Our Robotics Team Coach wrote: Hello Robotics Team, Congratulations on your hard work! We've written scripts for our presentation, made models of the school, worked on the technology piece that runs the robot and built a basic robot, all in 4 short sessions! The students have been working in small teams and parent volunteers have helped keep them on track!

Just a couple of updates: first, I have offered to have the students come in to the Lab before school (starting at 7:30 am) to work on their robot and get it ready for competition any morning for the next two weeks. Students can come by on any day. Second, our competition for both teams will be held on Saturday, November 7th, at Preuss School at UCSD. I don't have official times yet, but I will let you know as soon as our times are confirmed. Third, Thomas Mackey, a Robotics expert, will be here this Thursday (10/29) after school to help the teams with their robots and with programming them for the competition.

Feb 20, 2011. Monday, June 27 to Friday, August. Also two teen programs, one for grades 6 to 8, and one for grades 9 to 12. Extended day avail- able. Art Collaborations. Steven Mackey. Jazz & Blues. Todd Bashore Quartet, New. Brunswick Jazz Project, Hyatt. 2 Albany Street, New Brunswick.

If your child can stay after that would be terrific. I will be here with Thomas and any available team members until approximately 4:30 pm working on the robots. It will be a great way to get some expert advice about the robot. After the competition on November 7th our teams will know whether they qualified for the Championship (Dec. 5th at Legoland) or the Competition (Dec. 6th at Legoland).

I have registered our teams for both days; we will definitely attend either one or the other, and we will find out which one after November 7th. LEGO MINDSTORMS NXT is back and better than ever: new models, more customisable programming and all-new technologies! MINDSTORMS NXT 2.0 combines the versatility of the LEGO building system with all-new technologies, an intelligent microcomputer brick and intuitive drag-and-drop programming software.

The new 2.0 toolkit features everything you need to create your first robot in 30 minutes and then thousands of other robotics inventions that do what you want! Mindstorms originated from the programmable sensor blocks used in the line of educational toys. The first retail version of Lego Mindstorms was released in 1998 and marketed commercially as the Robotics Invention System (RIS).

The next version was released in 2006 as Lego Mindstorms NXT. The newest version, released in August 2009, is known as Lego Mindstorms NXT 2.0. The hardware and software roots of the Mindstorms Robotics Invention System kit go back to the programmable brick created at the MIT Media Lab. This brick was programmed in Brick Logo. The first visual programming environment, called LEGOsheets,[1] for this brick was created by the University of Colorado in 1994 and was based on AgentSheets.

The original Mindstorms Robotics Invention System kit contained two motors, two touch sensors, and one light sensor. The NXT version has three servo motors and one sensor each for touch, light, sound, and distance. Lego Mindstorms may be used to build a model of an embedded system with computer-controlled electromechanical parts. Many kinds of real-life embedded systems, from elevator controllers to industrial robots, may be modelled using Mindstorms. Mindstorms kits are also sold and used as an educational tool, originally through a partnership between Lego and the MIT Media Laboratory.[2][3] The educational version of the products is called Lego Mindstorms for Schools, and comes with the ROBOLAB GUI-based programming software, developed at Tufts University[4] using the National Instruments LabVIEW as an engine.

In addition, the shipped software can be replaced with third party firmware and/or programming languages, including some of the most popular ones used by professionals in the embedded systems industry, like Java and C. The only difference between the educational series, known as the 'Challenge Set', and the consumer series, known as the 'Inventor Set', is that it includes another light sensor and several more gearing options. Mindstorms is named after the book Mindstorms: Children, Computers, and Powerful Ideas by Seymour Papert.

Abstract The hexameric purine nucleoside phosphorylase from Bacillus subtilis (BsPNP233) displays great potential to produce nucleoside analogues in industry and can be exploited in the development of new anti-tumor gene therapies. In order to provide structural basis for enzyme and substrates rational optimization, aiming at those applications, the present work shows a thorough and detailed structural description of the binding mode of substrates and nucleoside analogues to the active site of the hexameric BsPNP233.

Here we report the crystal structure of BsPNP233 in the apo form and in complex with 11 ligands, including clinically relevant compounds. The crystal structure of six ligands (adenine, 2′deoxyguanosine, aciclovir, ganciclovir, 8-bromoguanosine, 6-chloroguanosine) in complex with a hexameric PNP are presented for the first time. Our data showed that free bases adopt alternative conformations in the BsPNP233 active site and indicated that binding of the co-substrate (2′deoxy)ribose 1-phosphate might contribute for stabilizing the bases in a favorable orientation for catalysis. The BsPNP233-adenosine complex revealed that a hydrogen bond between the 5′ hydroxyl group of adenosine and Arg 43* side chain contributes for the ribosyl radical to adopt an unusual C3’- endo conformation. The structures with 6-chloroguanosine and 8-bromoguanosine pointed out that the Cl 6 and Br 8 substrate modifications seem to be detrimental for catalysis and can be explored in the design of inhibitors for hexameric PNPs from pathogens.

