus pyogenes, suggested that this phosphonate may interact with amino acid residues near the binding site of δ 1-pyrroline-5carboxylic acid, thus blocking the substrate in a pocket and preventing its interaction with NADH. Because in higher plants the step catalyzed by δ 1-pyrroline-5-carboxylate reductase is shared by all pathways leading to proline synthesis, such a compound may represent a lead structure to be exploited for the design of new substances endowed with herbicidal activity.

Pest Management Science, Aug 21, 2009

BACKGROUND: Aiming at the rational design of new herbicides, the availability of the threedimensi... more BACKGROUND: Aiming at the rational design of new herbicides, the availability of the threedimensional structure of the target enzyme greatly enhances the optimisation of lead compounds and the design of derivatives with increased activity. Among the most widely exploited herbicide targets is glutamine synthetase. Recently, the structure of a cytosolic form of the maize enzyme has been described, making it possible to verify whether steric, electronic and hydrophobic features of a compound are in agreement with inhibitor-protein interaction geometry. RESULTS: Three series of compounds (aminophosphonates, hydroxyphosphonates and aminomethylenebisphosphonates) were evaluated as possible inhibitors of maize glutamine synthetase. Aminomethylenebisphosphonate derivatives substituted in the phenyl ring retained the inhibitory potential, whereas variations in the scaffold, i.e. the replacement of the second phosphonate moiety with a hydroxyl or an amino residue, resulted in a significant loss of activity. A kinetic characterisation showed a non-competitive mechanism against glutamate and an uncompetitive mechanism against ATP. A docking analysis suggested the mode of bisphosphonate binding to the active site. CONCLUSION: Results made it possible to define the features required to maintain or enhance the biological activity of these compounds, which represent lead structures to be further exploited for the design of new substances endowed with herbicidal activity.

Pest Management Science, 2001

A Penicillium chrysogenum strain was isolated for its ability to grow in minimal medium containin... more A Penicillium chrysogenum strain was isolated for its ability to grow in minimal medium containing the herbicide glyphosate as the only nitrogen source. The presence of concentrations up to 25 mM progressively stimulated the fungal growth rate, which was negligible in media lacking reduced nitrogen. However, glyphosate utilization never exceeded 1 mmol g À1 mycelial dry mass, and below a threshold concentration both herbicide uptake and fungal growth were subject to a lag phase, suggesting that the herbicide may enter the cell by either simple passive diffusion or inducible carriers. Amino acids, possible products of glyphosate breakdown, as well as ammonia, were found to replace the herbicide in restoring mycelial growth. Cells were devoid of detectable nitrate reductase activity, thus the isolate seems to be impaired in its ability to convert nitrate to ammonium. In vitro activity of 5-enol-pyruvyl-shikimate-3-phosphate synthase, the target site of glyphosate action, was highly sensitive to the herbicide. Fungal growth rate was considerably lower when the herbicide was also the only phosphorus source, whereas glyphosate utilization was substantially unaffected, suggesting an unusual route for its degradation. Herbicide metabolism was strongly reduced when other sources of organic nitrogen were made available.

Biology, May 6, 2023

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

Journal of Agricultural and Food Chemistry, Dec 13, 2017

Abenquines are natural N-acetylaminobenzoquinones bearing amino acids residues, which act as weak... more Abenquines are natural N-acetylaminobenzoquinones bearing amino acids residues, which act as weak inhibitors of the photosynthetic electron transport chain. Aiming to exploit the abenquine scaffold as a model for the synthesis of new herbicides targeting photosynthesis, fourteen new analogues were prepared by replacing the amino acid residue with benzylamines and the acetyl with different acyl groups. The synthesis was accomplished in three steps with a 68-95% overall yield from readily available 2,5dimethoxyaniline, acyl chlorides and benzyl amines. Key steps include (i) acylation of the aniline, (ii) oxidation, and (iii) oxidative addition of the benzylamino moiety. The compounds were assayed for their activity as Hill inhibitors, under basal, uncoupled or phosphorylating conditions, or excluding photosystem I. Four analogues showed high effectiveness (IC 50 = 0.1-0.4 µM), comparable with the commercial herbicide diuron (IC 50 = 0.3 µM). The data suggest that this class of compounds interfere at the reducing side of photosystem II, having protein D1 as the most probable target. Molecular docking studies with the plastoquinone binding site of Spinacia oleracea further strengthened this proposal.

