Phd in BIOCHEMISTRY

email: roberto.contestabile@uniroma1.it
phone: 06 49913176
building: CU020
room: Primo piano

Name and Surname: ROBERTO CONTESTABILE
Department of Biochemical Sciences
Address: Piazzale Aldo Moro 5, 00185 Roma
Telephone number: +39 06 49917575
E-mail: roberto.contestabile@uniroma1.it

EDUCATION
• Ph.D. Degree in Biochemistry at the University of Wales, Cardiff, UK, September 1997. Final dissertation on "Studies on aspartate aminotransferase and glutamate-1-semialdehyde aminomutase", supervisor Prof. Robert A. John.
• Biological Sciences Degree at Sapienza University of Rome, December 1992, final grade 110/110 cum laude. Dissertation on "Structure-function relationships in serine hydroxymethyltransferase", supervisor Prof. Francesco Bossa.

CURRENT POSITION
March 2022 Permanent position at Sapienza University of Rome, Faculty of Pharmacy and Medicine, Dept. of Biochemistry, as Full Professor of Molecular Biology.

PREVIOUS WORK EXPERIENCES, FELLOWSHIPS AND AWARDS
Jul. 2015 visiting professor at the Department of Chemistry, University of Manitoba, Winnipeg, invited by Prof. Jörg Stetefeld.
Dec 2010 Permanent position at Sapienza University of Rome, faculty of Pharmacy and Medicine, Dept. Biochemical Sciences, as associate professor.
Sep 2001 Permanent position at Sapienza University of Rome, School of Natural Sciences, Physics and Mathematics, Dept. of Biochemistry, as Researcher.
Sep. 2002 Invited researcher at the Max Planck Institute for Terrestrial Microbiology, Marburg, Germany, invited by Prof. Rudolf Thauer for a project on “Catalytic and Thermodynamic Properties of Tetrahydromethanopterin-dependent Serine Hydroxymethyltransferase from Methanococcus jannaschii”.
Feb. 2001 Invited researcher at the Virginia Commonwealth University, Richmond, VA, USA, invited by Prof. Verne Schirch to work on the catalytic mechanism of threonine aldolase.
Jan. 2000 Invited researcher at the Virginia Commonwealth University, Richmond, VA, USA, invited by Prof. Verne Schirch to work on the catalytic mechanism of serine hydroxymethyltransferase.
1999-2000 Postdoctoral fellowship (1 year) from the italian Pasteur Institute - Cenci Bolognetti Foundation to work on the project “Glutamate-1-semialdehyde aminomutase: a potential target of antibacterial drugs”.
Oct. 1998 Short-term contract (4 months) from the Dept. of Biochemical Sciences, Sapienza University of Rome, to work on the “Production and characterization of site-specific mutant forms of vitamin B6-dependent enzymes”.
1997-1998 Postdoctoral fellowship (1 year) from the Italian National Research Council (CNR) to work on the project “Origin of substrate specificity in glutamate semialdehyde-1-aminomutase: a key enzyme in chlorophyll biosynthesis”.
Apr. 1996 Award for the best oral presentation at the Annual Meeting of Ph.D. students organized by the British Biochemical Society at the University of Exeter.
1995-1997 Research Assistant position at the University of Wales, College of Cardiff, UK, to work on a project supported by the British Biotechnology and Biological Sciences Research Council and concerning the catalytic mechanism of glutamate-1-semialdehyde aminomutase.