Our data also corroborated the competitive inhibition mechanism of hexameric PNPs by tubercidin and suggested that the acyclic nucleoside ganciclovir is a better inhibitor for hexameric PNPs than aciclovir. Furthermore, comparative structural analyses indicated that the replacement of Ser 90 by a threonine in the B. Cereus hexameric adenosine phosphorylase (Thr 91) is responsible for the lack of negative cooperativity of phosphate binding in this enzyme.

Citation: de Giuseppe PO, Martins NH, Meza AN, dos Santos CR, Pereira HD, Murakami MT (2012) Insights into Phosphate Cooperativity and Influence of Substrate Modifications on Binding and Catalysis of Hexameric Purine Nucleoside Phosphorylases. PLoS ONE 7(9): e44282. Editor: Andreas Hofmann, Griffith University, Australia Received: June 18, 2012; Accepted: July 31, 2012; Published: September 5, 2012 Copyright: © Giuseppe et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the following research funding agencies: FAPESP [] (grants numbers 2007/00194-9, 2010/51890-8) and CNPq []. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist. Introduction Purine nucleoside phosphorylases (PNPs; EC 2.4.2.1) are versatile enzymes that catalyze the reversible phosphorolysis of purine (2′deoxy)ribonucleosides producing bases and (2′deoxy)ribose 1-phosphate. Their key role in the purine salvage pathway made PNPs attractive targets for drug design against several pathogens, such as Mycobacterium tuberculosis,, Plasmodium falciparum –, Trichomonas vaginalis – and Schistosoma mansoni,, which lacks the de novo pathway for purine nucleotides synthesis. Due to their catalytic function, PNPs have also been investigated for the synthesis of nucleoside analogues (NAs) and the activation of prodrugs in anti-cancer gene therapies. NAs can be used in the treatment of a range of human viral infections, such as those caused by HIV, herpesvirus and hepatitis B/C virus –. They are among the first cytotoxic molecules to be used in the treatment of cancer and have been studied as potential drugs against tuberculosis,, malaria,, trichomoniasis and schistosomiasis. The chemical synthesis of these compounds is generally a costly multistep process that includes several protection and deprotection stages,.

This has encouraged the development of new methods for the synthesis of NAs using PNPs and other enzymes as biocatalysts,,. The main advantages of this approach are the higher stereospecificity, regioselectivity and efficiency of enzymes, whose employment usually dispenses group protection and purification steps, optimizing the process. The differences in substrate specificity regarding trimeric and hexameric PNPs have allowed the development of suicide gene therapies strategies against solid tumors,. Trimeric PNPs are mainly found in mammalian species and are specific for guanine and hypoxanthine (2′-deoxy)ribonucleosides whereas hexameric PNPs are prevalent in bacteria and accept adenine as well as guanine and hypoxanthine (2′-deoxy)ribonucleosides as substrates. Thus, nontoxic adenosine analogues, which are poor substrates for human PNP, can be cleaved to cytotoxic bases specifically in tumor cells transfected with the bacterial hexameric PNP gene. Main advances in this field have been achieved with the E. In this context, the aim of the present work was to shed light on how a diverse set of substrate modifications affects its binding and catalysis by hexameric PNPs using a structural approach.

For this purpose, we choose the hexameric PNP (BsPNP233) from the model specie Bacillus subtilis, which displays great biotechnological potential to produce NAs, including the antiviral drug ribavirin. We have solved the crystal structure of BsPNP233 in the apo form and in complex with 11 ligands comprising sulfate, bases, natural nucleosides and NAs, including clinically relevant compounds. The crystal structure of six ligands (adenine, 2′deoxyguanosine, aciclovir, ganciclovir, 8-bromoguanosine, 6-chloroguanosine) in complex with a hexameric PNP are presented for the first time. Besides providing a broad structural basis for studies aiming at the rational design of BsPNP233 and its homologues for biotechnological applications, this work also bring new insights into the distinct kinetic models for phosphate binding in hexameric PNPs. Furthermore, the structural information showed here may also be instrumental for the development of new inhibitors against hexameric PNPs from pathogens such as Plasmodium falciparum and Trichomonas vaginalis,,, and for the combined design of both hexameric PNPs and prodrugs to improve specificity and efficiency of anti-cancer PNP gene therapies.

BsPNP233 Conserves the Quaternary Structure and Topology of Hexameric PNPs The crystal structure of BsPNP233 confirmed that it is a homohexamer with D 3 symmetry as observed for other hexameric PNPs (),. It was solved by X-ray crystallography in four distinct space groups ( P32 1, P2 12 12 1, P6 322 and H32).

The crystal contacts are similar in the crystal structures solved in P32 1, P2 12 12 1 and P6 322 but differ in the H32 space group. In the later, we observed additional crystallographic interfaces, resulting from a more compact crystal packing with a lower solvent content (41%) than crystals belonging to other space groups (∼56%) (). Free Purine Bases Adopt Alternative Conformations in the Active Site The crystal structures of BsPNP233 in complex with hypoxanthine (Hyp) and adenine (Ade) showed that the purine-binding site consists of residues Cys 91, Gly 92, Phe 159, Val 177 and Met 179. Hydrophobic interactions are predominant in the stabilization of both ligands ().

