Initial coordinates, molecular dynamics trajectories and representative snapshots resulting from the study "Structural determinants of ligand recognition by the human mitochondrial basic amino acids transporter SLC25A29. Insight from molecular dynamics simulations." by Pasquadibisceglie and Polticelli. The folders MD0-MD5 contain the parameter/topology (parm7) and initial coordinates (rst7) for the six molecular dynamics simulations. Moreover, a NetCDF trajectory "prod.nc" of the production phase is also included. The folder PDB_figures contains the PDB files used to produce the figures presented in the manuscript.
Grape pomaces have a wide and diverse antioxidant phenolics composition. Six Uruguayan red grape pomaces were evaluated in their phenolics composition, antioxidant capacity, and anti-inflammatory properties. Not only radical scavenging methods as DPPH· and ABTS·+ were employed but also ORAC and FRAP analyses were applied to assess the antioxidant potency of the extracts. The antioxidant reactivity of all extracts against hydroxyl radicals was assessed with ESR. The phenol profile of the most bioactive extract was analyzed by HPLC-MS, and a set of 57 structures were determined. To investigate the potential anti-inflammatory activity of the extracts, Nuclear Factor kappa-B (NF-κB) modulation was evaluated in the human colon cancer reporter cell line (HT-29-NF-κB-hrGFP). Our results suggest that Tannat grapes pomaces have higher phenolic content and antioxidant capacity compared to Cabernet Franc. These extracts inhibited TNF-alpha mediated NF-κB activation and IL-8 production when added to reporter cells. A molecular docking study was carried out to rationalize the experimental results allowing us to propose the proactive interaction between the NF-κB, the grape extracts phenols, and their putative anti-inflammatory bioactivity. The present findings show that red grape pomace constitutes a sustainable source of phenolic compounds, which may be valuable for pharmaceutical, cosmetic, and food industry applications.
ABSTRACT A mutation in the rsaL gene of Pseudomonas aeruginosa produces dramatically higher amounts of N -acyl homoserine lactone with respect to the wild type, highlighting the key role of this negative regulator in controlling quorum sensing (QS) in this opportunistic pathogen. The DNA binding site of the RsaL protein on the rsaL-lasI bidirectional promoter partially overlaps the binding site of the LasR protein, consistent with the hypothesis that RsaL and LasR could be in binding competition on this promoter. This is the first direct demonstration that RsaL acts as a QS negative regulator by binding to the lasI promoter.
A polyglutamine expansion of the N-terminal region of huntingtin (Htt) causes Huntington's disease, a severe neurodegenerative disorder. Htt huge multidomain structure, the presence of disordered regions, and the lack of sequence homologs of known structure, so far prevented structural studies of Htt, making the study of its structure-function relationships very difficult. In this work, the presence and location of five Htt ordered domains (named from Hunt1 to Hunt5) has been detected and the structure of these domains has been predicted for the first time using a combined threading/ab initio modeling approach. This work has led to the identification of a previously undetected HEAT repeats region in the Hunt3 domain. Furthermore, a putative function has been assigned to four out of the five domains. Hunt1 and Hunt5, displaying structural similarity with the regulatory subunit A of protein phosphatase 2A, are predicted to play a role in regulating the phosphorylation status of cellular proteins. Hunt2 and Hunt3 are predicted to be homologs of two yeast importins and to mediate vescicles transport and protein trafficking. Finally, a comprehensive analysis of the Htt interactome has been carried out and is discussed to provide a global picture of the Htt's structure-function relationships.