RESEARCH INTERESTS
The research activity of Prof. Roberto Contestabile concerns Biochemistry and Molecular Biology topics, with particular attention to: i) the mechanisms of regulation of vitamin B6 metabolism at the enzyme and transcriptional levels, in humans and bacteria; ii) the molecular basis of neurological diseases caused by defects of vitamin B6 metabolism; iii) one-carbon units metabolism and cancer metabolic reprogramming; iv) enzyme and cofactor catalysis and v) enzyme inhibition. These studies mainly focus on protein-ligand, protein-protein and protein-DNA interactions.
At the beginning of his carrier, Prof. Contestabile devoted his research activity to the study of pyridoxal 5’-phosphate (PLP)-dependent enzymes, contributing to the understanding of the catalytic and inhibition mechanism of several enzymes, through site-directed mutagenesis, rapid and steady-state kinetic studies. For the enzyme kinetics studies, the experience in the laboratory lead by Prof. Robert. A. John (University of Wales, Cardiff, UK) was of paramount importance. Prof. Contestabile has focused his interest on the catalytic mechanism of serine hydroxymethyltransferase (SHMT), a fundamental enzyme in one-carbon units metabolism. These studies were often carried out in collaboration with Prof. Verne Schirch (Virginia Commonwealth University, Richmond, VA, USA). SHMT plays an important role in the metabolic reprogramming of cancer cells and is a recognized target of chemotherapeutic agents. These characteristics of SHMT are at the basis of the fruitful collaboration with Prof. Francesca Cutruzzolà, Sapienza University of Rome, regarding studies on the role of this enzyme in cancer metabolism and the experimentation of inhibitors within three consecutive AIRC projects, which started in 2013 and are still in progress. In the last ten years, the scientific interest of Prof. Contestabile has also focused on the mechanism of regulation and homeostasis of vitamin B6 metabolism in bacteria and in humans.
With respect to bacteria, the investigations have concerned a family of PLP-dependent transcriptional regulators named MocR transcriptional factors, in particular, their general characterization, mechanism of action and specific interactions with the DNA promoter region. In this respect the research activity has concerned the PtsJ transcriptional repressor from the pathogen bacterium Salmonella typhimurium, the PdxR transcriptional activator from Bacillus clausii and the GabR transcriptional activator from Bacillus subtilis.
An intense research activity has been carried out on the mechanism of action, regulation and allosteric inhibition of Escherichia coli pyridoxal kinase (PLK) and pyridoxine 5’-phosphate oxidase (PNPO). In particular, these studies have concerned the mechanism of regulatory inhibition of these enzymes as a consequence of protein-ligand interactions (protein-pyridoxal and protein-PLP interactions). In humans, maintaining an adequate concentration of PLP in the cell is essential for the correct function of the central nervous system and the failure to do so is related to the onset of severe neurological disorders, such as epilepsy. In this regard, the research activity of Prof. Contestabile has focused on the molecular basis of PNPO deficiency, which is responsible for a rare form of neonatal epileptic encephalopathy. The research activity was carried out in collaboration with Prof. Martin Safo (Virginia Commonwealth University, Richmond, VA, USA), Prof. Tzu-Fun Fu (National Chen Kung University, Taiwan), Dr. Philippa B. Mills (University College of London, UK), Prof. Victoria Bunik (Lomonosov Moscow State University, Russia) and with the clinical team directed by Prof. Vincenzo Leuzzi (Sapienza University). The implication of human PLK variants in diabetes was investigated in collaboration with Dr. Fiammetta Vernì (Sapienza University of Rome) and Prof. Fabrizio Barbetti (University of Rome Tor Vergata).
Protein-protein interaction studies on the HemA and HemL enzymes, involved in tetrapyrroles biosynthesis, have been carried out in collaboration with Prof. Jörg Stetefeld (University of Manitoba, Winnipeg, Canada). Extensive studies on HemL (glutamate 1-semialdehyde aminomutase) have been carried out by Prof. Contestabile in the light of the potential role of this enzyme as target of antibacterial agents in the fight against pathogenic bacteria.
Prof. Contestabile has applied his knowledge on PLP-dependent enzymes to the biotechnological use of these enzymes in the semi-synthetic production of unnatural amino acids. This line of research, which was carried out in collaboration with Prof. Nediljco Budisa (Technische Universität Berlin, Germany and University of Manitoba, Canada), has interesting application developments regarding the metabolic engineering of microorganisms and the translational reprogramming at the ribosomal level.

Experimental Methodologies
Classical Molecular Biology and Biochemical methodologies. Site-directed mutagenesis. Recombinant DNA techniques. Protein expression in E. coli. Protein purification. Western blot. Steady-state and rapid enzyme kinetics. Absorbance, fluorescence and circular dichroism spectroscopy. Stopped-flow spectrophotometry and fluorimetry. Differential scanning fluorimetry. Quantitative, real-time PCR. Metabolic engineering, translational reprogramming, incorporation of non-canonical amino acids. HPLC analysis of small molecules and metabolites.