The BsPNP233-Ade binary complex was solved with (BsPNP233-Ade-SO 4) or without (BsPNP233-Ade) sulfate ion and represent the first of their kind to be reported for hexameric PNPs. Two subunits were observed in the asymmetric unit of both crystal structures and all of them exhibited clear density for the ligand in the active site ().

Superposition of BsPNP233-Ade and BsPNP233-Ade-SO 4 complexes showed a preferential orientation of Ade in the base-binding site, except in one case where it is rotated by 49° around an axis perpendicular to the base plane (). This alternative orientation is not followed by significant conformational changes in the active-site residues (); however, it alters the solvation of the active-site pocket.

In the alternative orientation, a crystallographic water molecule in the ribose-binding site is absent. This solvent molecule mediates a hydrogen bond between the AdeN 9 atom and the carbonyl group of Ser 90 in the presence of sulfate ion (). Comparison of free bases bound to the BsPNP233 active site. Structural comparison of a representative BsPNP233-Ade complex ( purple carbon atoms) with the BsPNP233-Hyp complex ( grey carbon atoms). The structure of the four BsPNP233-Ade complexes solved independently are superimposed.

The sulfate-free Ade-complexes are colored in purple (chain A) and pink (chain B) whereas the two independent complexes solved with sulfate bound (dataset I) are colored in orange (chain A) and blue (chain B). The structure of the Ade-complex where Ade presents an alternative conformation (carbon atoms in orange) is superimposed in the structure of Hyp-complex (carbon atoms in grey).

The surface of the glycerol molecule present at the Hyp-complex is shown to evidence the influence of this molecule in the position and orientation of Hyp in the active site. The hydrogen bonds are shown as dashed lines. Interestingly, the Hyp adopts an orientation similar to the alternative conformation of Ade (). In this case, a glycerol molecule is located in the ribose-binding site and seems to induce the displacement of Hyp, avoiding a steric clash with the HypN 9 atom. This observation, along with those described above, suggests that binding of the co-substrate ribose-1-phoshate might contribute for stabilizing the base in the favorable orientation for catalysis.

The Hydrogen Bond between the 5′ Hydroxyl Group of Ado and Arg 43* Side Chain Contributes for a Ribosyl C3′-endo Conformation The base moiety of adenosine (Ado) binds to the BsPNP233 active site in a very similar fashion to that seen in homologous PNPs (). However, the ribosyl group adopts a C3′- endo form instead of the C4′- endo or O4′ -exo conformations usually observed in Ado complexes with hexameric PNPs (PDB codes 3UAW,; 1ODI,; 1PK7,; 3U40,; 1Z37,; 1VHW, ) (). This unusual conformation may be explained by a hydrogen bond between the 5′ hydroxyl group of Ado and Arg 43* side chain (residues from the adjacent subunit are designated by an asterisk) not observed in other Ado complexes (). Typically, the 5′-OH group of Ado is found interacting with one or two water molecules not observed in BsPNP233-Ado complex, suggesting that the hydration of the active site may influence the ribosyl conformation.

The different conformations of Ado ribosyl radical. Structural superposition of BsPNP233-Ado ( magenta carbon atoms), B.

Cereus adenosine phosphorylase (BcAdoP)-Ado-SO 4 ( green carbon atoms, PDB code 3UAW ) and Entamoeba histolytica PNP-Ado ( cyan carbon atoms, PDB code 3U40 ) complexes. The different puckers adopted by the ribose moiety of adenosine are labeled and the hydrogen bonds involving the 5′-OH group of Ado in each complex are represented by dashed lines.

The sugar puckers were assigned by the pucker.py script of PyMOL. In sulfate/phosphate free complexes of hexameric PNPs with Ado, an O4′– exo conformation is usually found. However, in all complexes where the phosphate-binding site is occupied by a sulfate or phosphate, the ribosyl group shows a C4′– endo conformation, except for the Thermus thermophilus (TtPNP)-Ado complex (PDB code 1ODI, ). Since the side chain of Arg 43* participates in phosphate binding, the presence of this co-substrate and the hydration of the active site probably prevent the interaction between Ado 5′-OH group and Arg 43* side chain observed in BsPNP233-Ado complex favoring the ribose to adopt a C4′– endo conformation.

2′-deoxyguanosine Binding Mode Resembles to that Observed for Adenosine In the BsPNP233-(2′-deoxyguanosine) complex structure, the base of 2′-deoxyguanosine (dGuo) binds to the active site in a similar manner to that observed for Ado (). Neither the extra amino group at position 2 nor the carbonyl group at position 6 was observed making hydrogen bonds with the protein residues. A hydrogen bond between dGuoN 7 and Ser 202O γ atoms slightly rotates the base and brings the residue Ser 202 closer to the substrate (). The lack of the 2′-OH group in dGuo is counterbalanced by extra hydrophobic interactions between dGuoC 2′ and Glu 178 carbon atoms (). Comparisons between BsPNP233-dGuo and T.