Polyamine oxidases are key enzymes responsible of the polyamine interconversion metabolism in animal cells. Recently, a novel enzyme belonging to this class of enzymes has been characterized for its capability to oxidize preferentially spermine and designated as spermine oxidase. This is a flavin adenine dinucleotide-containing enzyme, and it has been expressed both in vitro and in vivo systems. The primary structure of mouse spermine oxidase (mSMO) was deduced from a cDNA clone (Image Clone 264769) recovered by a data base search utilizing the human counterpart of polyamine oxidases, PAOh1. The open reading frame predicts a 555-amino acid protein with a calculatedM r of 61,852.30, which shows a 95.1% identity with PAOh1. To understand the biochemical properties of mSMO and its structure/function relationship, the mSMO cDNA has been subcloned and expressed in secreted and secreted-tagged forms intoEscherichia coli BL21 DE3 cells. The recombinant enzyme shows an optimal pH value of 8.0 and is able to oxidize rapidly spermine to spermidine and 3-aminopropanal and fails to act upon spermidine and N 1-acetylpolyamines. The purified recombinant-tagged form enzyme (M r∼68,000) has K m and k catvalues of 90 μm and 4.5 s−1, respectively, using spermine as substrate at pH 8.0. Molecular modeling of mSMO protein based on maize polyamine oxidase three-dimensional structure suggests that the general features of maize polyamine oxidase active site are conserved in mSMO. Polyamine oxidases are key enzymes responsible of the polyamine interconversion metabolism in animal cells. Recently, a novel enzyme belonging to this class of enzymes has been characterized for its capability to oxidize preferentially spermine and designated as spermine oxidase. This is a flavin adenine dinucleotide-containing enzyme, and it has been expressed both in vitro and in vivo systems. The primary structure of mouse spermine oxidase (mSMO) was deduced from a cDNA clone (Image Clone 264769) recovered by a data base search utilizing the human counterpart of polyamine oxidases, PAOh1. The open reading frame predicts a 555-amino acid protein with a calculatedM r of 61,852.30, which shows a 95.1% identity with PAOh1. To understand the biochemical properties of mSMO and its structure/function relationship, the mSMO cDNA has been subcloned and expressed in secreted and secreted-tagged forms intoEscherichia coli BL21 DE3 cells. The recombinant enzyme shows an optimal pH value of 8.0 and is able to oxidize rapidly spermine to spermidine and 3-aminopropanal and fails to act upon spermidine and N 1-acetylpolyamines. The purified recombinant-tagged form enzyme (M r∼68,000) has K m and k catvalues of 90 μm and 4.5 s−1, respectively, using spermine as substrate at pH 8.0. Molecular modeling of mSMO protein based on maize polyamine oxidase three-dimensional structure suggests that the general features of maize polyamine oxidase active site are conserved in mSMO. Polyamine oxidase (PAO), 1The abbreviations used are: PAO, polyamine oxidase; FAD, flavin adenine dinucleotide; MPAO, maize polyamine oxidase; SMO, spermine oxidase; mSMO, mouse SMO; HT, His tag; PAOh1, human polyamine oxidase; spermidine, Spd; spermine, Spm; N 1-acetylSpm, N 1-acetyl derivative of spermine; N 1-acetylSpd, N 1-acetyl derivative of spermidine 1The abbreviations used are: PAO, polyamine oxidase; FAD, flavin adenine dinucleotide; MPAO, maize polyamine oxidase; SMO, spermine oxidase; mSMO, mouse SMO; HT, His tag; PAOh1, human polyamine oxidase; spermidine, Spd; spermine, Spm; N 1-acetylSpm, N 1-acetyl derivative of spermine; N 1-acetylSpd, N 1-acetyl derivative of spermidine a flavin adenine dinucleotide (FAD)-containing enzyme, catalyzes the oxidation of polyamines at the secondary amino group, giving different products according to the organism considered. In particular, vertebrate PAOs (EC 1.5.3.11) participate in the interconversion metabolism of polyamines, converting N 1-acetyl derivatives of spermine (N 1-acetylSpm) and spermidine (N 1-acetylSpd) into Spd and putrescine, respectively, plus 3-aminopropanal and H2O2, (1McIntire W.S. Hartman C. Davison V.L. Principle and Application of Quinoproteins. Marcel Dekker, Inc., New York1993: 97-171Google Scholar, 2Seiler N. Prog. Brain Res. 1995; 106: 333-344Crossref PubMed Scopus (123) Google Scholar, 3Den Munckhof R.J.M. Denyn M. Tigchelaar-Gutter W. Schipper R.G. Verhofstad A.A.J. Van Noorden C.J.F. Frederiks W.M. J. Histochem. Cytochem. 1995; 43: 1155-1162Crossref PubMed Scopus (28) Google Scholar). PAOs with similar characteristics occur in methylotrophic yeasts (4Cohen S.S. Cohen S.S. A Guide to the Polyamines. Oxford University Press, NY1998: 82Google Scholar, 5Nishikawa M. Hagishita T. Yurimoto H. Kato N. Sakai Y. Hatanaka T. FEBS Lett. 2000; 476: 150-154Crossref PubMed Scopus (23) Google Scholar) and amoebae (6Shukla O.P. Muller S. Walter R.D. Mol. Biochem. Parasitol. 