FUNDING
P.I. of projects funded by the Italian Pasteur Institute
P.I. and partecipant of projects funded by Sapienza University of Rome
Participant in AIRC projects
Participant in PRIN and FIRB projects

INVITED SPEAKER in several international (6) and national (4) scientific meetings.

TEACHING ACTIVITIES Biochemistry, Biochemical Methods and Enzymology in Biological Sciences, Industrial Chemistry and Biotechnology courses. Enzyme Kinetics in Biochemistry Master's degree.

SPOKEN LANGUAGES Italian, native language; English, proficient; French, basic.

SELECTED PUBLICATIONS (last 5 years)
1: Tramonti A, Donkor AK, Parroni A, Musayev FN, Barile A, Ghatge MS, Graziani
C, Alkhairi M, AlAwadh M, di Salvo ML, Safo MK, Contestabile R. Functional and
structural properties of pyridoxal reductase (PdxI) from Escherichia coli: a
pivotal enzyme in the vitamin B6 salvage pathway. FEBS J. 2023
Dec;290(23):5628-5651. doi: 10.1111/febs.16962. Epub 2023 Oct 2. PMID: 37734924.

2: Freda I, Exertier C, Barile A, Chaves-Sanjuan A, Vega MV, Isupov MN, Harmer
NJ, Gugole E, Swuec P, Bolognesi M, Scipioni A, Savino C, Di Salvo ML,
Contestabile R, Vallone B, Tramonti A, Montemiglio LC. Structural insights into
the DNA recognition mechanism by the bacterial transcription factor PdxR.
Nucleic Acids Res. 2023 Aug 25;51(15):8237-8254. doi: 10.1093/nar/gkad552. PMID:
37378428; PMCID: PMC10450172.

3: Pilesi E, Angioli C, Graziani C, Parroni A, Contestabile R, Tramonti A, Vernì
F. A gene-nutrient interaction between vitamin B6 and serine
hydroxymethyltransferase (SHMT) affects genome integrity in Drosophila. J Cell
Physiol. 2023 Jul;238(7):1558-1566. doi: 10.1002/jcp.31033. Epub 2023 May 14.
PMID: 37183313.

4: Babor J, Tramonti A, Nardella C, Deutschbauer A, Contestabile R, de Crécy-
Lagard V. 4'-Deoxypyridoxine disrupts vitamin B₆ homeostasis in
Escherichia coli K12 through combined inhibition of cumulative
B₆ uptake and PLP-dependent enzyme activity. Microbiology (Reading).
2023 Apr;169(4):001319. doi: 10.1099/mic.0.001319. PMID: 37040165; PMCID:
PMC10202323.

5: Rutkiewicz M, Nogues I, Witek W, Angelaccio S, Contestabile R, Ruszkowski M.
Insights into the substrate specificity, structure, and dynamics of plant
histidinol-phosphate aminotransferase (HISN6). Plant Physiol Biochem. 2023
Mar;196:759-773. doi: 10.1016/j.plaphy.2023.02.017. Epub 2023 Feb 10. PMID:
36842242.

6: Tramonti A, Ghatge MS, Babor JT, Musayev FN, di Salvo ML, Barile A, Colotti
G, Giorgi A, Paredes SD, Donkor AK, Al Mughram MH, de Crécy-Lagard V, Safo MK,
Contestabile R. Characterization of the Escherichia coli pyridoxal 5'-phosphate
homeostasis protein (YggS): Role of lysine residues in PLP binding and protein
stability. Protein Sci. 2022 Nov;31(11):e4471. doi: 10.1002/pro.4471. PMID:
36218140; PMCID: PMC9601805.

7: Nogués I, Sekula B, Angelaccio S, Grzechowiak M, Tramonti A, Contestabile R,
Ruszkowski M. Arabidopsis thaliana serine hydroxymethyltransferases: functions,
structures, and perspectives. Plant Physiol Biochem. 2022 Sep 15;187:37-49. doi:
10.1016/j.plaphy.2022.07.025. Epub 2022 Jul 31. PMID: 35947902.

