INSTITUTE OF BIOLOGY, MEDICINAL CHEMISTRY & BIOTECHNOLOGY
 
  Drug Discovery
  Computational Chemistry
  Molecular Analysis
  Organic and Organometallic Chemistry
  Medicinal Chemistry
  Structural Biology & Chemistry
  > Funded projects
  > Collaborations
  > Publications
  Molecular Endocrinology
  Signal Mediated Gene Expression
  Molecular & Cellular Ageing
  Biomedical Applications
  Holistic Approaches in Health
  Chemical Carcinogenesis and Genetic Toxicology
  Metabolic Engineering-Bioinformatics
  Biotechnology
  Enzyme and Synthetic Biotechnology
  Biomimetics & Nanobiotechnology

Structural Biology & Chemistry

The general theme that underlies the work of the team is functional and structural studies of proteins for the purpose of rational design, synthesis & discovery of novel therapeutic agents as well as for biotechnological applications.

The team utilizes recombinant DNA techniques, protein expression and advanced protein purification techniques in conjunction with biochemical binding assays, enzyme kinetics and X-ray protein crystallography, to study the three-dimensional structure and properties of proteins and protein-ligand complexes.

In depth understanding of the structure-function relationships, of protein targets of interest can guide both computational and synthetic chemists to the rational design, synthesis and discovery of novel therapeutic agents and other products of biotechnological interest. For this reason our team is in close collaboration with research groups specialized in the fields of computational chemistry, organic synthesis, biology, biotechnology and medicine.

 

Members

Research staff

Dr Spyros E. Zographos, Researcher B’ (2006-Present)

Current colleagues:
Dr Katerina E. Tsitsanou, BSc in Chemistry, PhD in Biology
Senior Post Doctoral Researcher (IBMCB-STHENOS)

Christina E. Drakou, BSc, MSc in Chemistry
PhD student, Dept. of Chemistry, University of Athens (IBMCB-PREVENT)

George N. Oikonomakos, BSc in Crop Production, MSc in Biochemistry
Research Technician (IBMCB-ChrysAnalys)

Elina Dima, BSc in Chemistry
MSc student, Faculty of Pharmacy, University of Athens

Evi Slavaki, BSc in Agriculture
MSc student, Dept. of Biotechnology, Agricultural University of Athens

Maria Kokkoti
Final year project student
Dept. of Food Technology, Technological Educational Institute of Athens

 

Dr Evangelia D. Chrysina, Research officer (2006- present)

Current colleagues:
Maria Dimarogona, MSc in Structural and Functional Engineering of Biomolecules, PhD in Chemical Engineering
Post-doctoral Researcher (external collaborator)

Maria-Despoina Charavgi, MSc in Chemistry
PhD student, School of Chemical Engineering, National Technical University of Athens (HRAKLEITOS II)

Michail Mamais, BSc, MSc in Chemistry
PhD student, Department of Chemistry, University of Athens (HRAKLEITOS II)

Maria Papakonstantinou, BSc in Chemistry
MSc student, Department of Chemistry, University of Athens

Anastasia Sotiropoulou, BSc in Chemistry
MSc student, Department of Chemistry, University of Athens

Maria Karamolegkou, BSc in Biology
MSc student, Department of Chemistry, University of Athens

Research Projects

 

OBP-structure-aided repellent discovery

(SE Zographos, K. Tsitsanou, C. Drakou & E. Dima)

OBP1.jpgMuch physiological and behavioral evidence has been provided suggesting that insect Odorant Binding Proteins (OBPs) are indispensable for odorant recognition and thus appealing targets for structure-based discovery and design of novel host-seeking disruptors. Despite the fact that more than 60 putative OBP-encoding genes have been identified in the malaria vector Anopheles gambiae, the crystal structures of only six of them are known. It is therefore clear that OBP structure determination constitutes the bottle-neck for structure-based approaches to mosquito repellent/attractant discovery.