1992; 51: 91-98Crossref PubMed Scopus (12) Google Scholar). On the contrary, plant (1McIntire W.S. Hartman C. Davison V.L. Principle and Application of Quinoproteins. Marcel Dekker, Inc., New York1993: 97-171Google Scholar), bacterial (7Tabor C.W. Tabor H. Annu. Rev. Biochem. 1984; 53: 749-790Crossref PubMed Scopus (3221) Google Scholar), and protozoan (8Kim B.G. Sobota A. Bitonti A.J. McCann P.P. Byers T.J. J. Protozool. 1987; 34: 278-284Crossref PubMed Scopus (27) Google Scholar) PAOs oxidize spermidine and spermine to 4-aminobutanol or N-(3-aminopropyl)-4-aminobutanol, respectively, plus 1,3-diaminopropane and H2O2. As these compounds cannot be converted directly to other polyamines, this class of PAOs generally is considered to be involved in the terminal catabolism of polyamines. Since PAOs play a crucial role in polyamine catabolism, these enzymes are important drug targets, and in fact, it has been shown that a number of polyamine analogues have an antitumor effect in different cell lines (9Pegg A.E. Hu R.H. Cancer Lett. 1995; 95: 247-252Crossref PubMed Scopus (25) Google Scholar, 10Bergeron R.J. Feng Y. Weimar W.R. McManis J.S. Dimova H. Porter C. Raisler B. Phanstiel O.A. J. Med. Chem. 1997; 40: 1475-1494Crossref PubMed Scopus (124) Google Scholar, 11Ha H.C. Woster P.M. Yager J.D. Casero Jr., R.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11557-11562Crossref PubMed Scopus (271) Google Scholar, 12Casero E.A. Woster P.M. J. Med. Chem. 2001; 44: 1-26Crossref PubMed Scopus (218) Google Scholar).As compared with large and detailed investigations on plant PAOs (1McIntire W.S. Hartman C. Davison V.L. Principle and Application of Quinoproteins. Marcel Dekker, Inc., New York1993: 97-171Google Scholar,13Smith T.A. Phytochemistry. 1976; 15: 633-636Crossref Scopus (35) Google Scholar, 14Smith T.A. Barker J.H. Zappia V. Pegg A.E. Progress in Polyamine Research: Novel Biochemical, Pharmacological and Clinical Aspects. Plenum Press, NY1988: 573-589Google Scholar, 15Federico R. Alisi C. Forlani F. Phytochemistry. 1989; 28: 45-46Crossref Scopus (36) Google Scholar, 16Angelini R. Federico R. Bonfante P. J. Plant Physiol. 1995; 145: 686-692Crossref Scopus (40) Google Scholar, 17Tavladoraki P. Schininà M.E. Cecconi F. Di Agostino S. Manera F. Rea G. Mariottini P. Federico R. Angelini R. FEBS Lett. 1998; 426: 62-66Crossref PubMed Scopus (89) Google Scholar, 18Binda C. Coda A. Angelini R. Federico R. Ascenzi P. Mattevi A. Structure. 1999; 7: 265-276Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 19Laurenzi M. Rea G. Federico R. Tavladoraki P. Angelini R. Planta. 1999; 208: 146-154Crossref Scopus (47) Google Scholar, 20Cervelli M. Tavladoraki P. Di Agostino S. Angelini R. Federico R. Mariottini P. Plant Physiol. Biochem. 2000; 38: 667-677Crossref Scopus (39) Google Scholar, 21Cervelli M. Cona A. Angelini R. Polticelli F. Federico R. Mariottini P. Eur. J. Biochem. 2001; 268: 3816-3838Crossref PubMed Scopus (55) Google Scholar, 22Šebela M. Radová A. Angelini R. Tavladoraki P. Frébort I. Pêc P. Plant Sci. 2001; 160: 197-207Crossref PubMed Scopus (113) Google Scholar), only little attention has been devoted to the animal counterpart (2Seiler N. Prog. Brain Res. 1995; 106: 333-344Crossref PubMed Scopus (123) Google Scholar, 23Hölttä E. Biochemistry. 1977; 16: 91-100Crossref PubMed Scopus (266) Google Scholar, 24Bolkenius F.N. Bey P. Seiler N. Biochim. Biophys. Acta. 1984; 838: 69-76Crossref Scopus (68) Google Scholar, 25Suzuki O. Matsumoto T. Katsumata Y. Experentia (Basel). 1984; 40: 838-839Crossref PubMed Scopus (45) Google Scholar, 26Libby P.R. Porter C.W. Biochem. Biophys. Res. Commun. 1985; 144: 528-535Crossref Scopus (21) Google Scholar, 27Tsukada T. Furusako S. Maekawa S. Hibasami H. Nakashima K. Int. J. Biochem. 1988; 20: 695-702Crossref PubMed Scopus (17) Google Scholar, 28Gasparyan V.K. Nalbandyan R.M. Biokhimiya. 1990; 55: 1632-1637PubMed Google Scholar). Recently, Wang et al. (29Wang Y. Devereux W. Woster P.M. Stewart T.M. Hacker A. Casero Jr, R.A. Cancer Res. 2001; 61: 5370-5373PubMed Google Scholar) and Vujcic et al. (30Vujcic S. Diegelman P. Bacchi C.J. Kramer D.L. Porter C.W. Biochem. J. 2002; 367: 665-675Crossref PubMed Scopus (187) Google Scholar) have reported the cloning and characterization of novel mammalian PAO enzymes capable of oxidizing preferentially Spm and for this reason named spermine oxidase (SMO) (30Vujcic S. Diegelman P. Bacchi C.J. Kramer D.L. Porter C.W. Biochem. J. 2002; 367: 665-675Crossref PubMed Scopus (187) Google Scholar). In particular, this enzyme was expressed in an in vitro transcription/translation system (29Wang Y. Devereux W. Woster P.M. Stewart T.M. Hacker A. Casero Jr, R.A. Cancer Res. 2001; 61: 5370-5373PubMed Google Scholar) and into transiently transfected human kidney cells (30Vujcic S. Diegelman P. Bacchi C.J. Kramer D.L. Porter C.W. Biochem. J. 2002; 367: 665-675Crossref PubMed Scopus (187) Google Scholar). Based on these studies, it was postulated that in