Publications by authors named "Andrea Mattevi"

159 Publications

How evolution dismantles and reassembles multienzyme complexes.

Authors:
Andrea Mattevi

Proc Natl Acad Sci U S A 2022 Jan 22;119(1). Epub 2021 Dec 22.

Department of Biology and Biotechnology, University of Pavia, 27100 Pavia, Italy

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http://dx.doi.org/10.1073/pnas.2120286118DOI Listing
January 2022

Structural and biochemical studies enlighten the unspecific peroxygenase from Hypoxylon sp. EC38 as an efficient oxidative biocatalyst.

ACS Catal 2021 Sep 2;11(18):11511-11525. Epub 2021 Sep 2.

Department of Biology and Biotechnology, University of Pavia, via Ferrata 9, 27100 Pavia, Italy.

Unspecific peroxygenases (UPO) are glycosylated fungal enzymes that can selectively oxidize C-H bonds. UPOs employ hydrogen peroxide as oxygen donor and reductant. With such an easy-to-handle co-substrate and without the need of a reducing agent, UPOs are emerging as convenient oxidative biocatalysts. Here, an unspecific peroxygenase from (UPO) was identified in an activity-based screen of six putative peroxygenase enzymes that were heterologously expressed in . The enzyme was found to tolerate selected organic solvents such as acetonitrile and acetone. UPO is a versatile catalyst performing various reactions, such as the oxidation of - and -alcohols, epoxidations and hydroxylations. Semi-preparative biotransformations were demonstrated for the non-enantioselective oxidation of racemic 1-phenylethanol (TON = 13000), giving the product with 88% isolated yield, and the oxidation of indole to give indigo (TON = 2800) with 98% isolated yield. UPO features a compact and rigid three-dimensional conformation that wraps around the heme and defines a funnel-shaped tunnel that leads to the heme iron from the protein surface. The tunnel extends along a distance of about 12 Å with a fairly constant diameter in its innermost segment. Its surface comprises both hydrophobic and hydrophilic groups for dealing with small-to-medium size substrates of variable polarities. The structural investigation of several protein-ligand complexes revealed that the active site of UPO is accessible to molecules of varying bulkiness and polarity with minimal or no conformational changes, explaining the relatively broad substrate scope of the enzyme. With its convenient expression system, robust operational properties, relatively small size, well-defined structural features, and diverse reaction scope, UPO is an exploitable candidate for peroxygenase-based biocatalysis.
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http://dx.doi.org/10.1021/acscatal.1c03065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7611678PMC
September 2021

Discovery, Biocatalytic Exploration and Structural Analysis of a 4-Ethylphenol Oxidase from Gulosibacter chungangensis.

Chembiochem 2021 Nov 30;22(22):3225-3233. Epub 2021 Sep 30.

Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, Via Ferrata 9, 27100, Pavia, Italy.

The vanillyl-alcohol oxidase (VAO) family is a rich source of biocatalysts for the oxidative bioconversion of phenolic compounds. Through genome mining and sequence comparisons, we found that several family members lack a generally conserved catalytic aspartate. This finding led us to study a VAO-homolog featuring a glutamate residue in place of the common aspartate. This 4-ethylphenol oxidase from Gulosibacter chungangensis (Gc4EO) shares 42 % sequence identity with VAO from Penicillium simplicissimum, contains the same 8α-N -histidyl-bound FAD and uses oxygen as electron acceptor. However, Gc4EO features a distinct substrate scope and product specificity as it is primarily effective in the dehydrogenation of para-substituted phenols with little generation of hydroxylated products. The three-dimensional structure shows that the characteristic glutamate side chain creates a closely packed environment that may limit water accessibility and thereby protect from hydroxylation. With its high thermal stability, well defined structural properties and high expression yields, Gc4EO may become a catalyst of choice for the specific dehydrogenation of phenolic compounds bearing small substituents.
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http://dx.doi.org/10.1002/cbic.202100457DOI Listing
November 2021

Baeyer-Villiger Monooxygenases and Their Mechanism of Oxygen Activation: From Microbes to Humans.

Biochemistry 2021 11 11;60(46):3419-3421. Epub 2021 May 11.

Department of Biology and Biotechnology, University of Pavia, via Ferrata 9, 27100 Pavia, Italy.

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http://dx.doi.org/10.1021/acs.biochem.1c00266DOI Listing
November 2021

Ancestral reconstruction of mammalian FMO1 enables structural determination, revealing unique features that explain its catalytic properties.

J Biol Chem 2021 Jan-Jun;296:100221. Epub 2020 Dec 25.