8: Mascolo E, Liguori F, Merigliano C, Schiano L, Gnocchini E, Pilesi E, Volonté
C, Di Salvo ML, Contestabile R, Tramonti A, Vernì F. Vitamin B6 rescues insulin
resistance and glucose-induced DNA damage caused by reduced activity of
Drosophila PI3K. J Cell Physiol. 2022 Sep;237(9):3578-3586. doi:
10.1002/jcp.30812. Epub 2022 Jun 9. PMID: 35678366; PMCID: PMC9545242.

9: Bunik V, Aleshin V, Nogues I, Kähne T, Parroni A, Contestabile R, Salvo ML,
Graf A, Tramonti A. Thiamine-dependent regulation of mammalian brain pyridoxal
kinase in vitro and in vivo. J Neurochem. 2022 Apr;161(1):20-39. doi:
10.1111/jnc.15576. Epub 2022 Feb 23. PMID: 35050500.

10: Spizzichino S, Boi D, Boumis G, Lucchi R, Liberati FR, Capelli D, Montanari
R, Pochetti G, Piacentini R, Parisi G, Paone A, Rinaldo S, Contestabile R,
Tramonti A, Paiardini A, Giardina G, Cutruzzolà F. Cytosolic localization and
in vitro assembly of human de novo thymidylate synthesis complex. FEBS J. 2022
Mar;289(6):1625-1649. doi: 10.1111/febs.16248. Epub 2021 Nov 12. PMID: 34694685;
PMCID: PMC9299187.

11: Boi D, Souvalidou F, Capelli D, Polverino F, Marini G, Montanari R, Pochetti
G, Tramonti A, Contestabile R, Trisciuoglio D, Carpinelli P, Ascanelli C, Lindon
C, De Leo A, Saviano M, Di Santo R, Costi R, Guarguaglini G, Paiardini A.
PHA-680626 Is an Effective Inhibitor of the Interaction between Aurora-A and
N-Myc. Int J Mol Sci. 2021 Dec 4;22(23):13122. doi: 10.3390/ijms222313122. PMID:
34884931; PMCID: PMC8658095.

12: Barile A, Mills P, di Salvo ML, Graziani C, Bunik V, Clayton P, Contestabile
R, Tramonti A. Characterization of Novel Pathogenic Variants Causing
Pyridox(am)ine 5'-Phosphate Oxidase-Dependent Epilepsy. Int J Mol Sci. 2021 Nov
6;22(21):12013. doi: 10.3390/ijms222112013. PMID: 34769443; PMCID: PMC8584306.

13: Tramonti A, Cuyàs E, Encinar JA, Pietzke M, Paone A, Verdura S, Arbusà A,
Martin-Castillo B, Giardina G, Joven J, Vazquez A, Contestabile R, Cutruzzolà F,
Menendez JA. Metformin Is a Pyridoxal-5'-phosphate (PLP)-Competitive Inhibitor
of SHMT2. Cancers (Basel). 2021 Aug 9;13(16):4009. doi: 10.3390/cancers13164009.
PMID: 34439169; PMCID: PMC8393646.

14: Tramonti A, Contestabile R, Florio R, Nardella C, Barile A, Di Salvo ML. A
Novel, Easy Assay Method for Human Cysteine Sulfinic Acid Decarboxylase. Life
(Basel). 2021 May 14;11(5):438. doi: 10.3390/life11050438. PMID: 34068845;
PMCID: PMC8153620.

15: Tramonti A, Nardella C, di Salvo ML, Barile A, D'Alessio F, de Crécy-Lagard
V, Contestabile R. Knowns and Unknowns of Vitamin B₆ Metabolism in
Escherichia coli. EcoSal Plus. 2021
Apr;9(2):10.1128/ecosalplus.ESP-0004-2021. doi:
10.1128/ecosalplus.ESP-0004-2021. PMID: 33787481; PMCID: PMC8995791.