addition to the traditional interconversion pathway in which Spm is first acetylated by spermidine/spermineN 1-acetyltransferase and then oxidized by PAO, mammalian cells contain an enzyme capable of directly oxidizing Spm to Spd (30Vujcic S. Diegelman P. Bacchi C.J. Kramer D.L. Porter C.W. Biochem. J. 2002; 367: 665-675Crossref PubMed Scopus (187) Google Scholar).Data base searching analysis using PAOh1 cDNA sequence recovered a mouse cDNA clone (Image Clone 264769) corresponding to the murine counterpart, which was supplied by the United Kingdom Medical Research Council Human Genome Mapping Resource Centre (Cambridge, United Kingdom) consortium and herein defined as mSMO. To enhance the knowledge of enzymology of mammalian SMO and shed light on the structure/function relationship of this enzyme, the mSMO cDNA was further subcloned and expressed in secreted and secreted-tagged forms into Escherichia coli BL21 DE3 cells. This paper describes the expression and the main biochemical features of mouse spermine oxidase. Notwithstanding the low amino acid sequence homology shown by the animal and plant PAOs (45% sequence homology between MPAO and mSMO), molecular modeling of mSMO based on MPAO three-dimensional structure (18Binda C. Coda A. Angelini R. Federico R. Ascenzi P. Mattevi A. Structure. 1999; 7: 265-276Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar) suggests that the general features of MPAO active site are conserved in mSMO.DISCUSSIONPlant or animal recombinant PAOs have been never reported to be expressed in E. coli cells to our knowledge. This is the first report of a vertebrate polyamine oxidase overexpressed in secreted and secreted-tagged forms in such a heterologous system. SMO recombinant enzymes are targeted to the periplasmic membrane compartment, and the catalytically active proteins are expressed at a level of ∼6 units/liter of culture broth. The data obtained for the secreted recombinant mSMO perfectly match the ones obtained for the secreted-tagged enzyme form.The analysis of the expressed mSMO protein gave an improved understanding of the biochemical features of this enzyme, since the purified recombinant enzyme was able to oxidize only Spm and failed to act upon Spd and the N 1-acetyl derivatives. The enzyme specificity reported herein is in agreement with the one described in transfected human cells by Vujcic et al.(30Vujcic S. Diegelman P. Bacchi C.J. Kramer D.L. Porter C.W. Biochem. J. 2002; 367: 665-675Crossref PubMed Scopus (187) Google Scholar).The precise nature of the reaction products and the cleavage position on the Spm substrate are those typical of an animal PAO enzyme. In line with this finding, the specific inhibition of mSMO enzyme activity was obtained with MDL72527 but not with pargyline.The absorption spectrum of the native enzyme showed the typical three-banded spectrum for flavoproteins. Furthermore, the addition of the Spm substrate in anaerobic conditions resulted in a dramatic absorbance decrease, while reoxygenation of the enzyme restored the initial spectrum, indicating the involvement of a FAD group in the catalytic cycle.An analysis of the molecular model of mSMO as compared with MPAO three-dimensional structure suggests that the catalytic tunnel of the former enzyme is wider. It is tempting to speculate that the preference for Spm over Spd as a substrate observed for mSMO is linked to the different shape of the catalytic tunnel in which the short Spd substrate would be bound in a "floppy" fashion, thus rendering less efficient the enzyme catalysis, which has been hypothesized to rely on an "in register" binding of the substrates.The substitution of Glu-62 by a His residue in mSMO can provide an explanation for the peculiar pH dependence of the activity. In fact, it can be reasonably assumed that His-62 is partially protonated at pH values lower than 7.0. Thus, the binding of the cationic polyamine substrates would be unfavorable and enzyme activity would be very low as observed experimentally. At pH values higher than 7.0, His-62 would deprotonate, thus facilitating substrate binding and leading to an increase in enzyme activity, which is maximal at pH values of ∼8.0.In conclusion, in mammalian cells, polyamine catabolism seems to be mediated by the activity of two enzymes, PAO and the novel SMO, described in this work (30Vujcic S. Diegelman P. Bacchi C.J. Kramer D.L. Porter C.W. Biochem. J. 2002; 367: 665-675Crossref PubMed Scopus (187) Google Scholar). The precise significance of both polyamine oxidase activities in the metabolism of polyamines remains to be established, particularly regarding the role of each enzyme in regulating polyamine concentrations in mammalian tissues in relation with growth processes and malignant transformation. Polyamine oxidase (PAO), 1The abbreviations used are: PAO, polyamine oxidase; FAD, flavin adenine dinucleotide; MPAO, maize polyamine oxidase; SMO, spermine oxidase; mSMO, mouse