We have recently proposed OBPs as valuable molecular targets for the structure-based discovery and design of disruptors of normal olfactory and host seeking mosquito behavior (Tsitsanou et al, 2012). Significantly, we have now developed and successfully validated a Ligand- & OBP-structure-aided Discovery Protocol that led to identification of novel bioactive leads, attesting the applicability of the OBP-structure-aided discovery method (Tsitsanou et al, 2013a). Therefore, the study on OBPs of A. gambiae and the determination of their 3D-structures and binding specificities could help us understand the molecular basis of odorant detection by OBPs, in this and other anthropophilic species, towards the development of new effective agents for the prevention of mosquito-borne diseases.

 

Collaborators

ENAROMaTIC consortium: 2GR, 6EU, 1 USA and 1 Nigerian partner and Novartis A.G., Janssen PMP and Key Organics Ltd. (www.enaromatic.org)
Dr. Kostas Iatrou, Insect Molecular Genetics and Biotechnology Laboratory, NCRS Demokritos
Prof. Elias Eliopoulos, Dept. of Agricultural Biotechnology, Agricultural University of Athens
Lecturer Trias Thireou, Dept. of Agricultural Biotechnology, Agricultural University of Athens
Dr. M. Zervou, Molecular Analysis Team, IBMCB
Dr. P. Zoumpoulakis, Molecular Analysis Team, IBMCB
Dr. T. Calogeropoulou, Medicinal Chemistry Team, IBMCB
Prof. V. Roussis, School of Pharmacy, University of Athens
Dr. Antonios Michaelakis, Laboratory of Agricultural Entomology, Benaki Phytopathological Institute
Ass. Prof. M. Senay Sengul, Dept. of Molecular Biology and Genetics, Gaziosmanpasa University, Turkey

 

Publications

Tsitsanou KE, Drakou CE, Thireou T, Gruber AV, Kythreoti G, Azem A, Fessas D, Eliopoulos E, Iatrou K, Zographos SE (2013) The crystal and solution studies of the "Plus-C" odorant binding protein 48 from Anopheles gambiae: Control of binding specificity through 3D domain-swapping. Journal of Biological Chemistry 288(46): 33427-33438

Tsitsanou KE, Thireou T, Drakou CE, Koussis K, Keramioti MV, Leonidas DD, Eliopoulos E, Iatrou K, Zographos SE (2012) Anopheles gambiae odorant binding protein crystal complex with the synthetic repellent DEET: implications for structure-based design of novel mosquito repellents. Cellular and Molecular Life Sciences 69(2): 283-297

Return to Research Activies

 

hCINAP: Regulation of RNA processing in eukaryotes

(SE Zographos, C. Drakou)

SBCG50Y_CINAP.jpghuman Coilin Interacting Nuclear ATPase Protein (hCINAP) directly interacts with coilin, a marker protein of Cajal Bodies (CBs), nuclear organelles involved in the maturation of small nuclear ribonucleoproteins (Santama et al, 2005). hCINAP has previously been designated as an adenylate kinase (AK6), but it exhibits structural features characteristic of ATPase/GTPase proteins (Walker motifs A and B) and also intrinsic ATPase activity. Recently we have reported the first high-resolution structure of hCINAP in complex with the substrate ADP and the structure of the ternary complex hCINAP-Mg2+ADP-PO43-. Structural analysis suggested a functional role for His79 in the Walker B motif. We have shown that in vivo expression of hCINAP-H79G in human cells is toxic and drastically deregulates the number and appearance of CBs. Our findings suggest that hCINAP may not simply regulate nucleotide homeostasis, but may have broader functionality, including control of CBs assembly (Drakou et al, 2012). hCINAP has also been reported to be an important regulator of Ribosomal Protein-HDM2-p53 pathway and a potential anticancer drug target (Zhang et al, 2013). The detailed understanding of hCINAP function and its interaction with ligands and protein partners will give new insights into basic steps in eukaryotic gene expression, such as regulation of RNA processing, and assess its potential as a novel pharmacological target to combat cancer.