Vaginalis PNP (TvPNP)-(2′-deoxyinosine) complexes showed that the deoxyribosyl group of both ligands conserves the binding mode, whereas the base assumes a little different orientation induced by the dGuoN 7-Ser 202O γ hydrogen bond exclusively observed in BsPNP233-dGuo complex (). The binding mode of 2′ deoxyguanosine and 2-fluoradenosine. Representation of the BsPNP233 residues that interact with dGuo showing as spheres the atoms involved in hydrophobic interactions with dGuo C 2′ atom. Structural alignment between the dGuo-complex (carbon atoms in orange) and the Ado-complex (carbon atoms in grey). C The structure of dGuo-complex and TvPNP-(2′-deoxyinosine) complex (carbon atoms in green, PDB code 1Z39, ) are superimposed. Representation of F-Ado complex showing the residues involved in van der Waals interactions with F 2 atom ( light blue sphere).

Structural comparison of F-Ado complex (carbon atoms in yellow) with the Ado-complex (carbon atoms in grey). The structures of F-Ado complex, EcPNP-F-Ado-PO 4 complex (carbon atoms in pink, PDB code 1PK9 ) and TvPNP-F-Ado complex (carbon atoms in green, PDB code 1Z35, ) are superimposed. In all panels hydrogen bonds are represented by dashed lines and color coded according to their respective structures. The Cl 6 Substituent of 6-chloroguanosine Induces a Ribose C3′-exo Conformation and May Prevent Catalysis The NA 6-chloroguanosine (Cl-Guo) can be used for the synthesis of 2-amino-6-chloro-9-(2,3-dideoxy-3-fluoro-beta-D-erythro-pentofuranosyl)purine, a compound with anti-HBV effects.

In addition, the free base 6-chloroguanine is an inhibitor of the trimeric PNP from Schistosoma mansoni. Here we report the first crystal structure of a PNP in complex with Cl-Guo. The molecule Cl-Guo displays a similar binding mode to that observed for dGuo (). Synology Surveillance Station 6 License Cracked Tooth. However, as the chlorine van der Waals radius is larger than that of oxygen, the Cl 6 substituent pushes the base in the direction of the ribosyl moiety to avoid steric clashes with Gly 92C α, Val 205C γ2, and Asp 203O δ1 atoms.

This base displacement induces the ribosyl group to adopt an unusual C3′- exo conformation. The influence of Cl 6 and Br 8 modifications in catalysis and nucleoside binding. Structural alignment of Cl-Guo complex ( pink carbon atoms) and dGuo complex ( cyan carbon atoms). Spheres represent the van der Waals radius of Cl 6, Gly 92C α, Asp 203O δ1 and Val 205C γ2 atoms.

Superposition of Cl-Guo complex and EcPNP-Ado-PO 4 complex ( yellow carbon atoms, PDB code 1PK7 ). Spheres represent the van der Waals radius of Cl 6 and EcPNP Asp 204O δ1 atoms to highlight the steric conflict imposed by the Cl 6 atom. The Br-Guo complex (carbon atoms in green), dGuo complex (carbon atoms in orange) and sulfate complex (carbon atoms in magenta, form IV, chain B) structures are superimposed. The sphere represents the van der Waals radius of Br 8 and the dashed lines represent hydrogen bonds colored according to the respective structures. Structural comparison of Br-Guo complex and the trimeric HsPNP-Guo-SO 4 complex ( purple carbon atoms, PDB code 1RFG, ). The spheres represent the van der Waals radius of Br 8 and HsPNP Thr 242O γ1 atoms.

The dashed circle has the same radius of Br 8 and indicates the steric clash that would occur if BrGuo was placed at the Guo position in the HsPNP active site. The C3′- exo pucker was already observed in the nucleoside 9-β-D-xylofuranosyladenine bound to EcPNP (PDB code 1PR6, ) and it is considered incompatible with the sugar conformation required for PNP catalysis. Moreover, structural comparisons with the EcPNP-Ado-PO 4 complex (PDB code 1PK7, ) showed that the chlorine atom may prevent the Asp 203 side chain to approach to the N 7 atom to donate a proton during catalysis (). Thus, these findings suggest that Cl-Guo as well as other NAs with 6-substituents heavier than chlorine cannot be cleaved by BsPNP233 and other hexameric PNPs. Corroboration of the Competitive Inhibition Mechanism of Hexameric PNP by Tubercidin Tubercidin (7-deazaadenosine) is an adenosine analogue which presents antiviral, antischistosomal and antifungal properties as well as antitumor activity –.

Furthermore, tubercidin and other 7-deazapurine nucleosides are inhibitors of EcPNP,. TBN presented an interaction mode very similar to that seen for the natural substrate adenosine (). Slightly differences were observed in its ribosyl moiety that assumed an O4′– exo pucker instead of the C3′- exo conformation of Ado in complex with BsPNP233 (). The C 7 substituent in TBN makes hydrophobic and van der Waals interactions with residues Cys 91 and Ser 202. The ribosyl moiety is stabilized by a conserved network of hydrogen bonds involving His 4*, Glu 180 and Arg 87 side chains and by hydrophobic interactions with Glu 178 (C α and C β atoms) and Met 179C γ atom (). Our structural data corroborate the competitive inhibition mechanism of hexameric PNP by TBN defined by in vitro studies. The substitution of N 7 by a carbon prevents the protonation step of the N 7 atom required for catalysis, making TBN a non-cleavable adenosine analogue by EcPNP and probably by other PNPs.