SMO; HT, His tag; PAOh1, human polyamine oxidase; spermidine, Spd; spermine, Spm; N 1-acetylSpm, N 1-acetyl derivative of spermine; N 1-acetylSpd, N 1-acetyl derivative of spermidine 1The abbreviations used are: PAO, polyamine oxidase; FAD, flavin adenine dinucleotide; MPAO, maize polyamine oxidase; SMO, spermine oxidase; mSMO, mouse SMO; HT, His tag; PAOh1, human polyamine oxidase; spermidine, Spd; spermine, Spm; N 1-acetylSpm, N 1-acetyl derivative of spermine; N 1-acetylSpd, N 1-acetyl derivative of spermidine a flavin adenine dinucleotide (FAD)-containing enzyme, catalyzes the oxidation of polyamines at the secondary amino group, giving different products according to the organism considered. In particular, vertebrate PAOs (EC 1.5.3.11) participate in the interconversion metabolism of polyamines, converting N 1-acetyl derivatives of spermine (N 1-acetylSpm) and spermidine (N 1-acetylSpd) into Spd and putrescine, respectively, plus 3-aminopropanal and H2O2, (1McIntire W.S. Hartman C. Davison V.L. Principle and Application of Quinoproteins. Marcel Dekker, Inc., New York1993: 97-171Google Scholar, 2Seiler N. Prog. Brain Res. 1995; 106: 333-344Crossref PubMed Scopus (123) Google Scholar, 3Den Munckhof R.J.M. Denyn M. Tigchelaar-Gutter W. Schipper R.G. Verhofstad A.A.J. Van Noorden C.J.F. Frederiks W.M. J. Histochem. Cytochem. 1995; 43: 1155-1162Crossref PubMed Scopus (28) Google Scholar). PAOs with similar characteristics occur in methylotrophic yeasts (4Cohen S.S. Cohen S.S. A Guide to the Polyamines. Oxford University Press, NY1998: 82Google Scholar, 5Nishikawa M. Hagishita T. Yurimoto H. Kato N. Sakai Y. Hatanaka T. FEBS Lett. 2000; 476: 150-154Crossref PubMed Scopus (23) Google Scholar) and amoebae (6Shukla O.P. Muller S. Walter R.D. Mol. Biochem. Parasitol. 1992; 51: 91-98Crossref PubMed Scopus (12) Google Scholar). On the contrary, plant (1McIntire W.S. Hartman C. Davison V.L. Principle and Application of Quinoproteins. Marcel Dekker, Inc., New York1993: 97-171Google Scholar), bacterial (7Tabor C.W. Tabor H. Annu. Rev. Biochem. 1984; 53: 749-790Crossref PubMed Scopus (3221) Google Scholar), and protozoan (8Kim B.G. Sobota A. Bitonti A.J. McCann P.P. Byers T.J. J. Protozool. 1987; 34: 278-284Crossref PubMed Scopus (27) Google Scholar) PAOs oxidize spermidine and spermine to 4-aminobutanol or N-(3-aminopropyl)-4-aminobutanol, respectively, plus 1,3-diaminopropane and H2O2. As these compounds cannot be converted directly to other polyamines, this class of PAOs generally is considered to be involved in the terminal catabolism of polyamines. Since PAOs play a crucial role in polyamine catabolism, these enzymes are important drug targets, and in fact, it has been shown that a number of polyamine analogues have an antitumor effect in different cell lines (9Pegg A.E. Hu R.H. Cancer Lett. 1995; 95: 247-252Crossref PubMed Scopus (25) Google Scholar, 10Bergeron R.J. Feng Y. Weimar W.R. McManis J.S. Dimova H. Porter C. Raisler B. Phanstiel O.A. J. Med. Chem. 1997; 40: 1475-1494Crossref PubMed Scopus (124) Google Scholar, 11Ha H.C. Woster P.M. Yager J.D. Casero Jr., R.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11557-11562Crossref PubMed Scopus (271) Google Scholar, 12Casero E.A. Woster P.M. J. Med. Chem. 2001; 44: 1-26Crossref PubMed Scopus (218) Google Scholar). As compared with large and detailed investigations on plant PAOs (1McIntire W.S. Hartman C. Davison V.L. Principle and Application of Quinoproteins. Marcel Dekker, Inc., New York1993: 97-171Google Scholar,13Smith T.A. Phytochemistry. 1976; 15: 633-636Crossref Scopus (35) Google Scholar, 14Smith T.A. Barker J.H. Zappia V. Pegg A.E. Progress in Polyamine Research: Novel Biochemical, Pharmacological and Clinical Aspects. Plenum Press, NY1988: 573-589Google Scholar, 15Federico R. Alisi C. Forlani F. Phytochemistry. 1989; 28: 45-46Crossref Scopus (36) Google Scholar, 16Angelini R. Federico R. Bonfante P. J. Plant Physiol. 1995; 145: 686-692Crossref Scopus (40) Google Scholar, 17Tavladoraki P. Schininà M.E. Cecconi F. Di Agostino S. Manera F. Rea G. Mariottini P. Federico R. Angelini R. FEBS Lett. 1998; 426: 62-66Crossref PubMed Scopus (89) Google Scholar, 18Binda C. Coda A. Angelini R. Federico R. Ascenzi P. Mattevi A. Structure. 1999; 7: 265-276Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 19Laurenzi M. Rea G. Federico R. Tavladoraki P. Angelini R. Planta. 1999; 208: 146-154Crossref Scopus (47) Google Scholar, 20Cervelli M. Tavladoraki P. Di Agostino S. Angelini R. Federico R. Mariottini P. Plant Physiol. Biochem. 2000; 38: 667-677Crossref Scopus (39) Google Scholar, 21Cervelli M. Cona A. Angelini R. Polticelli F. Federico R. Mariottini P. Eur. J. Biochem. 2001; 268: 3816-3838Crossref PubMed Scopus (55) Google Scholar, 22Šebela M. Radová A. Angelini R. Tavladoraki P. Frébort I. Pêc P. Plant Sci. 2001; 160: 197-207Crossref PubMed Scopus (113) Google Scholar), only little attention has been devoted to the animal counterpart (2Seiler N. Prog. Brain Res. 1995; 106: 333-344Crossref PubMed Scopus (123) Google Scholar, 23Hölttä E. Biochemistry. 