 

Collaborators

Ass. Prof. N. Santama, Department of Biological Sciences, University of Cyprus
Prof. A.I. Lamond, Welcome Trust Biocentre, University of Dundee, UK

 

Publications

Drakou CE, Malekkou A, Hayes JM, Lederer CW, Leonidas DD, Oikonomakos NG, Lamond AI, Santama N, Zographos SE (2012) hCINAP is an atypical mammalian nuclear adenylate kinase with an ATPase motif: structural and functional studies. Proteins 80(1): 206-220

Santama N, Ogg SC, Malekkou A, Zographos SE, Weis K, Lamond AI (2005) Characterization of hCINAP, a novel coilin-interacting protein encoded by a transcript from the transcription factor TAFIID(32) locus. Journal of Biological Chemistry 280(43): 36429-36441

 

Return to Research Activies

 

Mechanism of Glycogen Phosphorylase inhibition by naturally occurring hypoglycemic agents

(SE Zographos, K. Tsitsanou)

TOC4.jpgA number of natural compounds with hypoglycemic activity have been identified as potent inhibitors of Glycogen phosphorylase targeting the catalytic site (iminosugars; Oikonomakos et al, 2006), the allosteric site (pentacyclic triterpenes and FR258900 from Fungus No. 138,354; Wen et al, 2008, Tiraidis et al, 2007) and the inhibitor (caffeine) site of the enzyme (flavonoids; Oikonomakos et al, 2000). These compounds provide new scaffolds that can be exploited by means of chemical modifications to yield new potent inhibitors of glycogen phosphorylase with improved anti-hyperglycemic properties. To elucidate the inhibition mechanism of flavonoids, we have recently performed structural, kinetic and QM/MM-PBSA calculations of three natural flavonoid derivatives that led us to propose a QM/MM-PBSA protocol for prediction of flavanoid binding at the inhibitor or allosteric site of GP (Tsitsanou et al, 2013b). The nature of flavanoid phenyl ring substituents are determined as critical to preferred GP binding site, and require consideration in future structure-activity relationship (SAR) studies, before embarking on more time-consuming and costly experimental evaluation.
We now intend to obtain more structural and kinetic data of glycogen phosphorylase in complex with more than 100 natural flavonoid derivatives. The essential binding properties of specific compounds will be analyzed in an effort to provide rationalizations for the affinities of these compounds, carry on a more detailed SAR study and to exploit the molecular interactions that might give rise to better inhibitors, potential agents for controlling hyperglycaemia in T2D.

 

Collaborators

Ass. Prof. D. Leonidas, Dept. of Biochemistry & Biotechnology, University of Thessaly
Lecturer JM. Hayes, School of Forensic and Investigative Sciences, Univ. of Central Lancashire, UK
Prof. A. Kato, Dept. of Hospital Pharmacy, University of Toyama, Japan
Prof. L. Agius, Dept. of Diabetes, University of Newcastle, UK

 

Featured Publications

Tsitsanou KE, Hayes JM, Keramioti M, Mamais M, Oikonomakos NG, Kato A, Leonidas DD, Zographos SE (2013) Sourcing the affinity of flavonoids for the glycogen phosphorylase inhibitor site via crystallography, kinetics and QM/MM-PBSA binding studies: Comparison of chrysin and flavopiridol. Food Chem Toxicol 61, 14-27

Wen XA, Sun HB, Liu J, Cheng KG, Zhang P, Zhang LY, Hao J, Ni PZ, Zographos SE, Leonidas DD, Alexacou KM, Gimisis T, Hayes JM & Oikonomakos NG (2008) Naturally occurring pentacyclic triterpenes as inhibitors of glycogen phosphorylase: Synthesis, structure-activity relationships, and X-ray crystallographic studies. J Med Chem 51, 3540-3554

Tiraidis C, Alexacou KM, Zographos SE, Leonidas DD, Gimisis T & Oikonomakos NG (2007) FR258900, a potential anti-hyperglycemic drug, binds at the allosteric site of glycogen phosphorylase. Protein Sci 16, 1773-1782

Oikonomakos NG, Tiraidis C, Leonidas DD, Zographos SE, Kristiansen M, Jessen CU, Norskov-Lauritsen L & Agius L (2006) Iminosugars as potential inhibitors of glycogenolysis: Structural insights into the molecular basis of glycogen phosphorylase inhibition. J Med Chem 49, 5687-5701

 

Return to Research Activies

 

Development of dissociated glucocorticoids

(SE Zographos, K. Tsitsanou)