Ganciclovir Inhibits Both Trimeric and Hexameric PNPs Ganciclovir (GCV) is an acyclic NA used to treat cytomegalovirus infections. It is also used together with herpes simplex virus thymidine kinase in a suicide gene therapy system that has been studied for the treatment of hepatocellular carcinoma. GCV is an inhibitor of the human PNP (trimeric) and probably has inhibitory effects on hexameric PNPs as well. Our structural data support this hypothesis revealing that GCV binds to the nucleoside binding site of BsPNP233 (). The binding mode of acyclic nucleosides.

Stick representation of GCV bound in the BsPNP233 active site. Structural comparison of GCV-complex ( blue carbon atoms) with dGuo-complex ( orange carbon atoms). C and D show the stick representation of the two conformations of ACV (ACV 1 and ACV 2) bound to the BsPNP233 active site. The structures of GCV-complex ( grey) and ACV 1,2-complex ( green carbon atoms) are superimposed.

Structural alignment of ACV 1,2-complex with HsPNP-ACV complex ( pink carbon atoms, PDB code 1PWY ). In all panels dashed lines indicate hydrogen bonds and are color coded according to their respective complexes.

The guanine moiety of GCV conserves the position observed for the 2′-deoxyguanosine base but it is rotated by about 10° to accommodate the acyclic chain in the ribose-binding site (). A water molecule mediates hydrogen bonds between the ligand O 6 atom and the side chains of Ser 202 and Asp 203. The N 7 atom interacts with Ser 202O γ and Gly 92N atoms, and the base is stabilized by hydrophobic contacts with Ser 90, Cys 91, Ser 202 and Phe 159 ().

Interestingly, the three oxygens of the acyclic radical occupy similar positions to those observed for the three oxygens of dGuo ribosyl group, mimetizing its binding mode (). From the three hydrogen bonds observed for dGuo ribosyl moiety, the GCV acyclic radical conserves two, involving the His 4* and Glu 180 side chains. Moreover, the C 4′ atom of the GCV acyclic moiety preserves the hydrophobic interactions with Met 179C β and Met 179C γ atoms performed by the dGuo C 3′ atom (). Therefore our data indicate that GCV is also a competitive inhibitor for hexameric PNPs. Aciclovir Acyclic Chain Adopts Two Conformations in the BsPNP233 Ribosyl Binding Site Aciclovir (ACV) is an antiviral drug used to treat herpes virus infections and has modest inhibitory effects on human PNP.

Here, we present for the first time the crystal structure of a hexameric PNP with ACV. This structure revealed differences in the aciclovir binding mode, which can be explored for drug design targeting hexameric PNPs from pathogens such as P. Falciparum and T. Aciclovir binds to the BsPNP233 nucleoside binding site and is stabilized by hydrophobic interactions and a hydrogen-bonding network mediated by solvent molecules (). Interestingly, the acyclic tail assumes two alternative conformations that, seen simultaneously, resemble the conformation observed for the ganciclovir acyclic radical ().

In one of these conformations, the 3′ hydroxyl group of ACV is attached to the carboxyl group of Glu 180 side chain while the carbon atoms make hydrophobic contacts with the main chain of Glu 178 and with the Met 179C β and Met 179C γ atoms (). A phosphate ion, modeled with half occupancy based on difference maps, also makes a hydrogen bond with the ligand 3′ hydroxyl group (). The other conformation is stabilized by a hydrogen bond between the 3′-OH group of ACV and the His 4* side chain (). The ACV guanine moiety assumes a different position and orientation from that observed for GCV (), getting closer to the Phe 159 side chain. The main chain of Cys 91 and the side chain of Val 177 also contribute with hydrophobic interactions to the base (). The O 6 atom makes water mediated hydrogen bonds with the Asp 203 side chain and with the Phe 159 carbonyl oxygen ().

The same is observed for N 1 and N 2 atoms, which interact through a water molecule with the Gln 158 carbonyl oxygen; for N 7 atom, which makes water mediated hydrogen bonds with Asp 203 side chain, and; for N 9 atom, whose interaction with both Ser 90 and Ser 202 hydroxyl groups is also mediated by a solvent molecule (). Structural comparison between BsPNP233-ACV and human PNP (HsPNP)-ACV (PDB code 1PWY, ) complexes showed differences in the binding mode. In the HsPNP-ACV complex, the base N 1, N 2, N 7 and O 6 atoms interact directly with active-site residues through hydrogen bonds. In addition, the acyclic chain adopts a different conformation, which is stabilized by hydrophobic interactions with Phe 200 side chain and Ala 116/Ala 117 main chains (). To investigate if differences in the interaction mode of aciclovir with BsPNP233 and HsPNP may result in different binding affinities, we estimated the strength of protein–ligand interactions using the rerank score function of MOLEGRO.