1977; 16: 91-100Crossref PubMed Scopus (266) Google Scholar, 24Bolkenius F.N. Bey P. Seiler N. Biochim. Biophys. Acta. 1984; 838: 69-76Crossref Scopus (68) Google Scholar, 25Suzuki O. Matsumoto T. Katsumata Y. Experentia (Basel). 1984; 40: 838-839Crossref PubMed Scopus (45) Google Scholar, 26Libby P.R. Porter C.W. Biochem. Biophys. Res. Commun. 1985; 144: 528-535Crossref Scopus (21) Google Scholar, 27Tsukada T. Furusako S. Maekawa S. Hibasami H. Nakashima K. Int. J. Biochem. 1988; 20: 695-702Crossref PubMed Scopus (17) Google Scholar, 28Gasparyan V.K. Nalbandyan R.M. Biokhimiya. 1990; 55: 1632-1637PubMed Google Scholar). Recently, Wang et al. (29Wang Y. Devereux W. Woster P.M. Stewart T.M. Hacker A. Casero Jr, R.A. Cancer Res. 2001; 61: 5370-5373PubMed Google Scholar) and Vujcic et al. (30Vujcic S. Diegelman P. Bacchi C.J. Kramer D.L. Porter C.W. Biochem. J. 2002; 367: 665-675Crossref PubMed Scopus (187) Google Scholar) have reported the cloning and characterization of novel mammalian PAO enzymes capable of oxidizing preferentially Spm and for this reason named spermine oxidase (SMO) (30Vujcic S. Diegelman P. Bacchi C.J. Kramer D.L. Porter C.W. Biochem. J. 2002; 367: 665-675Crossref PubMed Scopus (187) Google Scholar). In particular, this enzyme was expressed in an in vitro transcription/translation system (29Wang Y. Devereux W. Woster P.M. Stewart T.M. Hacker A. Casero Jr, R.A. Cancer Res. 2001; 61: 5370-5373PubMed Google Scholar) and into transiently transfected human kidney cells (30Vujcic S. Diegelman P. Bacchi C.J. Kramer D.L. Porter C.W. Biochem. J. 2002; 367: 665-675Crossref PubMed Scopus (187) Google Scholar). Based on these studies, it was postulated that in addition to the traditional interconversion pathway in which Spm is first acetylated by spermidine/spermineN 1-acetyltransferase and then oxidized by PAO, mammalian cells contain an enzyme capable of directly oxidizing Spm to Spd (30Vujcic S. Diegelman P. Bacchi C.J. Kramer D.L. Porter C.W. Biochem. J. 2002; 367: 665-675Crossref PubMed Scopus (187) Google Scholar). Data base searching analysis using PAOh1 cDNA sequence recovered a mouse cDNA clone (Image Clone 264769) corresponding to the murine counterpart, which was supplied by the United Kingdom Medical Research Council Human Genome Mapping Resource Centre (Cambridge, United Kingdom) consortium and herein defined as mSMO. To enhance the knowledge of enzymology of mammalian SMO and shed light on the structure/function relationship of this enzyme, the mSMO cDNA was further subcloned and expressed in secreted and secreted-tagged forms into Escherichia coli BL21 DE3 cells. This paper describes the expression and the main biochemical features of mouse spermine oxidase. Notwithstanding the low amino acid sequence homology shown by the animal and plant PAOs (45% sequence homology between MPAO and mSMO), molecular modeling of mSMO based on MPAO three-dimensional structure (18Binda C. Coda A. Angelini R. Federico R. Ascenzi P. Mattevi A. Structure. 1999; 7: 265-276Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar) suggests that the general features of MPAO active site are conserved in mSMO. DISCUSSIONPlant or animal recombinant PAOs have been never reported to be expressed in E. coli cells to our knowledge. This is the first report of a vertebrate polyamine oxidase overexpressed in secreted and secreted-tagged forms in such a heterologous system. SMO recombinant enzymes are targeted to the periplasmic membrane compartment, and the catalytically active proteins are expressed at a level of ∼6 units/liter of culture broth. The data obtained for the secreted recombinant mSMO perfectly match the ones obtained for the secreted-tagged enzyme form.The analysis of the expressed mSMO protein gave an improved understanding of the biochemical features of this enzyme, since the purified recombinant enzyme was able to oxidize only Spm and failed to act upon Spd and the N 1-acetyl derivatives. The enzyme specificity reported herein is in agreement with the one described in transfected human cells by Vujcic et al.(30Vujcic S. Diegelman P. Bacchi C.J. Kramer D.L. Porter C.W. Biochem. J. 2002; 367: 665-675Crossref PubMed Scopus (187) Google Scholar).The precise nature of the reaction products and the cleavage position on the Spm substrate are those typical of an animal PAO enzyme. In line with this finding, the specific inhibition of mSMO enzyme activity was obtained with MDL72527 but not with pargyline.The absorption spectrum of the native enzyme showed the typical three-banded spectrum for flavoproteins. Furthermore, the addition of the Spm substrate in anaerobic conditions resulted in a dramatic absorbance decrease, while reoxygenation of the enzyme restored the initial spectrum, indicating the involvement of a FAD group in the catalytic cycle.An