Molecular Enzymology Group, University of Groningen, Groningen, the Netherlands. Electronic address:

Mammals rely on the oxidative flavin-containing monooxygenases (FMOs) to detoxify numerous and potentially deleterious xenobiotics; this activity extends to many drugs, giving FMOs high pharmacological relevance. However, our knowledge regarding these membrane-bound enzymes has been greatly impeded by the lack of structural information. We anticipated that ancestral-sequence reconstruction could help us identify protein sequences that are more amenable to structural analysis. As such, we hereby reconstructed the mammalian ancestral protein sequences of both FMO1 and FMO4, denoted as ancestral flavin-containing monooxygenase (AncFMO)1 and AncFMO4, respectively. AncFMO1, sharing 89.5% sequence identity with human FMO1, was successfully expressed as a functional enzyme. It displayed typical FMO activities as demonstrated by oxygenating benzydamine, tamoxifen, and thioanisole, drug-related compounds known to be also accepted by human FMO1, and both NADH and NADPH cofactors could act as electron donors, a feature only described for the FMO1 paralogs. AncFMO1 crystallized as a dimer and was structurally resolved at 3.0 Å resolution. The structure harbors typical FMO aspects with the flavin adenine dinucleotide and NAD(P)H binding domains and a C-terminal transmembrane helix. Intriguingly, AncFMO1 also contains some unique features, including a significantly porous and exposed active site, and NADPH adopting a new conformation with the 2'-phosphate being pushed inside the NADP binding domain instead of being stretched out in the solvent. Overall, the ancestrally reconstructed mammalian AncFMO1 serves as the first structural model to corroborate and rationalize the catalytic properties of FMO1.
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http://dx.doi.org/10.1074/jbc.RA120.016297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7948450PMC
August 2021

A lonely electron blocks incoming pairs.

J Biol Chem 2021 Jan-Jun;296:100294. Epub 2021 Feb 12.

Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, Pavia, Italy. Electronic address:

Electron bifurcation exploits high energetic states to drive unfavorable single electron reactions and determining the overall mechanism governing these electron transfers represents an arduous task. Using extensive stopped-flow spectroscopy and kinetic simulations, Sucharitakul et al. now explore the bifurcation mechanism of the electron transfer flavoprotein EtfAB from the anaerobic gut bacterium Acidaminococcus fermentans. Strikingly, they illustrated that catalysis is orchestrated by a negatively charged radical, α-FAD, that inhibits further reductions and features an atypical inverted kinetic isotope effect. These results provide additional insight behind electron transfers that are prevalent within multienzyme governed reactions.
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http://dx.doi.org/10.1016/j.jbc.2021.100294DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7948957PMC
July 2021

The multipurpose family of flavoprotein oxidases.

Enzymes 2020 18;47:63-86. Epub 2020 Jul 18.

Department of Biology and Biotechnology, University of Pavia, Pavia, Italy.

This chapter represents a journey through flavoprotein oxidases. The purpose is to excite the reader curiosity regarding this class of enzymes by showing their diverse applications. We start with a brief overview on oxidases to then introduce flavoprotein oxidases and elaborate on the flavin cofactors, their redox and spectroscopic characteristics, and their role in the catalytic mechanism. The six major flavoprotein oxidase families will be described, giving examples of their importance in biology and their biotechnological uses. Specific attention will be given to a few selected flavoprotein oxidases that are not extensively discussed in other chapters of this book. Glucose oxidase, cholesterol oxidase, 5-(hydroxymethyl)furfural (HMF) oxidase and methanol oxidase are four examples of oxidases belonging to the GMC-like flavoprotein oxidase family and that have been shown to be valuable biocatalysts. Their structural and mechanistic features and recent enzyme engineering will be discussed in details. Finally we give a look at the current trend in research and conclude with a future outlook.
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http://dx.doi.org/10.1016/bs.enz.2020.05.002DOI Listing
December 2020

An Elegant Four-Helical Fold in NOX and STEAP Enzymes Facilitates Electron Transport across Biomembranes-Similar Vehicle, Different Destination.

Acc Chem Res 2020 09 20;53(9):1969-1980. Epub 2020 Aug 20.

Department of Biology and Biotechnology 'L. Spallanzani', University of Pavia, 27100 Pavia, Italy.

The ferric reductase superfamily comprises several oxidoreductases that use an intracellular electron source to reduce an extracellular acceptor substrate. NADPH oxidases (NOXs) and six-transmembrane epithelial antigen of the prostate enzymes (STEAPs) are iconic members of the superfamily. NOXs produce extracellular reactive oxygen species that exert potent bactericidal activities and trigger redox-signaling cascades that regulate cell division and differentiation. STEAPs catalyze the reduction of extracellular iron and copper which is necessary for the bioavailability of these essential elements. Both NOXs and STEAPs are present as multiple isozymes with distinct regulatory properties and physiological roles. Despite the important roles of NOXs and STEAPs in human physiology and despite their wide involvement in diseases like cancer, their mode of action at the molecular level remained incompletely understood for a long time, in part due to the absence of high-resolution models of the complete enzymes. Our two laboratories have elucidated the three-dimensional structures of NOXs and STEAPs, providing key insight into their mechanisms and evolution. The enzymes share a conserved transmembrane helical domain with an eye-catching hourglass shape. On the extracellular side, a heme prosthetic group is at the bottom of a pocket where the substrate (O in NOX, chelated iron or copper in STEAP) is reduced. On the intracellular side, the inner heme of NOX and the FAD of STEAP are bound to topological equivalent sites. This is a rare case where critical amino acid substitutions and local conformational changes enable a cofactor (heme vs FAD) swap between two structurally and functionally conserved scaffolds. The catalytic core of these enzymes is completed by distinct cytosolic NADPH-binding domains that are topologically unrelated (a ferredoxin reductase-like flavoprotein domain in NOX and a FH:NADP-like domain in STEAP), feature different quaternary structures, and underlie specific regulatory mechanisms. Despite their differences, these domains all establish electron-transfer chains that direct the electrons from NADPH to the transmembrane domain. The multistep nature of the process and the chemical nature of the products pose considerable problems in the enzymatic assays. We learned that great care must be exerted in the validation of a candidate inhibitor. Multiple orthogonal assays are required to rule out off-target effects such as ROS-scavenging activities or nonspecific interference with the enzyme redox chain. The structural analysis of STEAP/NOX enzymes led us to further notice that their transmembrane heme-binding topology is shared by other enzymes. We found that the core domain of the cytochrome subunits of the mitochondrial complex III and photosynthetic cytochrome 6f are closely related to NOXs and STEAPs and likely arose from the same ancestor protein. This observation expands the substrate portfolio of the superfamily since cytochromes act on ubiquinone. The rigidly packed helices of the NOX/STEAP/cytochrome domain contrast with the more malleable membrane proteins like ion channels or amino-acid transporters, which undergo large conformational changes to allow passage of relatively large metabolites. This notion of a rigid hourglass scaffold found an unexpected confirmation in the observation, revealed by structural comparisons, that an helical bundle identical to the NOX/STEAP/cytochrome enzymes is featured by a de novo designed heme-binding protein, PS1. Apparently, nature and protein designers have independently converged to this fold as a versatile scaffold for heme-mediated reactions. The challenge is now to uncover the molecular mechanisms that implement the isozyme-specific regulation of the enzyme functions and develop much needed inhibitors and modulators for chemical biology and drug design studies.
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http://dx.doi.org/10.1021/acs.accounts.0c00400DOI Listing
September 2020