16: Mascolo E, Liguori F, Stufera Mecarelli L, Amoroso N, Merigliano C, Amadio
S, Volonté C, Contestabile R, Tramonti A, Vernì F. Functional Inactivation of
DrosophilaGCK Orthologs Causes Genomic Instability and Oxidative
Stress in a Fly Model of MODY-2. Int J Mol Sci. 2021 Jan 18;22(2):918. doi:
10.3390/ijms22020918. PMID: 33477627; PMCID: PMC7831483.

17: Barile A, Battista T, Fiorillo A, di Salvo ML, Malatesta F, Tramonti A,
Ilari A, Contestabile R. Identification and characterization of the pyridoxal
5'-phosphate allosteric site in Escherichia coli pyridoxine 5'-phosphate
oxidase. J Biol Chem. 2021 Jan-Jun;296:100795. doi: 10.1016/j.jbc.2021.100795.
Epub 2021 May 18. PMID: 34019876; PMCID: PMC8215295.

18: Schipp CJ, Ma Y, Al-Shameri A, D'Alessio F, Neubauer P, Contestabile R,
Budisa N, di Salvo ML. An Engineered Escherichia coli Strain with Synthetic
Metabolism for in-Cell Production of Translationally Active Methionine
Derivatives. Chembiochem. 2020 Dec 11;21(24):3525-3538. doi:
10.1002/cbic.202000257. Epub 2020 Oct 13. PMID: 32734669; PMCID: PMC7756864.

19: Nardella C, Barile A, di Salvo ML, Milano T, Pascarella S, Tramonti A,
Contestabile R. Interaction of Bacillus subtilis GabR with the gabTD promoter:
role of repeated sequences and effect of GABA in transcriptional activation.
FEBS J. 2020 Nov;287(22):4952-4970. doi: 10.1111/febs.15286. Epub 2020 Mar 20.
PMID: 32147931.

20: Nogués I, Tramonti A, Angelaccio S, Ruszkowski M, Sekula B, Contestabile R.
Structural and kinetic properties of serine hydroxymethyltransferase from the
halophytic cyanobacterium Aphanothece halophytica provide a rationale for salt
tolerance. Int J Biol Macromol. 2020 Sep 15;159:517-529. doi:
10.1016/j.ijbiomac.2020.05.081. Epub 2020 May 15. PMID: 32417544.

21: Barile A, Nogués I, di Salvo ML, Bunik V, Contestabile R, Tramonti A.
Molecular characterization of pyridoxine 5'-phosphate oxidase and its pathogenic
forms associated with neonatal epileptic encephalopathy. Sci Rep. 2020 Aug
12;10(1):13621. doi: 10.1038/s41598-020-70598-7. PMID: 32788630; PMCID:
PMC7424515.

22: Boyko AI, Artiukhov AV, Kaehne T, di Salvo ML, Bonaccorsi di Patti MC,
Contestabile R, Tramonti A, Bunik VI. Isoforms of the DHTKD1-Encoded
2-Oxoadipate Dehydrogenase, Identified in Animal Tissues, Are not Observed upon
the Human DHTKD1 Expression in Bacterial or Yeast Systems. Biochemistry (Mosc).
2020 Aug;85(8):920-929. doi: 10.1134/S0006297920080076. PMID: 33045952.

23: Contestabile R, di Salvo ML, Bunik V, Tramonti A, Vernì F. The multifaceted
role of vitamin B₆ in cancer: Drosophila as a model system to
investigate DNA damage. Open Biol. 2020 Mar;10(3):200034. doi:
10.1098/rsob.200034. Epub 2020 Mar 25. PMID: 32208818; PMCID: PMC7125957.

24: Di Salvo ML, Fesko K, Phillips RS, Contestabile R. Editorial: PLP-Dependent
Enzymes: Extraordinary Versatile Catalysts and Ideal Biotechnological Tools for
the Production of Unnatural Amino Acids and Related Compounds. Front Bioeng
Biotechnol. 2020 Feb 11;8:52. doi: 10.3389/fbioe.2020.00052. PMID: 32117932;
PMCID: PMC7026007.