analysis of the molecular model of mSMO as compared with MPAO three-dimensional structure suggests that the catalytic tunnel of the former enzyme is wider. It is tempting to speculate that the preference for Spm over Spd as a substrate observed for mSMO is linked to the different shape of the catalytic tunnel in which the short Spd substrate would be bound in a "floppy" fashion, thus rendering less efficient the enzyme catalysis, which has been hypothesized to rely on an "in register" binding of the substrates.The substitution of Glu-62 by a His residue in mSMO can provide an explanation for the peculiar pH dependence of the activity. In fact, it can be reasonably assumed that His-62 is partially protonated at pH values lower than 7.0. Thus, the binding of the cationic polyamine substrates would be unfavorable and enzyme activity would be very low as observed experimentally. At pH values higher than 7.0, His-62 would deprotonate, thus facilitating substrate binding and leading to an increase in enzyme activity, which is maximal at pH values of ∼8.0.In conclusion, in mammalian cells, polyamine catabolism seems to be mediated by the activity of two enzymes, PAO and the novel SMO, described in this work (30Vujcic S. Diegelman P. Bacchi C.J. Kramer D.L. Porter C.W. Biochem. J. 2002; 367: 665-675Crossref PubMed Scopus (187) Google Scholar). The precise significance of both polyamine oxidase activities in the metabolism of polyamines remains to be established, particularly regarding the role of each enzyme in regulating polyamine concentrations in mammalian tissues in relation with growth processes and malignant transformation. Plant or animal recombinant PAOs have been never reported to be expressed in E. coli cells to our knowledge. This is the first report of a vertebrate polyamine oxidase overexpressed in secreted and secreted-tagged forms in such a heterologous system. SMO recombinant enzymes are targeted to the periplasmic membrane compartment, and the catalytically active proteins are expressed at a level of ∼6 units/liter of culture broth. The data obtained for the secreted recombinant mSMO perfectly match the ones obtained for the secreted-tagged enzyme form. The analysis of the expressed mSMO protein gave an improved understanding of the biochemical features of this enzyme, since the purified recombinant enzyme was able to oxidize only Spm and failed to act upon Spd and the N 1-acetyl derivatives. The enzyme specificity reported herein is in agreement with the one described in transfected human cells by Vujcic et al.(30Vujcic S. Diegelman P. Bacchi C.J. Kramer D.L. Porter C.W. Biochem. J. 2002; 367: 665-675Crossref PubMed Scopus (187) Google Scholar). The precise nature of the reaction products and the cleavage position on the Spm substrate are those typical of an animal PAO enzyme. In line with this finding, the specific inhibition of mSMO enzyme activity was obtained with MDL72527 but not with pargyline. The absorption spectrum of the native enzyme showed the typical three-banded spectrum for flavoproteins. Furthermore, the addition of the Spm substrate in anaerobic conditions resulted in a dramatic absorbance decrease, while reoxygenation of the enzyme restored the initial spectrum, indicating the involvement of a FAD group in the catalytic cycle. An analysis of the molecular model of mSMO as compared with MPAO three-dimensional structure suggests that the catalytic tunnel of the former enzyme is wider. It is tempting to speculate that the preference for Spm over Spd as a substrate observed for mSMO is linked to the different shape of the catalytic tunnel in which the short Spd substrate would be bound in a "floppy" fashion, thus rendering less efficient the enzyme catalysis, which has been hypothesized to rely on an "in register" binding of the substrates. The substitution of Glu-62 by a His residue in mSMO can provide an explanation for the peculiar pH dependence of the activity. In fact, it can be reasonably assumed that His-62 is partially protonated at pH values lower than 7.0. Thus, the binding of the cationic polyamine substrates would be unfavorable and enzyme activity would be very low as observed experimentally. At pH values higher than 7.0, His-62 would deprotonate, thus facilitating substrate binding and leading to an increase in enzyme activity, which is maximal at pH values of ∼8.0. In conclusion, in mammalian cells, polyamine catabolism seems to be mediated by the activity of two enzymes, PAO and the novel SMO, described in this work (30Vujcic S. Diegelman P. Bacchi C.J. Kramer D.L. Porter C.W. Biochem. J. 2002; 367: 665-675Crossref PubMed Scopus (187) Google Scholar). The precise significance of both polyamine oxidase activities in the metabolism of polyamines remains to be established, particularly regarding the role of each enzyme in regulating polyamine concentrations in mammalian tissues in relation with growth processes and malignant transformation.