According to this analysis, ACV presented similar predicted binding affinities in both complexes, which was slightly higher (lower rerank score value) for the BsPNP233 complex (). The same analysis was performed for GCV whose predicted binding affinity was considerable higher than that observed for ACV (). This result indicates that GCV is a better inhibitor for hexameric PNPs than ACV. Structural Basis of Distinct Kinetic Models for Phosphate Binding in Hexameric PNPs The asymmetric unit of the BsPNP233 crystal structure belonging to the H32 space group presented a catalytic dimer whose protomers adopt an open and a closed conformation, respectively (). The electron density map clearly showed a tetrahedral molecule in the active site of both subunits (). As the crystallization condition was phosphate free and contained high concentrations of ammonium sulfate, we modeled sulfate ions in both sites.

Structural basis of distinct kinetic models for phosphate binding in hexameric PNPs. Structural superposition of BsPNP233-sulfate open ( green) and closed ( pink) conformations with the BcAdoP-Ado complex ( yellow, PDB code 3UAW, ). The cartoon representation highlights the conformational differences observed in the main chain of the β9-α7 loop and the N-terminal portion of helix α7 in the three structures. Dashed lines represent hydrogen bonds and follow the color code of their respective structures. The surface representation of BsPNP233 Phe 220 in the closed conformation ( pink) and of the BcAdoP Thr 91 evidence the steric hindrance imposed by the Thr 91C γ2 atom to that Phe 220 rotamer. The surface representation of BsPNP233 Phe 220 and Ser 90 in the closed conformation shows that the Ser 90 side chain allows the Phe 220 side chain to perform the conformational change needed for the closed conformation takes place.

The open and closed conformations of BsPNP233-sulfate complex were already observed in EcPNP-sulfate/phosphate structures and have been associated with two dissociation constants that characterize phosphate binding to EcPNP,. The closed conformation is defined by a disruption of helix α7 and subsequent displacement of its N-terminal portion and the precedent loop towards the active site (). This conformation seems to be triggered by the interaction of Arg 24 side chain with phosphate and results in an approximation of Arg 216 to the catalytic residue Asp 203 (). As BsPNP233 protomers are able to adopt open and closed conformations like EcPNP subunits, this suggests that the negative cooperativity of phosphate binding demonstrated for EcPNP is also applied for BsPNP233. Comparison between BsPNP233-sulfate and Bacillus cereus adenosine phosphorylase (BcAdoP)-sulfate complexes (PDB codes 3UAV, 3UAW, 3UAX, 3UAY, 3UAZ, ) showed that BcAdoP assumes an intermediate conformation where only the first turn of helix α7 is disrupted ().

In the BcAdoP-sulfate complex structure (PDB code 3UAW, ), Arg 217 (corresponding to BsPNP233-Arg 216) points to the active site but it is not able to approach Asp 204 (BsPNP233-Asp 203) such as BsPNP233-Arg 216 (). The apparent inability of BcAdoP to adopt the closed conformation seems to be caused by a steric hindrance imposed by Thr 91 to the conformational change that Phe 221 (BsPNP233-Phe 220) undergoes for the closed conformation being achieved (). In BsPNP233 and EcPNP this threonine residue is replaced by a serine, which allows Phe 220 side chain to adopt the rotamer observed in the closed conformation (). These analyses suggest that the negative cooperativity model of phosphate binding displayed by EcPNP cannot be applied for BcAdoP, as BcAdoP apparently presents only one conformational state. This hypothesis is supported by functional studies, which showed that BcAdoP obeys Michaelis–Menten kinetics. A previous work reported that BsPNP233 is specific for 6-aminopurine nucleosides. However, Xie and coworkers recently showed that BsPNP233 (named PNP 702) exhibits a broad substrate specificity and present comparable activity towards both guanosine (6-oxopurine nucleoside) and adenosine (6-aminopurine nucleoside).

Our structural data is in agreement with Xie and coworkers data indicating that BsPNP233 conserves the same catalytic mechanism proposed for EcPNP, where catalysis occurs in the closed conformation (). Conclusion This report provided a broad description of how the hexameric PNP from B. Subtilis interacts with natural substrates and the impact of modifications in such substrates on binding and catalysis. The structural analysis reported here can be instrumental for studies aiming to optimize BsPNP233 or other hexameric PNPs for biotechnological applications such as industrial synthesis of nucleoside analogues or gene therapy against solid tumors.

An initiative of this sort has been taken for E. Coli PNP to optimize the cleavage of the prodrug Me( talo)-MeP-R with great success. The crystal structure of six ligands (adenine, 2′deoxyguanosine, aciclovir, ganciclovir, 8-bromoguanosine and 6-chloroguanosine) in complex with a hexameric PNP are presented for the first time. The information extracted from these structures can be extended to homologous hexameric PNPs to help the development of new inhibitors against pathogens such as T. Vaginalis and P. Falciparum as well as new prodrugs for gene therapies against tumors,. In addition, our results and comparative analyses shed light on distinct kinetic models for phosphate binding in hexameric PNPs.

According to our model the substitution of the conserved residue Ser 90 by a threonine disrupts the open/close mechanism of hexameric PNPs subunits, which results in the loss of the negative cooperativity of phosphate binding.