25: Ruszkowski M, Sekula B, Ruszkowska A, Contestabile R, Nogues I, Angelaccio
S, Szczepaniak A, Dauter Z. Structural basis of methotrexate and pemetrexed
action on serine hydroxymethyltransferases revealed using plant models. Sci Rep.
2019 Dec 23;9(1):19614. doi: 10.1038/s41598-019-56043-4. PMID: 31873125; PMCID:
PMC6928210.

26: Barile A, Tramonti A, di Salvo ML, Nogués I, Nardella C, Malatesta F,
Contestabile R. Allosteric feedback inhibition of pyridoxine 5'-phosphate
oxidase from Escherichia coli. J Biol Chem. 2019 Oct
25;294(43):15593-15603. doi: 10.1074/jbc.RA119.009697. Epub 2019 Sep 4. PMID:
31484724; PMCID: PMC6816108.

27: Mascolo E, Barile A, Mecarelli LS, Amoroso N, Merigliano C, Massimi A,
Saggio I, Hansen T, Tramonti A, Di Salvo ML, Barbetti F, Contestabile R, Vernì
F. The expression of four pyridoxal kinase (PDXK) human variants in Drosophila
impacts on genome integrity. Sci Rep. 2019 Oct 2;9(1):14188. doi:
10.1038/s41598-019-50673-4. PMID: 31578392; PMCID: PMC6775053.

28: Guiducci G, Paone A, Tramonti A, Giardina G, Rinaldo S, Bouzidi A, Magnifico
MC, Marani M, Menendez JA, Fatica A, Macone A, Armaos A, Tartaglia GG,
Contestabile R, Paiardini A, Cutruzzolà F. The moonlighting RNA-binding activity
of cytosolic serine hydroxymethyltransferase contributes to control
compartmentalization of serine metabolism. Nucleic Acids Res. 2019 May
7;47(8):4240-4254. doi: 10.1093/nar/gkz129. PMID: 30809670; PMCID: PMC6486632.

29: Nardella C, Boi D, di Salvo ML, Barile A, Stetefeld J, Tramonti A,
Contestabile R. Isolation of a Complex Formed Between Acinetobacter
baumannii HemA and HemL, Key Enzymes of Tetrapyrroles Biosynthesis. Front
Mol Biosci. 2019 Feb 26;6:6. doi: 10.3389/fmolb.2019.00006. PMID: 30863751;
PMCID: PMC6399207.

SELECTED BOOKS
1. Angelaccio, S., Contestabile, R., Di Giovine, P. and Bossa, F. (2000) “Role of Y65 and E57 in Escherichia coli serine hydroxymethyltransferase”. In Biochemistry and Molecular Biology of Vitamin B6 and PQQ-dependent Proteins (A. Iriarte, H. M. Kagan and M. Martinez-Carrion Eds), Birkhäuser Verlag, Basel, Switzerland, pp. 167-170.
2. John, R.A., Khayer, K., Jenn, T., Akhtar, M. , Gani, D. and Contestabile, R. (2000) “Reactions of glutamate 1-semialdehyde aminomutase with R- and S-enantiomers of a novel, mechanism-based inhibitor, 2,3-diaminopropyl sulphate”. In Biochemistry and Molecular Biology of Vitamin B6 and PQQ-dependent Proteins (A. Iriarte, H. M. Kagan and M. Martinez-Carrion Eds), Birkhäuser Verlag, Basel, Switzerland, pp. 285-288.
3. Raboni S, Contestabile R, Spyrakis F, Campanini B, Amadasi A, Bettati S, Peracchi A and Mozzarelli A (2010) “Pyridoxal 5'-Phosphate-Dependent Enzymes: Catalysis, Conformation and Genomics”, in Comprehensive Natural Products Chemistry.
4. Ghatge, M.S., di Salvo, M.L., Contestabile, R., Karve, S., Schirch, V., Safo. M.K. Molecular Defects of Vitamin B6 Metabolism Associated with Neonatal Epileptic Encephalopathy (2012) pp.267-290. In Miscellanea on Encephalopathies - A Second Look - vol. 2. ISBN:9789535105589.
5. Bonaccorsi, Contestabile, di Salvo (2012) Metodologie biochimiche. Casa Editrice Ambrosiana. ISBN:9788808183293