Glucocorticoids (GCs) are essential steroid hormones that regulate a variety of physiological processes including development, metabolism, homeostasis and cell death. The biological actions of GCs are mediated through the glucocorticoid receptor (GR), a ligand-activated transcription factor. Interestingly, activation of GR is associated with favourable prognosis and treatment of estrogen receptor-positive breast cancer and Acute Lymphoblastic Leukaemia (Dordelmann et al, 1999; Pan et al, 2011). In this context, GR-induced inhibition of NFKB activity is of clinical importance. However, the clinical use of glucocorticoids is limited by serious side-effects due to e.g. glucocorticoid responsive elements (GREs)-mediated activation of gluconeogenesis leading to severe hyperglycemia. It is therefore important to discover/develop “dissociated glucocorticoids” (DGCs) i.e. glucocorticoids that suppress NFKB activity but display little or no GRE-mediated activation and examine their synergism with established anticancer drugs (Biggadike et al, 2009; Madauss et al, 2008). 
The crystal structures of DGCs-bound GR complexes will allow lead improvement by structure-based approaches and unravel the molecular determinants of DGC action.

 

Collaborators

All members of the Molecular analysis, Medicinal Chemistry, Organic and Organometallic Chemistry and Molecular Endocrinology Teams of IBMCB (Project STHENOS 2013-2015)

 

Return to Research Activies

 

Cfd1 and Nbp35: Regulation of ciliogenesis

(SE Zographos, E. Slavaki)

Nucleotide binding proteins Cfd1 and Nbp35 (NUBPs)are MRP/MinD-type P-loop NTPases with sequence similarity to bacterial division site-determining proteins and are highly conserved essential proteins in eukaryotes. They have been implicated in the assembly of cytosolic iron-sulfur proteins and iron homeostasis (Netz et al, 2007; Netz et al, 2012; Stehling et al, 2008) and the regulation of centriole duplication in mammalian cells (Christodoulou et al, 2006). A current study reveals that NUBPs are also components of the basal body and have significant effects on the formation of primary cilia, acting as negative regulators for ciliogenesis. Primary cilia are microtubule-based nanomachines involved in mechano- and chemo-sensory functions and signal transduction in animal cells. Anomalies in their structure and function are at the heart of many ciliary diseases, serious, multi-system genetic disorders causing low quality of life and early death. Understanding the structure-function relationship of the Nubps and their interacting proteins (Kypri et al, 2013), could offer critical insights in the molecular pathology of ciliary disease.
The goal of this project is to generate crystals of Cfd1 and Nbp35 and solve their 3D-structures in both free and in complex with their substrates. Given the involvement of these proteins as regulators in diverse and seemingly unrelated cellular functions, deciphering their structure and their interactions may be key to understanding their mode of action in these processes including, but not limited, to pathogenic pathways of ciliopathies.

 

Collaborators

Ass. Prof. N. Santama, Department of Biological Sciences, University of Cyprus
Prof. José M. Valpuesta, Centro Nacional de Biotecnología, Spain

 

Return to Research Activies

 

Kinetic characterization of mushroom tyrosinase modulators

(SE Zographos, M. Kokkoti)

Tyrosinase, is responsible for the undesired enzymatic browning of fruits and vegetables {Martinez, M. V.; Whitaker, J. R. The biochemistry and control of enzymatic browning. Trends Food Sci. Technol. 1995, 6, 195-200. (22)} However, besides this role in undesired browning, the activity of tyrosinase is needed in other cases (raisins, cocoa, fermented tea leaves) where it produces distinct organoleptic properties. Furthermore, tyrosinase inhibitors are becoming increasingly important in the cosmetic industry due to their skin-whitening effects. A number of tyrosinase inhibitors are reported from both natural and synthetic sources, but only a few of them are used as skin-whitening agents, primarily due to various safety concerns {Maeda, K.; Fukuda, M. In Vitro effectiveness of several whitening cosmetic components in human melanocytes. J. Soc. Cosmet. Chem. 1991, 42, 361-368}.

Our specific task in this collaborative project is the detailed enzyme kinetic analysis of the newly synthesized ligands as a part of an integrated approach towards the development of new compounds that can be used as additives of food, beverage and cosmetics.