Crystallographic snapshots of UDP-glucuronic acid 4-epimerase ligand binding, rotation, and reduction.

J Biol Chem 2020 08 13;295(35):12461-12473. Epub 2020 Jul 13.

Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, Pavia, Italy

UDP-glucuronic acid is converted to UDP-galacturonic acid en route to a variety of sugar-containing metabolites. This reaction is performed by a NAD-dependent epimerase belonging to the short-chain dehydrogenase/reductase family. We present several high-resolution crystal structures of the UDP-glucuronic acid epimerase from The geometry of the substrate-NAD interactions is finely arranged to promote hydride transfer. The exquisite complementarity between glucuronic acid and its binding site is highlighted by the observation that the unligated cavity is occupied by a cluster of ordered waters whose positions overlap the polar groups of the sugar substrate. Co-crystallization experiments led to a structure where substrate- and product-bound enzymes coexist within the same crystal. This equilibrium structure reveals the basis for a "swing and flip" rotation of the pro-chiral 4-keto-hexose-uronic acid intermediate that results from glucuronic acid oxidation, placing the C4' atom in position for receiving a hydride ion on the opposite side of the sugar ring. The product-bound active site is almost identical to that of the substrate-bound structure and satisfies all hydrogen-bonding requirements of the ligand. The structure of the apoenzyme together with the kinetic isotope effect and mutagenesis experiments further outlines a few flexible loops that exist in discrete conformations, imparting structural malleability required for ligand rotation while avoiding leakage of the catalytic intermediate and/or side reactions. These data highlight the double nature of the enzymatic mechanism: the active site features a high degree of precision in substrate recognition combined with the flexibility required for intermediate rotation.
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http://dx.doi.org/10.1074/jbc.RA120.014692DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7458814PMC
August 2020

Rational Redesign of Monoamine Oxidase A into a Dehydrogenase to Probe ROS in Cardiac Aging.

ACS Chem Biol 2020 07 30;15(7):1795-1800. Epub 2020 Jun 30.

Department of Biology and Biotechnology, University of Pavia, Milan, Italy.

Cardiac senescence is a typical chronic frailty condition in the elderly population, and cellular aging is often associated with oxidative stress. The mitochondrial-membrane flavoenzyme monoamine oxidase A (MAO A) catalyzes the oxidative deamination of neurotransmitters, and its expression increases in aged hearts. We produced recombinant human MAO A variants at Lys305 that play a key role in O reactivity leading to HO production. The K305Q variant is as active as the wild-type enzyme, whereas K305M and K305S have 200-fold and 100-fold lower values and similar . Under anaerobic conditions, K305M MAO A was normally reduced by substrate, whereas reoxidation by O was much slower but could be accomplished by quinone electron acceptors. When overexpressed in cardiomyoblasts by adenoviral vectors, the K305M variant showed enzymatic turnover similar to that of the wild-type but displayed decreased ROS levels and senescence markers. These results might translate into pharmacological treatments as MAO inhibitors may attenuate cardiomyocytes aging.
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http://dx.doi.org/10.1021/acschembio.0c00366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8009472PMC
July 2020

Diphenylene Iodonium Is a Noncovalent MAO Inhibitor: A Biochemical and Structural Analysis.

ChemMedChem 2020 08 10;15(15):1394-1397. Epub 2020 Jun 10.

Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, Via Ferrata 9, 27100, Pavia, Italy.