New Phytologist, Aug 1, 2001

ABSTRACT Summary • Isolation and biochemical characterization is reported here of 5-enol-pyruvyl-... more ABSTRACT Summary • Isolation and biochemical characterization is reported here of 5-enol-pyruvyl-shikimate-3-phosphate (EPSP) synthase, the enzyme that catalyses the sixth step in the common prechorismate pathway of aromatic amino acid biosynthesis and the target of the widely used herbicide glyphosate, from the cyanobacterium Spirulina platensis. • Homogeneous enzyme preparations were obtained by ammonium sulphate fractionation, anion-exchange and substrate-elution chromatography, and chromatofocusing. Protein characterization was carried out by conventional kinetic analysis, PAGE and gel permeation. • A 2800-fold purification was achieved, with a recovery of 20% of initial activity. Unusually low apparent affinities for both substrates, phosphoenolpyruvate and shikimate-3-phosphate, did not correspond to decreased glyphosate sensitivity. During SDS-PAGE, the protein migrated as a single band corresponding to a molecular mass of c. 49 kDa. The behaviour of the protein upon gel permeation chromatography under nondenaturing conditions was, however, consistent with a mass of c. 91 kDa. • The native enzyme appears to be homodimeric, a remarkable feature that has not been previously reported for EPSP synthases from either cyanobacteria or higher plants. The presence of mono- and dimeric EPSP synthases could represent an important tool for cyanobacterial classification.

Plant Physiology and Biochemistry, Mar 1, 2000

Glutamine synthetase (EC 6.3.1.2) was purified and characterized from leaf protoplast-derived sus... more Glutamine synthetase (EC 6.3.1.2) was purified and characterized from leaf protoplast-derived suspension cultured cells of Nicotiana plumbaginifolia. Maximal specific activity observed reflected a purification of over 1 500-fold, with a yield of about 40 %. The native protein appeared to be an octamer, with subunits of a molecular mass of 37 kDa. Subcellular fractionation experiments accounted for a cytosolic localization of the enzyme. No evidence for multiple enzyme forms was found following either anion-exchange chromatography or native polyacrylamide gel electrophoresis. Results also suggest that in the absence of intercellular transport, type-1 glutamine synthetase may function preferentially to assimilate ammonia from primary nitrate reduction. © 2000 Éditions scientifiques et médicales Elsevier SAS Ammonia assimilation / cultured cells / glutamine synthetase isoenzymes / Nicotiana plumbaginifolia / subunit composition GS(-1,-2), glutamine synthetase (cytosolic, plastidial)

Amino Acids, Jul 9, 2011

Compounds able to interfere with amino acid biosynthesis have the potential to inhibit cell growt... more Compounds able to interfere with amino acid biosynthesis have the potential to inhibit cell growth. In both prokaryotic and eukaryotic microorganisms, unless an ornithine cyclodeaminase is present, the activity of 1-pyrroline-5-carboxylate (P5C) reductase is mandatory to proline production, and the enzyme inhibition should result in amino acid starvation, blocking in turn protein synthesis. The ability of some substituted derivatives of aminomethylenebisphosphonic acid and its analogues to interfere with the activity of the enzyme from the human pathogen Streptococcus pyogenes was investigated. Several compounds were able to suppress activity in the micromolar range of concentrations, with a mechanism of uncompetitive type with respect to the substrate P5C and non-competitive with respect to the electron donor NAD(P)H. The actual occurrence of

Journal of Plant Physiology, 1997

Two isoforms of the shikimate pathway enzyme 5-enol-pyruvyl-shikimate-3-phosphate synthase, previ... more Two isoforms of the shikimate pathway enzyme 5-enol-pyruvyl-shikimate-3-phosphate synthase, previously purified from maize cultured cells and both found to be functionally located in the plastid while showing a different pattern of expression during the culture growth cycle, were characterized with respect to physical and functional properties. A high degree of similarity was found as to their structural features, with the only exception of a slight difference in molecular mass. Both enzyme activities were extremely susceptible to the inhibition brought about by the herbicide glyphosate, and not subjected to feedback regulation by aromatic amino acids or shikimate pathway intermediates. A more pronounced difference was evident in thermal stability, catalytic efficiency as judged from the comparison of catalytic constants, affinities for the two substrates and activation energy values. The isozyme detectable in actively proliferating cells, when the plant cell demand for aromatic amino acids increases, proved to be the more stable and efficient. Data are consistent with the hypothesis of an isoform-based mechanism of enzyme level modulation in plant aromatic metabolism.