 

Collaborators

Dr. P. Zoumpoulakis, Molecular Analysis Team, IBMCB
Dr. T. Calogeropoulou, Medicinal Chemistry Team, IBMCB
Dr. N. Chondrogianni, Molecular & Cellular Ageing Team, IBMCB

 

Return to Research Activies

 

New antidiabetics, Protein structure and drug design

(ED Chrysina, M. Mamais, M. Papakonstantinou, A. Sotiropoulou, M. Karamolegkou)

The biomedical target: The number of people suffering from type 2 diabetes (T2D) is on the increase; hence, there is strong demand for the discovery of new effective antidiabetic agents with improved specificity. Glycogen is a major contributor to high blood glucose levels in the bloodstream. Given the importance of glycogen metabolism, attention has been drawn on the investigation of enzymes directly implicated in the corresponding metabolic pathway that control glycogen synthesis and degradation.

Aim of the study: glycogen phosphorylase (GP), a validated target of key importance in glycogen metabolism, has been employed for in vitro screening of potential inhibitors using biochemical assays and X-ray protein crystallography. Our work employs rabbit muscle GP (RMGPb) the hepatic isoenzyme that shares > 80 % homology (100 % identity at the active site).

Results obtained: More than 150 inhibitors targeting the catalytic, the inhibitor and the new allosteric site of GP have been tested for their potency against RMGPb, Most of the analogues studied until present are based on the information derived by the 3D structure of RMGPb in complex with α-D-glucose (lead compound) and its derivatives N-acetyl-β-D-glucopyranosylamine (Ki of 32 μM) and naphthoyl analogues of β-D-glucopyranosylurea (Ki of 350 nM). The in depth knowledge we acquired from the 3D structure of the complexes studied until present enabled us to achieve low nM range inhibitors synthesized by our colleagues in Athens and Hungary.

Collaborations

Calogeropoulou Theodora, Koufaki Maria, Papahatjis Demetris, Zervou Maria, Zoumpoulakis Panagiotis, Drs, Institute of Biology, Medicinal Chemistry and Biotechnology, NHRF

Emiris Ioannis, Professor, Department of Informatics & Telecommunications, University of Athens
Gimisis Thanasis, Associate Professor, Lab. of Organic Chemistry, Department of Chemistry, University of Athens
Mikros Emmanouel, Professor Department of Pharmaceutical Chemistry, University of Athens
Perrea Despoina, Professor, Laboratory of Experimental Surgery & Surgical Research “N.S. Christeas”, Medical School, University of Athens, Greece

Gergely Pal, Professor, Department of Medical Chemistry, University of Debrecen, Hungary
Krimm Isabelle, Dr, Institut des Sciences Analytiques, UMR CNRS 5280, France
Lancelin Jean-Marc, Professor, Institut des Sciences Analytiques, UMR CNRS 5280, Université Claude-Bernard Lyon 1, France
Markovitsi Dimitra, CNRS Research Director, Head of the Francis Perrin Laboratory, URA CEA-CNRS 2453, France
Praly Jean-Pierre, Dr, Institut de Chimie et Biochimie Moleculaires et Supramoleculaires, UMR-CNRS 5246, Université Claude-Bernard Lyon 1, France
Somsák Laszlo, Professor, Department of Organic Chemistry, University of Debrecen, Hungary
Vidal Sebastien, Dr, Institut de Chimie et Biochimie Moleculaires et Supramoleculaires Laboratoire de Chimie Organique 2 - Glycochimie UMR-CNRS 5246

University of Maryland-USA
MacKerell, Jr. Alexander, Professor, Department of Pharmaceutical Sciences, University of Maryland, USA
Srivastava Amrita, Department of Chemistry, Indian Institute of Technology, Madras, India

 

Selected publications

  • Chrysina, E.D., Kosmopoulou, M.N., Tiraidis, C., Kardakaris, R., Bischler, N., Leonidas, D.D., Hadady, Z., Somsak, L., Docsa, T., Gergely, P. and Oikonomakos, N.G. (2005). Kinetic and crystallographic studies on 2-(b-D-glucopyranosyl-5-methyl-1, 3, 4-oxadiazole, -benzothiazole, and –benzimidazole, inhibitors of muscle glycogen phosphorylase b. Evidence for a new binding site. Protein Sci. 14, 873-888 [DOI: 10.1110/ps.041216105]