Diphenylene iodonium (DPI) is known for its inhibitory activities against many flavin- and heme-dependent enzymes, and is often used as an NADPH oxidase inhibitor. We probed the efficacy of DPI on two well-known drug targets, the human monoamine oxidases MAO A and B. UV-visible spectrophotometry and steady-state kinetics experiments demonstrate that DPI acts as a competitive and reversible MAO inhibitor with K values of 1.7 and 0.3 μM for MAO A and MAO B, respectively. Elucidation of the crystal structure of human MAO B bound to the inhibitor revealed that DPI binds deeply in the active-site cavity to establish multiple hydrophobic interactions with the surrounding side chains and the flavin. These data prove that DPI is a genuine MAO inhibitor and that the inhibition mechanism does not involve a reaction with the reduced flavin. This binding and inhibitory activity against the MAOs, two major reactive oxygen species (ROS)-producing enzymes, will have to be carefully considered when interpreting experiments that rely on DPI for target validation and chemical biology studies on ROS functions.
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http://dx.doi.org/10.1002/cmdc.202000264DOI Listing
August 2020

Photoinduced monooxygenation involving NAD(P)H-FAD sequential single-electron transfer.

Nat Commun 2020 05 25;11(1):2600. Epub 2020 May 25.

Institute for Molecular Microbiology and Biotechnology, University of Münster, Corrensstr. 3, 48149, Münster, Germany.

Light-dependent or light-stimulated catalysis provides a multitude of perspectives for implementation in technological or biomedical applications. Despite substantial progress made in the field of photobiocatalysis, the number of usable light-responsive enzymes is still very limited. Flavoproteins have exceptional potential for photocatalytic applications because the name-giving cofactor intrinsically features light-dependent reactivity, undergoing photoreduction with a variety of organic electron donors. However, in the vast majority of these enzymes, photoreactivity of the enzyme-bound flavin is limited or even suppressed. Here, we present a flavoprotein monooxygenase in which catalytic activity is controllable by blue light illumination. The reaction depends on the presence of nicotinamide nucleotide-type electron donors, which do not support the reaction in the absence of light. Employing various experimental approaches, we demonstrate that catalysis depends on a protein-mediated photoreduction of the flavin cofactor, which proceeds via a radical mechanism and a transient semiquinone intermediate.
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http://dx.doi.org/10.1038/s41467-020-16450-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7248105PMC
May 2020

Targeting the scaffolding role of LSD1 (KDM1A) poises acute myeloid leukemia cells for retinoic acid-induced differentiation.

Sci Adv 2020 04 8;6(15):eaax2746. Epub 2020 Apr 8.

Department of Experimental Oncology, European Institute of Oncology (IEO), IRCCS, Via Adamello 16, Milan 20139, Italy.

The histone demethylase LSD1 is deregulated in several tumors, including leukemias, providing the rationale for the clinical use of LSD1 inhibitors. In acute promyelocytic leukemia (APL), pharmacological doses of retinoic acid (RA) induce differentiation of APL cells, triggering degradation of the PML-RAR oncogene. APL cells are resistant to LSD1 inhibition or knockout, but targeting LSD1 sensitizes them to physiological doses of RA without altering of PML-RAR levels, and extends survival of leukemic mice upon RA treatment. The combination of RA with LSD1 inhibition (or knockout) is also effective in other non-APL, acute myeloid leukemia (AML) cells. Nonenzymatic activities of LSD1 are essential to block differentiation, while RA with targeting of LSD1 releases a differentiation gene expression program, not strictly dependent on changes in histone H3K4 methylation. Integration of proteomic/epigenomic/mutational studies showed that LSD1 inhibitors alter the recruitment of LSD1-containing complexes to chromatin, inhibiting the interaction between LSD1 and the transcription factor GFI1.
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http://dx.doi.org/10.1126/sciadv.aax2746DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7141832PMC
April 2020

Approaching boiling point stability of an alcohol dehydrogenase through computationally-guided enzyme engineering.

Elife 2020 03 31;9. Epub 2020 Mar 31.

Molecular Enzymology Group, University of Groningen, Groningen, Netherlands.

Enzyme instability is an important limitation for the investigation and application of enzymes. Therefore, methods to rapidly and effectively improve enzyme stability are highly appealing. In this study we applied a computational method (FRESCO) to guide the engineering of an alcohol dehydrogenase. Of the 177 selected mutations, 25 mutations brought about a significant increase in apparent melting temperature (Δ ≥ +3 °C). By combining mutations, a 10-fold mutant was generated with a of 94 °C (+51 °C relative to wild type), almost reaching water's boiling point, and the highest increase with FRESCO to date. The 10-fold mutant's structure was elucidated, which enabled the identification of an activity-impairing mutation. After reverting this mutation, the enzyme showed no loss in activity compared to wild type, while displaying a of 88 °C (+45 °C relative to wild type). This work demonstrates the value of enzyme stabilization through computational library design.
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http://dx.doi.org/10.7554/eLife.54639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7164962PMC
March 2020

A closer look into NADPH oxidase inhibitors: Validation and insight into their mechanism of action.

Redox Biol 2020 05 15;32:101466. Epub 2020 Feb 15.

Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, Via Ferrata 9, 27100, Pavia, Italy. Electronic address:

NADPH-oxidases (NOXs) purposefully produce reactive-oxygen-species (ROS) and are found in most kingdoms of life. The seven human NOXs are each characterized by a specific expression profile and a fine regulation to spatio-temporally tune ROS concentration in cells and tissues. One of the best known roles for NOXs is in host protection against pathogens but ROS themselves are important second messengers involved in tissue regeneration and the modulation of pathways that induce and sustain cell proliferation. As such, NOXs are attractive pharmacological targets in immunomodulation, fibrosis and cancer. We have studied an extensive number of available NOX inhibitors, with the specific aim to identify bona fide ligands versus ROS-scavenging molecules. Accordingly, we have established a comprehensive platform of biochemical and biophysical assays. Most of the investigated small molecules revealed ROS-scavenging and/or assay-interfering properties to various degrees. A few compounds, however, were also demonstrated to directly engage one or more NOX enzymes. Diphenylene iodonium was found to react with the NOXs' flavin and heme prosthetic groups to form stable adducts. We also discovered that two compounds, VAS2870 and VAS3947, inhibit NOXs through the covalent alkylation of a cysteine residue. Importantly, the amino acid involved in covalent binding was found to reside in the dehydrogenase domain, where the nicotinamide ring of NADPH is bound. This work can serve as a springboard to guide further development of bona fide ligands with either agonistic or antagonistic properties toward NOXs.
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http://dx.doi.org/10.1016/j.redox.2020.101466DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7042484PMC
May 2020

Tranylcypromine-Based LSD1 Inhibitors: Structure-Activity Relationships, Antiproliferative Effects in Leukemia, and Gene Target Modulation.

ChemMedChem 2020 04 14;15(7):643-658. Epub 2020 Feb 14.

Department of Drug Chemistry and Technologies, Sapienza University of Rome, P. le A. Moro 5, 00185, Rome, Italy.

LSD1 is a lysine demethylase highly involved in initiation and development of cancer. To design highly effective covalent inhibitors, a strategy is to fill its large catalytic cleft by designing tranylcypromine (TCP) analogs decorated with long, hindered substituents. We prepared three series of TCP analogs, carrying aroyl- and arylacetylamino (1 a-h), Z-amino acylamino (2 a-o), or double-substituted benzamide (3 a-n) residues at the C4 or C3 position of the phenyl ring. Further fragments obtained by chemical manipulation applied on the TCP scaffold (compounds 4 a-i) were also prepared. When tested against LSD1, most of 1 and 3 exhibited IC values in the low nanomolar range, with 1 e and 3 a,d,f,g being also the most selective respect to monoamine oxidases. In MV4-11 AML and NB4 APL cells compounds 3 were the most potent, displaying up to sub-micromolar cell growth inhibition against both cell lines (3 a) or against NB4 cells (3 c). The most potent compounds in cellular assays were also able to induce the expression of LSD1 target genes, such as GFI-1b, ITGAM, and KCTD12, as functional read-out for LSD1 inhibition. Mouse and human intrinsic clearance data highlighted the high metabolic stability of compounds 3 a, 3 d and 3 g. Further studies will be performed on the new compounds 3 a and 3 c to assess their anticancer potential in different cancer contexts.
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http://dx.doi.org/10.1002/cmdc.201900730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7125024PMC
April 2020

Publisher Correction: Ancestral-sequence reconstruction unveils the structural basis of function in mammalian FMOs.

Nat Struct Mol Biol 2020 Feb;27(2):222

Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, Pavia, Italy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41594-020-0378-8DOI Listing
February 2020

Ancestral-sequence reconstruction unveils the structural basis of function in mammalian FMOs.

Nat Struct Mol Biol 2020 01 23;27(1):14-24. Epub 2019 Dec 23.

Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, Pavia, Italy.

Flavin-containing monooxygenases (FMOs) are ubiquitous in all domains of life and metabolize a myriad of xenobiotics, including toxins, pesticides and drugs. However, despite their pharmacological importance, structural information remains bereft. To further our understanding behind their biochemistry and diversity, we used ancestral-sequence reconstruction, kinetic and crystallographic techniques to scrutinize three ancient mammalian FMOs: AncFMO2, AncFMO3-6 and AncFMO5. Remarkably, all AncFMOs could be crystallized and were structurally resolved between 2.7- and 3.2-Å resolution. These crystal structures depict the unprecedented topology of mammalian FMOs. Each employs extensive membrane-binding features and intricate substrate-profiling tunnel networks through a conspicuous membrane-adhering insertion. Furthermore, a glutamate-histidine switch is speculated to induce the distinctive Baeyer-Villiger oxidation activity of FMO5. The AncFMOs exhibited catalysis akin to human FMOs and, with sequence identities between 82% and 92%, represent excellent models. Our study demonstrates the power of ancestral-sequence reconstruction as a strategy for the crystallization of proteins.
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http://dx.doi.org/10.1038/s41594-019-0347-2DOI Listing
January 2020

Deciphering the enzymatic mechanism of sugar ring contraction in UDP-apiose biosynthesis.

Nat Catal 2019 Nov;2(12):1115-1123

Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria.