The hexameric purine nucleoside phosphorylase from Bacillus subtilis (BsPNP233) displays great potential to produce nucleoside analogues in industry and can be exploited in the development of new anti-tumor gene therapies. In order to provide structural basis for enzyme and substrates rational optimization, aiming at those applications, the present work shows a thorough and detailed structural description of the binding mode of substrates and nucleoside analogues to the active site of the hexameric BsPNP233.

Here we report the crystal structure of BsPNP233 in the apo form and in complex with 11 ligands, including clinically relevant compounds. The crystal structure of six ligands (adenine, 2′deoxyguanosine, aciclovir, ganciclovir, 8-bromoguanosine, 6-chloroguanosine) in complex with a hexameric PNP are presented for the first time. Our data showed that free bases adopt alternative conformations in the BsPNP233 active site and indicated that binding of the co-substrate (2′deoxy)ribose 1-phosphate might contribute for stabilizing the bases in a favorable orientation for catalysis. The BsPNP233-adenosine complex revealed that a hydrogen bond between the 5′ hydroxyl group of adenosine and Arg 43* side chain contributes for the ribosyl radical to adopt an unusual C3’- endo conformation.

The structures with 6-chloroguanosine and 8-bromoguanosine pointed out that the Cl 6 and Br 8 substrate modifications seem to be detrimental for catalysis and can be explored in the design of inhibitors for hexameric PNPs from pathogens. Our data also corroborated the competitive inhibition mechanism of hexameric PNPs by tubercidin and suggested that the acyclic nucleoside ganciclovir is a better inhibitor for hexameric PNPs than aciclovir. Furthermore, comparative structural analyses indicated that the replacement of Ser 90 by a threonine in the B. Cereus hexameric adenosine phosphorylase (Thr 91) is responsible for the lack of negative cooperativity of phosphate binding in this enzyme. Introduction Purine nucleoside phosphorylases (PNPs; EC 2.4.2.1) are versatile enzymes that catalyze the reversible phosphorolysis of purine (2′deoxy)ribonucleosides producing bases and (2′deoxy)ribose 1-phosphate. Their key role in the purine salvage pathway made PNPs attractive targets for drug design against several pathogens, such as Mycobacterium tuberculosis,, Plasmodium falciparum –, Trichomonas vaginalis – and Schistosoma mansoni,, which lacks the de novo pathway for purine nucleotides synthesis.

Due to their catalytic function, PNPs have also been investigated for the synthesis of nucleoside analogues (NAs) and the activation of prodrugs in anti-cancer gene therapies. NAs can be used in the treatment of a range of human viral infections, such as those caused by HIV, herpesvirus and hepatitis B/C virus –. They are among the first cytotoxic molecules to be used in the treatment of cancer and have been studied as potential drugs against tuberculosis,, malaria,, trichomoniasis and schistosomiasis. The chemical synthesis of these compounds is generally a costly multistep process that includes several protection and deprotection stages,. This has encouraged the development of new methods for the synthesis of NAs using PNPs and other enzymes as biocatalysts,,. The main advantages of this approach are the higher stereospecificity, regioselectivity and efficiency of enzymes, whose employment usually dispenses group protection and purification steps, optimizing the process.

The differences in substrate specificity regarding trimeric and hexameric PNPs have allowed the development of suicide gene therapies strategies against solid tumors,. Trimeric PNPs are mainly found in mammalian species and are specific for guanine and hypoxanthine (2′-deoxy)ribonucleosides whereas hexameric PNPs are prevalent in bacteria and accept adenine as well as guanine and hypoxanthine (2′-deoxy)ribonucleosides as substrates. Thus, nontoxic adenosine analogues, which are poor substrates for human PNP, can be cleaved to cytotoxic bases specifically in tumor cells transfected with the bacterial hexameric PNP gene. Main advances in this field have been achieved with the E. In this context, the aim of the present work was to shed light on how a diverse set of substrate modifications affects its binding and catalysis by hexameric PNPs using a structural approach.

For this purpose, we choose the hexameric PNP (BsPNP233) from the model specie Bacillus subtilis, which displays great biotechnological potential to produce NAs, including the antiviral drug ribavirin. We have solved the crystal structure of BsPNP233 in the apo form and in complex with 11 ligands comprising sulfate, bases, natural nucleosides and NAs, including clinically relevant compounds. The crystal structure of six ligands (adenine, 2′deoxyguanosine, aciclovir, ganciclovir, 8-bromoguanosine, 6-chloroguanosine) in complex with a hexameric PNP are presented for the first time.

Besides providing a broad structural basis for studies aiming at the rational design of BsPNP233 and its homologues for biotechnological applications, this work also bring new insights into the distinct kinetic models for phosphate binding in hexameric PNPs. Furthermore, the structural information showed here may also be instrumental for the development of new inhibitors against hexameric PNPs from pathogens such as Plasmodium falciparum and Trichomonas vaginalis,,, and for the combined design of both hexameric PNPs and prodrugs to improve specificity and efficiency of anti-cancer PNP gene therapies. BsPNP233 Conserves the Quaternary Structure and Topology of Hexameric PNPs The crystal structure of BsPNP233 confirmed that it is a homohexamer with D 3 symmetry as observed for other hexameric PNPs (),. It was solved by X-ray crystallography in four distinct space groups ( P32 1, P2 12 12 1, P6 322 and H32). The crystal contacts are similar in the crystal structures solved in P32 1, P2 12 12 1 and P6 322 but differ in the H32 space group. In the later, we observed additional crystallographic interfaces, resulting from a more compact crystal packing with a lower solvent content (41%) than crystals belonging to other space groups (∼56%) ().