Members of the major facilitator superfamily of transporters (MFS) play an essential role in many physiological processes such as development, neurotransmission, and signaling. Aberrant functions of MFS proteins are associated with several diseases, including cancer, schizophrenia, epilepsy, amyotrophic lateral sclerosis and Alzheimer’s disease. MFS transporters are also involved in multidrug resistance in bacteria and fungi. The structures of most MFS members, especially those of members with significant physiological relevance, are yet to be solved. The lack of structural and functional information impedes our detailed understanding, and thus the pharmacological targeting, of these transporters. To improve our knowledge on the mechanistic principles governing the function of MSF members, molecular dynamics (MD) simulations were performed on the inward-facing and outward-facing crystal structures of the human ferroportin homologue from the Gram-negative bacterium Bdellovibrio bacteriovorus (BdFpn). Several simulations with an excess of iron ions were also performed to explore the relationship between the protein’s dynamics and the ligand recognition mechanism. The results reinforce the existence of the alternating-access mechanism already described for other MFS members. In addition, the reorganization of salt bridges, some of which are conserved in several MFS members, appears to be a key molecular event facilitating the conformational change of the transporter.
Arabidopsis thaliana has four genes with close homology to human histone H3 lysine 4 demethylase (HsLSD1), a component of various transcriptional corepressor complexes that often also contain histone deacetylases and the corepressor protein CoREST. All four Arabidopsis proteins contain a flavin amine oxidase domain and a SWIRM domain, the latter being present in a number of proteins involved in chromatin regulation. Here, we describe the heterologous expression and biochemical characterization of one of these Arabidopsis proteins (AtLSD1) and show that, similarly to HsLSD1, it has demethylase activity toward mono- and dimethylated Lys4 but not dimethylated Lys9 and Lys27 of histone 3. Modeling of the AtLSD1 three-dimensional structure using the HsLSD1 crystal structure as a template revealed a high degree of conservation of the residues building up the active site and some important differences. Among these differences, the most prominent is the lack of the HsLSD1 Tower domain, which has been shown to interact with CoREST and to be indispensable for HsLSD1 demethylase activity. This observation, together with AtLSD1 peculiar surface electrostatic potential distribution, suggests that the molecular partners of AtLSD1 are probably different from those of the human orthologue.
SMO (spermine oxidase) and APAO (acetylpolyamine oxidase) are flavoenzymes that play a critical role in the catabolism of polyamines. Polyamines are basic regulators of cell growth and proliferation and their homoeostasis is crucial for cell life since dysregulation of polyamine metabolism has been linked with cancer. In vertebrates SMO specifically catalyses the oxidation of spermine, whereas APAO displays a wider specificity, being able to oxidize both N1-acetylspermine and N1-acetylspermidine, but not spermine. The molecular bases of the different substrate specificity of these two enzymes have remained so far elusive. However, previous molecular modelling, site-directed mutagenesis and biochemical characterization studies of the SMO enzyme–substrate complex have identified Glu216–Ser218 as a putative active site hot spot responsible for SMO substrate specificity. On the basis of these analyses, the SMO double mutants E216L/S218A and E216T/S218A have been produced and characterized by CD spectroscopy and steady-state and rapid kinetics experiments. The results obtained demonstrate that mutation E216L/S218A endows SMO with N1-acetylspermine oxidase activity, uncovering one of the structural determinants that confer the exquisite and exclusive substrate specificity of SMO for spermine. These results provide the theoretical bases for the design of specific inhibitors either for SMO or APAO.