D-Apiose is a -branched pentose sugar important for plant cell wall development. Its biosynthesis as UDP-D-apiose involves decarboxylation of the UDP-D-glucuronic acid precursor coupled to pyranosyl-to-furanosyl sugar ring contraction. This unusual multistep reaction is catalyzed within a single active site by UDP-D-apiose/UDP-D-xylose synthase (UAXS). Here, we decipher the UAXS catalytic mechanism based on crystal structures of the enzyme from , molecular dynamics simulations expanded by QM/MM calculations, and mutational-mechanistic analyses. Our studies show how UAXS uniquely integrates a classical catalytic cycle of oxidation and reduction by a tightly bound nicotinamide coenzyme with retro-aldol/aldol chemistry for the sugar ring contraction. They further demonstrate that decarboxylation occurs only after the sugar ring opening and identify the thiol group of Cys100 in steering the sugar skeleton rearrangement by proton transfer to and from the C3'. The mechanistic features of UAXS highlight the evolutionary expansion of the basic catalytic apparatus of short-chain dehydrogenases/reductases for functional versatility in sugar biosynthesis.
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http://dx.doi.org/10.1038/s41929-019-0382-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6914363PMC
November 2019

On the mechanism of calcium-dependent activation of NADPH oxidase 5 (NOX5).

FEBS J 2020 06 20;287(12):2486-2503. Epub 2019 Dec 20.

Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, Italy.

It is now accepted that reactive oxygen species (ROS) are not only dangerous oxidative agents but also chemical mediators of the redox cell signaling and innate immune response. A central role in ROS-controlled production is played by the NADPH oxidases (NOXs), a group of seven membrane-bound enzymes (NOX1-5 and DUOX1-2) whose unique function is to produce ROS. Here, we describe the regulation of NOX5, a widespread family member present in cyanobacteria, protists, plants, fungi, and the animal kingdom. We show that the calmodulin-like regulatory EF-domain of NOX5 is partially unfolded and detached from the rest of the protein in the absence of calcium. In the presence of calcium, the C-terminal lobe of the EF-domain acquires an ordered and more compact structure that enables its binding to the enzyme dehydrogenase (DH) domain. Our spectroscopic and mutagenesis studies further identified a set of conserved aspartate residues in the DH domain that are essential for NOX5 activation. Altogether, our work shows that calcium induces an unfolded-to-folded transition of the EF-domain that promotes direct interaction with a conserved regulatory region, resulting in NOX5 activation.
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http://dx.doi.org/10.1111/febs.15160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7317449PMC
June 2020

Editorial overview: Biological catalysis at the cross-roads of signaling and metabolism.

Curr Opin Struct Biol 2019 12 22;59:iii-v. Epub 2019 Oct 22.

Department of Biology and Biotechnology, University of Pavia, Via Ferrata 1, 27100 Pavia, Italy. Electronic address:

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http://dx.doi.org/10.1016/j.sbi.2019.09.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7083092PMC
December 2019

Impact of Mutations on NPAC Structural Dynamics: Mechanistic Insights from MD Simulations.

J Chem Inf Model 2019 09 21;59(9):3927-3937. Epub 2019 Aug 21.

ICRM-CNR , Via Mario Bianco 9 , 20131 Milano , Italy.

NPAC is a cytokine-like nuclear factor involved in chromatin modification and regulation of gene expression. In humans, the C-terminal domain of NPAC has the conserved structure of the β-hydroxyacid dehydrogenases (β-HAD) protein superfamily, which forms a stable tetrameric core scaffold for demethylase enzymes and organizes multiple sites for chromatin interactions. In spite of the close structural resemblance to other β-HAD family members, the human NPAC dehydrogenase domain lacks a highly conserved catalytic lysine, substituted by a methionine. The reintroduction of the catalytic lysine by M437 K mutation results in a significant decrease of stability of the tetramer. Here, we have computationally investigated the molecular determinants of the functional differences between methionine and lysine-containing NPAC proteins. We find that the single mutation can determine strong consequences in terms of dynamics, stability, and ultimately ability to assemble in supramolecular complexes: the higher stability and lower flexibility of the methionine variant structurally preorganizes the monomer for tetramerization, whereas lysine increases flexibility and favors conformations that, while catalytically active, are not optimal for tetrameric assembly. We combine structure-dynamics analysis to an evolutionary study of NPAC sequences, showing that the methionine mutation occurs in a specifically flexible region of the lysine-containing protein, flanked by two domains that concentrate most of the stabilizing interactions. In our model, such separation of stability nuclei and flexible regions appears to favor the functional innovability of the protein.
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http://dx.doi.org/10.1021/acs.jcim.9b00588DOI Listing
September 2019

Structure and mechanisms of ROS generation by NADPH oxidases.

Curr Opin Struct Biol 2019 12 30;59:91-97. Epub 2019 Apr 30.

Department of Biology and Biotechnology, University of Pavia, Via Ferrata 1, 27100 Pavia, Italy. Electronic address:

NADPH oxidases (NOXs) are integral membrane enzymes that produce reactive oxygen species. Humans have seven NOX enzymes that feature a very similar catalytic core but distinct regulatory mechanisms. The recent structural elucidation of the NOX catalytic domains has been a step forward in the field. NADPH, FAD, and two hemes form a linear array of redox cofactors that transfer electrons across to the two sides of the membrane. Oxygen is reduced through an unusual outer sphere mechanism that does not involve any covalent intermediate with the heme iron. Several recent studies have expanded the roles of NOXs in cell signaling, innate immune response, and cell proliferation including oncogenic transformation. This work reinforces NOX-generated ROS as powerful signaling molecules. A challenging question is to understand the specific mechanisms of enzyme regulation and to harness the growing insight on NOXs' structure and biochemistry to generate more powerful small-molecule modulators of NOX activities.
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http://dx.doi.org/10.1016/j.sbi.2019.03.001DOI Listing
December 2019

Histone deacetylases as an epigenetic pillar for the development of hybrid inhibitors in cancer.