Pdb Ids: 1XKX, 1XL0, 1XL1

  • Archontis, G., Watson, K.A., Xie, Q., Andreou, G., Chrysina, E.D., Zographos, S.E., Oikonomakos, N.G., and Karplus, M. (2005) Molecular recognition and relative binding of glucopyranose spirohydantoin analogues to glycogen phosphorylase: a free energy perturbation study. Proteins: Structure, Function, and Bioinformatics 61, 984-998 [DOI: 10.1002/prot.20641]
  • +Watson, K.A., +Chrysina, E.D., Tsitsanou K.E., Zographos, S.E., Gregoriou, M., Archontis, G., Fleet, G.W.J., and Oikonomakos, N.G. (2005) Kinetic and crystallographic studies of glucopyranose spirohydantoin and glucopyranosylamine analogues inhibitors of glycogen phosphorylase. Proteins: Structure, Function, and Bioinformatics 61, 966-983 (+Equal Contribution) [DOI: 10.1002/prot.20653]

Pdb Ids: 1FS4, 1FTQ, 1FTW, 1FTY, 1FU4, 1FU7, 1FU8

  • Somsák, L., Czifrák, K., Tóth, M., Bokor, E., *Chrysina, E.D., Alexacou, K.M., Hayes, J.M., Tiraidis, C., Lazoura, E., Leonidas, D.D., Zographos, S.E., Oikonomakos, N.G. (2008). New Inhibitors of Glycogen Phosphorylase as Potential Antidiabetic Agents. Curr. Med. Chem. 15, 2933-2983 [DOI: 10.2174/092986708786848659]
  • *Chrysina, E.D., Bokor, E., Alexacou, K-M., Charavgi, M-D., Oikonomakos, G.N., Zographos, S.E., Leonidas, D.D., Oikonomakos, N.G., *Somsák, L. (2009). Amide-1,2,3-triazole bioisosterism: the glycogen phosphorylase case. Tetrahedron Asymmetry 20, 733-740 [DOI: 10.1016/j.tetasy.2009.03.021]

Pdb IDs: 3G2I, 3G2H, 3G2K, 3G2J, 3G2L, 3G2N

  • Benltifa, M., Hayes, J., Vidal, S. Gueyrard, D., Goekjian, P.G., *Praly, J-P, Kizilis, G., Tiraidis, C., Alexacou, K-M., *Chrysina, E.D., Zographos, S.E., Leonidas, D.D., Archontis, G., Oikonomakos, N.G. (2009). Glucose-based spiro-isoxazolines: A new family of potent glycogen phosphorylase inhibitors. Bioorg. Med. Chem. 17, 7368-7380 [DOI: 10.1016/j.bmc.2009.08.060]

Pdb IDs: 2QRM, 2QRQ, 2QRP, 2QRH, 2QRG

  • *Chrysina, E.D. The prototype of glycogen phosphorylase (2010) Mini Rev. Med. Chem. 10, 1093-10101 [DOI: 10.2174/1389557511009011093]
  • *Chrysina, E.D., Chajistamatiou, A., Chegkazi, M. (2011) From structure-based to knowledge-based drug design through x-ray protein crystallography: sketching glycogen phosphorylase binding sites. Curr. Med. Chem. 18, 2620-22629 [DOI: 10.2174/092986711795933632]
  • Nagy, V., Felföldi, N., Kónya, B., Praly, J.-P., Docsa, T., Gergely, P., *Chrysina, E.D., Tiraidis, C., Kosmopoulou, M.N., Alexacou, K.-M., Konstantakaki, M., Leonidas, D.D., Zographos, S.E., Oikonomakos, N.G., Kozmon, S., Tvaroška,I., Somsák, L. (2012) N-(4-Substituted-benzoyl)-N'-(β-D-glucopyranosyl)ureas as inhibitors of glycogen phosphorylase: synthesis and evaluation by kinetic, crystallographic, and molecular modelling methods Bioorg. Med. Chem. 20, 1801-1816 [DOI: 10.1016/j.bmc.2011.12.059]