Springer eBooks, 2019

The accumulation of proline is a conserved response of plants to abiotic stress conditions. Moreo... more The accumulation of proline is a conserved response of plants to abiotic stress conditions. Moreover, the activation of proline metabolism takes place during the plant response to some pathogens. Although these responses are well documented, the molecular and physiological functions of proline accumulation under stress are still a matter of debate. The biochemical pathways that lead to proline accumulation and its functions in regulating development are described in the cognate chapter “Proline Metabolism and Its Functions in Development and Tolerance to Stress”. In this chapter, we will describe the potential roles assigned to proline accumulation, dissecting the data coming from in vitro/in silico and in vivo approaches and those coming from bacterial or unicellular eukaryotes and plants. With this, we aim to present a clear view of the evidence related to the molecular and physiological functions of proline accumulation under stress conditions in plants. In recent years, the understanding of the regulation of proline accumulation at transcriptional level under stress conditions in plants has been increased considerably, yet little is known about the possible occurrence of post-translational regulatory mechanisms. We will integrate this knowledge with the potential roles of proline accumulation to see whether it contributes to comprehending which roles might be physiologically more relevant.

Journal of Medicinal Chemistry, Sep 14, 2005

A new group of potent inhibitors of glutamine synthetase was designed and synthesized. The X-ray ... more A new group of potent inhibitors of glutamine synthetase was designed and synthesized. The X-ray structure of bacterial glutamine synthetase complexed with phosphinothricin was used for computer-aided structure-based design of the inhibitors, in which the methyl group of phosphinothricin was chosen as the modification site. Amino and hydroxyl moieties were introduced into the phosphinic acid portion of the lead molecule to interact with ammonium binding site in the active cleft of the enzyme. Designed compounds were synthesized in enantiomerically pure form analogous to l-glutamic acid. In vitro kinetic studies with Escherichia coli glutamine synthetase confirmed the biological activity of the designed inhibitors, which with K(i) values in the micromolar range (K(i) = 0.59 microM for the most potent compound 2) appear to be slightly weaker inhibitors or equipotent to phosphinothricin.

Zenodo (CERN European Organization for Nuclear Research), Jan 21, 2021

The availability of molecular markers able to distinguish drug-type from fiber-type Cannabis sati... more The availability of molecular markers able to distinguish drug-type from fiber-type Cannabis sativa cultivars would allow fast and cheap analysis of any plant specimen, including seeds and leaves. Several approaches to this issue have been described, mainly using polymorphisms in the genes coding for tetrahydrocannabinol acid synthase or cannabidiolic acid synthase. Some studies reported sequencing of these genes from small groups of hemp varieties belonging to both chemotypes, showing the occurrence of specific DNA signatures. However, the effectiveness of the corresponding primers to discriminate among chemotypes has been validated on a limited number of cultivars, or not tested at all. Here we report a thorough in silico analysis of available gene sequences for both synthases, showing the existence of hypervariable regions at 3' and 5' ends. This notwithstanding, some possible signatures were identified, and 12 putatively specific primer pairs were designed and tested on 16 fibertype and 11 drug-type varieties. In most cases inconsistent results were obtained, further strengthening the high genetic variability of these genes in hemp germplasm, yet some highly informative polymorphisms were identified. Potentiality and perspectives of this approach are discussed.

Dual choice feeding tests were performed to verify a preference of forager honeybees for artifici... more Dual choice feeding tests were performed to verify a preference of forager honeybees for artificial nectars supplemented with various amino acids. Proline-containing nectar was preferred over a solution containing only sugars. Daily individual consumption was initially higher also for alanine-containing nectar, but the difference became not statistically significant afterwards. On the contrary, a negative response was found for serine. When the test was carried out with two nectars enriched with different amino acids, the same preference hierarchy was evident

Giornale botanico italiano, 1995

... of an acetolactate decarboxy-lase has been demonstrated in several bacteria species; on ... o... more ... of an acetolactate decarboxy-lase has been demonstrated in several bacteria species; on ... of four plant species were fractionated by adsorption chromatography onto a hydroxyapatite column. ... providing fkrther evidence that the plant enzyme catalysed also acetoin synthesis as a ...