Curr Opin Chem Biol 2019 06 12;50:89-100. Epub 2019 Apr 12.

Department of Chemistry and Technologies of Drugs, Sapienza University of Rome, P. le A. Moro 5, 00185 Rome, Italy. Electronic address:

The polypharmacology strategy of multi-targeting drugs acting on different biological pathways is capturing the researchers' attention, particularly in cancer. The simultaneous inhibition of two or more targets by drug combination or by a single 'hybrid molecule' can provide improved therapeutic efficacy when compared to the one-target inhibitors. In this regard, because of their multiple anticancer effects, histone deacetylase inhibitors have become a privileged tool for the development of hybrid drugs. The clinical trials of two multi-acting chimeras, HDAC/EGFR/HER2 and HDAC/PI3K inhibitors, encouraged the design of novel hybrids, such as compounds 22a (LSD1/HDAC) and 16a (CDK4/JAK1/HDAC), which showed superior anticancer effects than single-targeting agents or their combination both in cellular and mouse models.
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http://dx.doi.org/10.1016/j.cbpa.2019.03.002DOI Listing
June 2019

A Tail-Based Mechanism Drives Nucleosome Demethylation by the LSD2/NPAC Multimeric Complex.

Cell Rep 2019 04;27(2):387-399.e7

Department of Biology and Biotechnology "Lazzaro Spallanzani," University of Pavia, via Ferrata 9, 27100 Pavia, Italy. Electronic address:

LSD1 and LSD2 are homologous histone demethylases with opposite biological outcomes related to chromatin silencing and transcription elongation, respectively. Unlike LSD1, LSD2 nucleosome-demethylase activity relies on a specific linker peptide from the multidomain protein NPAC. We used single-particle cryoelectron microscopy (cryo-EM), in combination with kinetic and mutational analysis, to analyze the mechanisms underlying the function of the human LSD2/NPAC-linker/nucleosome complex. Weak interactions between LSD2 and DNA enable multiple binding modes for the association of the demethylase to the nucleosome. The demethylase thereby captures mono- and dimethyl Lys4 of the H3 tail to afford histone demethylation. Our studies also establish that the dehydrogenase domain of NPAC serves as a catalytically inert oligomerization module. While LSD1/CoREST forms a nucleosome docking platform at silenced gene promoters, LSD2/NPAC is a multifunctional enzyme complex with flexible linkers, tailored for rapid chromatin modification, in conjunction with the advance of the RNA polymerase on actively transcribed genes.
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http://dx.doi.org/10.1016/j.celrep.2019.03.061DOI Listing
April 2019

Side-Chain Pruning Has Limited Impact on Substrate Preference in a Promiscuous Enzyme.

ACS Catal 2018 Dec 30;8(12):11648-11656. Epub 2018 Oct 30.

Department of Biology and Biotechnology, University of Pavia, Via Ferrata 1, 27100, Pavia, Italy.

Detoxifying enzymes such as flavin-containing monooxygenases deal with a huge array of highly diverse xenobiotics and toxic compounds. In addition to being of high physiological relevance, these drug-metabolizing enzymes are useful catalysts for synthetic chemistry. Despite the wealth of studies, the molecular basis of their relaxed substrate selectivity remains an open question. Here, we addressed this issue by applying a cumulative alanine mutagenesis approach to cyclohexanone monooxygenase from , a flavin-dependent Baeyer-Villiger monooxygenase which we chose as a model system because of its pronounced thermostability and substrate promiscuity. Simultaneous removal of up to eight noncatalytic active-site side chains including four phenylalanines had no effect on protein folding, thermostability, and cofactor loading. We observed a linear decrease in activity, rather than a selectivity switch, and attributed this to a less efficient catalytic environment in the enlarged active-site space. Time-resolved kinetic studies confirmed this interpretation. We also determined the crystal structure of the enzyme in complex with a mimic of the reaction intermediate that shows an unaltered overall protein conformation. These findings led us to propose that this cyclohexanone monooxygenase may lack a distinct substrate selection mechanism altogether. We speculate that the main or exclusive function of the protein shell in promiscuous enzymes might be the stabilization and accessibility of their very reactive catalytic intermediates.
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http://dx.doi.org/10.1021/acscatal.8b03793DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6345240PMC
December 2018

Characterization of a thermostable flavin-containing monooxygenase from Nitrincola lacisaponensis (NiFMO).

Appl Microbiol Biotechnol 2019 Feb 4;103(4):1755-1764. Epub 2019 Jan 4.

Groningen Enzyme and Cofactor Collection (GECCO), University of Groningen, Nijenborgh 4, 9747AG, Groningen, The Netherlands.