Pdb IDs: 2QNB, 2QLM, 2QLN, 2QN3, 2QN7, 2QN8, 2QN9

  • Goyard, D., Baron, M., Skourti, P.V., Chajistamatiou, A.S., Docsa, T., Gergely, P., *Chrysina, E.D., Praly, J.-P., Vidal., S. (2012) Synthesis of 1,2,3-triazoles from azido xylose and 5-thioxylose: Evaluation of the xylose scaffold for the design of potential glycogen phosphorylase inhibitors Carbohydrate Res. 364, 28-40 [DOI:10.1016/j.carres.2012.09.020]

 

Front covers in peer-reviewed scientific journals

DOI:10.1016/j.bmc.2005.08.010

DOI: 10.2174/1570180054405839

   

DOI: 10.1016/j.bmc.2006.01.045

DOI: 10.2174/092986711795933632

 

Return to Research Activies

 

Structural studies of Biocatalysts to shed light on their mechanism of action

(ED Chrysina, M Dimarogona, M-D Charavgi)

The targets: Xylanases have been the focus of considerable research in recent decades owing to their extensive use in a variety of industrial applications in biotechnology and pharmacology. Similarly, due to the extended interest in exploiting thermostable enzymes in the production of second-generation biofuels, (GEs) have been under investigation. GEs are suggested to be responsible for cleaving the ester bond between the aromatic alcohols of lignin and the carboxyl group of 4-O-methyl-D-glucuronic acid residues in glucuronoxylan, thus constituting an attractive target for industrial applications also as a source of fine chemicals for pharmaceutical industries. However, the function and biochemical properties of GEs have not yet been elucidated, as there is lack of direct evidence on their physiological role.

Aim of the study: Structural studies of two new enzymes a GH10 xylanase from Fusarium oxysporum (FoXyn10a), and a GE from Sporotrichum thermophile, (StGE), and its S213A mutant were performed to shed light on their calalytic mechanism.

Results obtained: We determined the structure of FoXyn10a at 1.94 Å resolution from crystals grown to the tetragonal lattice, spacegroup P41212 with five molecules per asymmetric unit in the frame a PhD thesis under my co-supervision. Alignment of FoXyn10a with sequence and structural homologues denoted an atypically long loop connecting strand 6b and helix 6 that was only present in one other GH10 xylanase, the structure of which is not known. This structural feature may be of functional importance, with potential implications in the catalytic efficiency of the enzyme β-D-glucopyranuronate at high resolution also in the frame of a PhD thesis currently in progress (in collab. with School of Chemical Engineering, NTUA). These are the first crystal structures of a thermophilic GE esterase both unliganded and bound to a substrate analogue unraveling the organization of the catalytic triad residues & their neighbors lining the active site

      

Xylanase, FoXyn10a                                                                      Glucuronoyl esterase, StGE2

 

Collaborators

Christakopoulos Paul, Associate Professor, School of Chemical Engineering, NTUA
Topakas Evangelos, Lecturer, School of Chemical Engineering, NTUA

Selected publications

  1. Charavgi, M., Dimarogona, M., Topakas, E., Christakopoulos, P., *Chrysina, E.D. (2013) The crystal structure of a novel glucuronoyl esterase from Myceliophthora thermophila gives new insights on its role as a potential biocatalyst. Acta Crystallogr. D69, 63-73[DOI: 10.1107/S0907444912042400]

Pdb IDs: 4G4G, 4G4I, 4G4J

  1. Dimarogona, M., Topakas, E., Christakopoulos, P., *Chrysina, E.D. (2012) A new crystal structure of a Fusarium oxysporum GH10 xylanase reveals the presence of an extended loop on top of the catalytic cleft Acta Crystallogr. D68, 735-742

Pdb IDs: 3U7B [DOI: 10.1107/S0907444912007044]

 

Return to Research Activies

 

 
 

 

 

 

 
 

 

   
       

 

 

National Hellenic Research Foundation (NHRF), 48 Vassileos Constantinou Ave., 11635 Athens, Greece, Tel. +302107273700, Fax. +302107246618