Computational and structural biotechnology journal, 2022

Graphical abstract

Journal of Agricultural and Food Chemistry, Jan 6, 2006

A series of phosphinothricin derivatives with a modified methyl group, designed on the basis of t... more A series of phosphinothricin derivatives with a modified methyl group, designed on the basis of the crystal structure of the complex formed by the inhibitor and the target enzyme from Salmonella typhimurium, were evaluated as potential inhibitors of plant glutamine synthetase. These compounds were previously shown to be equipotent or slightly weaker inhibitors to the lead compound against the bacterial enzyme. Because of the presence in higher plants of at least two enzyme forms with different subcellular localization and possible separate metabolic functions, plastidial and cytosolic glutamine synthetases were purified to electrophoretic homogeneity from spinach chloroplasts and cultured tobacco cells, respectively. Kinetic analysis confirmed the ability of the phosphinothricin analogues to inhibit both isoenzymes in the micromolar range, with a mechanism of a competitive type with respect to glutamate. Interestingly, some of them exerted a differential effect against either the two plant isoforms, or against the plant versus the bacterial enzyme.

Research in Microbiology, Mar 1, 2006

The enzyme responsible for the hydrolysis of phosphonoacetic acid, a non-biogenic C-P compound, w... more The enzyme responsible for the hydrolysis of phosphonoacetic acid, a non-biogenic C-P compound, was purified to electrophoretic homogeneity from a wild-type strain of Penicillium oxalicum. A 50-fold enrichment was obtained by a combination of anion exchange, hydrophobic interaction and MonoQ-fast protein liquid chromatography, with a yield of one-third of the initial activity. A characterization of the protein showed both similarities and differences with respect to the well-characterized bacterial counterpart. The fungal phosphonoacetate hydrolase is a 43-kDa monomeric protein showing low affinity toward its substrate and high sensitivity to even mildly acidic pH values. Enzyme activity neither required nor was stimulated by the presence of divalent cations. Polyclonal antibodies were raised in mouse against the purified protein, allowing the study of enzyme induction as a function of the phosphate status of the cell. Peptide mass mapping led to the determination of about 20% of the primary structure. Despite the biochemical differences, amino acid alignment showed a high degree of similarity of the fungal hydrolase with the few sequences available to date for the bacterial enzyme. The possible physiological role of a phosphonoacetate hydrolase is discussed.

Environmental Microbiology, Jan 23, 2017

Running head: Polyphosphonate degradation by a cyanobacterium Originality-Significance Statement ... more Running head: Polyphosphonate degradation by a cyanobacterium Originality-Significance Statement Capability of metabolising the polyphosphonate diethylenetriaminepenta(methylenephosphonic) acid (DTPMP) by the freshwater cyanobacterium Anabaena variabilis has been studied in detail. Based on 31 P NMR measurements of crude extracts, a pathway for the biodegradation of this xenobiotic bearing five methylenephosphonic groups is proposed. This mechanism differs at least in part from the commonly accepted route for organophosphonate breakdown, which relies on the activity of the inducible C-P lyase complex. Because of the ability to hydrolyze multiple carbon-to-phosphorus bonds in a single molecule, this prokaryotic microalga may be considered a model species for studying the biodegradation of phosphonates. Summary Cyanobacteria, the only prokaryotes capable of oxygenic photosynthesis, play a major role in carbon, nitrogen and phosphorus global cycling. Under conditions of increased P availability and nutrient loading, some cyanobacteria are capable of blooming, rapidly multiplying and possibly altering the ecological structure of the ecosystem. Because of their ability of using non-conventional P sources, these microalgae can be used for bioremediation purposes. Under this perspective, the metabolization of the polyphosphonate diethylenetriaminepenta(methylenephosphonic) acid (DTPMP) by the strain CCALA 007 of Anabaena variabilis was investigated using 31 P NMR analysis. Results showed a quantitative breakdown of DTPMP by cell-free extracts from cyanobacterial cells grown in the absence of any phosphonate. The identification of intermediates and products allowed us to propose a unique and new biodegradation pathway in which the formation of (Nacetylaminomethyl)phosphonic acid represents a key step. This hypothesis was strengthened by the results obtained by incubating cell-free extracts with pathway intermediates. When Anabaena cultures were grown in the presence of the phosphonate, or phosphorus-starved before the extraction, significantly higher biodegradation rates were found.