The flavin-containing monooxygenases (FMOs) play an important role in drug metabolism but they also have a high potential in industrial biotransformations. Among the hitherto characterized FMOs, there was no thermostable representative, while such biocatalyst would be valuable for FMO-based applications. Through a targeted genome mining approach, we have identified a gene encoding for a putative FMO from Nitrincola lacisaponensis, an alkaliphilic extremophile bacterium. Herein, we report the biochemical and structural characterization of this newly discovered bacterial FMO (NiFMO). NiFMO can be expressed as active and soluble enzyme at high level in Escherichia coli (90-100 mg/L of culture). NiFMO is relatively thermostable (melting temperature (T) of 51 °C), displays high organic solvent tolerance, and accepts a broad range of substrates. The crystal structure of NiFMO was solved at 1.8 Å resolution, which allows future structure-based enzyme engineering. Altogether, NiFMO represents an interesting newly discovered enzyme with the appropriate features to develop into an industrially applied biocatalyst.
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http://dx.doi.org/10.1007/s00253-018-09579-wDOI Listing
February 2019

Development of alkyl glycerone phosphate synthase inhibitors: Structure-activity relationship and effects on ether lipids and epithelial-mesenchymal transition in cancer cells.

Eur J Med Chem 2019 Feb 28;163:722-735. Epub 2018 Nov 28.

Department of Chemistry and Technologies of Drugs, Sapienza University of Rome, P. le A. Moro 5, 00185, Rome, Italy. Electronic address:

In aggressive tumors, alkylglyceronephosphate synthase (AGPS) controls cellular ether phospholipid utilization and metabolism to promote cancer cell proliferation and motility. SAR studies on the first-in-class AGPS inhibitor 1, discovered by our group, led to the 2,6-difluoro analog 2i which showed higher binding affinity than 1in vitro. In 231MFP cancer cells, 2i reduced ether lipids levels and cell migration rate. When tested in PC-3 and MDA-MB-231 cancer cells, 2i specifically impaired epithelial to mesenchymal transition (EMT) by modulating E-cadherin, Snail and MMP2 expression levels. Moreover, the combination of siRNAs against AGPS and 2i provided no additive effect, confirming that the modulation of 2i on EMT specifically relies on AGPS inhibition. Finally, this compound also affected cancer cell proliferation especially in MDA-MB-231 cells expressing higher AGPS level, whereas it provided negligible effects on MeT5A, a non-tumorigenic cell line, thus showing cancer specificity.
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http://dx.doi.org/10.1016/j.ejmech.2018.11.050DOI Listing
February 2019

The Extreme Structural Plasticity in the CYP153 Subfamily of P450s Directs Development of Designer Hydroxylases.

Biochemistry 2018 12 16;57(48):6701-6714. Epub 2018 Nov 16.

Department of Biology and Biotechnology , University of Pavia , via Ferrata 9 , Pavia 27100 , Italy.

CYP153s are bacterial class I P450 enzymes traditionally described as alkane hydroxylases with a high terminal regioselectivity. They have been more recently shown to also catalyze hydroxylations at nonactivated carbon atoms of small heterocycles. The aim of our work was to perform an extensive characterization of this subfamily in order to deliver a toolbox of CYP153 enzymes for further development as biocatalysts. Through the screening of recently sequenced bacterial genomes, 20 CYP153s were selected, comprising 17 single monooxygenase domains and three multidomain variants, where the monooxygenase domain is naturally fused to its redox partners in a single polypeptide chain. The 20 novel variants were heterologously expressed, and their activity was screened toward octane and small heterocycles. A more extended substrate characterization was then performed on three representative candidates, and their crystal structures were unveiled and compared with those of the known CYP153A7 and CYP153A33. The tested enzymes displayed a wide range of activities, ranging from Ω and Ω-1 hydroxylations of lauric acid to indigo-generating indole modification. The comparative analysis highlighted a conserved architecture and amino acid composition of the catalytic core close to the heme, while showing a huge degree of structural plasticity and flexibility in those regions hosting the substrate recognition sites. Although dealing with this type of conformational variability adds a layer of complexity and difficulty to structure-based protein engineering, such diversity in substrate acceptance and recognition promotes the investigated CYP153s as a prime choice for tailoring designer hydroxylases.
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http://dx.doi.org/10.1021/acs.biochem.8b01052DOI Listing
December 2018

Cryo-EM structures of human STEAP4 reveal mechanism of iron(III) reduction.

Nat Commun 2018 10 18;9(1):4337. Epub 2018 Oct 18.

Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.

Enzymes of the six-transmembrane epithelial antigen of the prostate (STEAP) family reduce Fe and Cu ions to facilitate metal-ion uptake by mammalian cells. STEAPs are highly upregulated in several types of cancer, making them potential therapeutic targets. However, the structural basis for STEAP-catalyzed electron transfer through an array of cofactors to metals at the membrane luminal side remains elusive. Here, we report cryo-electron microscopy structures of human STEAP4 in absence and presence of Fe-NTA. Domain-swapped, trimeric STEAP4 orients NADPH bound to a cytosolic domain onto axially aligned flavin-adenine dinucleotide (FAD) and a single b-type heme that cross the transmembrane-domain to enable electron transfer. Substrate binding within a positively charged ring indicates that iron gets reduced while in complex with its chelator. These molecular principles of iron reduction provide a basis for exploring STEAPs as therapeutic targets.
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http://dx.doi.org/10.1038/s41467-018-06817-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6194020PMC
October 2018
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