Comparison of free bases bound to the BsPNP233 active site. The BsPNP233-Ade binary complex was solved with (BsPNP233-Ade-SO 4) or without (BsPNP233-Ade) sulfate ion and represent the first of their kind to be reported for hexameric PNPs. Two subunits were observed in the asymmetric unit of both crystal structures and all of them exhibited clear density for the ligand in the active site ().

Superposition of BsPNP233-Ade and BsPNP233-Ade-SO 4 complexes showed a preferential orientation of Ade in the base-binding site, except in one case where it is rotated by 49° around an axis perpendicular to the base plane (). This alternative orientation is not followed by significant conformational changes in the active-site residues (); however, it alters the solvation of the active-site pocket. In the alternative orientation, a crystallographic water molecule in the ribose-binding site is absent. This solvent molecule mediates a hydrogen bond between the AdeN 9 atom and the carbonyl group of Ser 90 in the presence of sulfate ion ().

Interestingly, the Hyp adopts an orientation similar to the alternative conformation of Ade (). In this case, a glycerol molecule is located in the ribose-binding site and seems to induce the displacement of Hyp, avoiding a steric clash with the HypN 9 atom.

This observation, along with those described above, suggests that binding of the co-substrate ribose-1-phoshate might contribute for stabilizing the base in the favorable orientation for catalysis. The Hydrogen Bond between the 5′ Hydroxyl Group of Ado and Arg 43* Side Chain Contributes for a Ribosyl C3′-endo Conformation The base moiety of adenosine (Ado) binds to the BsPNP233 active site in a very similar fashion to that seen in homologous PNPs (). However, the ribosyl group adopts a C3′- endo form instead of the C4′- endo or O4′ -exo conformations usually observed in Ado complexes with hexameric PNPs (PDB codes 3UAW,; 1ODI,; 1PK7,; 3U40,; 1Z37,; 1VHW, ) (). This unusual conformation may be explained by a hydrogen bond between the 5′ hydroxyl group of Ado and Arg 43* side chain (residues from the adjacent subunit are designated by an asterisk) not observed in other Ado complexes (). Typically, the 5′-OH group of Ado is found interacting with one or two water molecules not observed in BsPNP233-Ado complex, suggesting that the hydration of the active site may influence the ribosyl conformation. The different conformations of Ado ribosyl radical. In sulfate/phosphate free complexes of hexameric PNPs with Ado, an O4′– exo conformation is usually found.

However, in all complexes where the phosphate-binding site is occupied by a sulfate or phosphate, the ribosyl group shows a C4′– endo conformation, except for the Thermus thermophilus (TtPNP)-Ado complex (PDB code 1ODI, ). Since the side chain of Arg 43* participates in phosphate binding, the presence of this co-substrate and the hydration of the active site probably prevent the interaction between Ado 5′-OH group and Arg 43* side chain observed in BsPNP233-Ado complex favoring the ribose to adopt a C4′– endo conformation. 2′-deoxyguanosine Binding Mode Resembles to that Observed for Adenosine In the BsPNP233-(2′-deoxyguanosine) complex structure, the base of 2′-deoxyguanosine (dGuo) binds to the active site in a similar manner to that observed for Ado (). Neither the extra amino group at position 2 nor the carbonyl group at position 6 was observed making hydrogen bonds with the protein residues.

A hydrogen bond between dGuoN 7 and Ser 202O γ atoms slightly rotates the base and brings the residue Ser 202 closer to the substrate (). The lack of the 2′-OH group in dGuo is counterbalanced by extra hydrophobic interactions between dGuoC 2′ and Glu 178 carbon atoms (). Comparisons between BsPNP233-dGuo and T. Vaginalis PNP (TvPNP)-(2′-deoxyinosine) complexes showed that the deoxyribosyl group of both ligands conserves the binding mode, whereas the base assumes a little different orientation induced by the dGuoN 7-Ser 202O γ hydrogen bond exclusively observed in BsPNP233-dGuo complex (). The Cl 6 Substituent of 6-chloroguanosine Induces a Ribose C3′-exo Conformation and May Prevent Catalysis The NA 6-chloroguanosine (Cl-Guo) can be used for the synthesis of 2-amino-6-chloro-9-(2,3-dideoxy-3-fluoro-beta-D-erythro-pentofuranosyl)purine, a compound with anti-HBV effects. In addition, the free base 6-chloroguanine is an inhibitor of the trimeric PNP from Schistosoma mansoni.

Here we report the first crystal structure of a PNP in complex with Cl-Guo. The molecule Cl-Guo displays a similar binding mode to that observed for dGuo (). However, as the chlorine van der Waals radius is larger than that of oxygen, the Cl 6 substituent pushes the base in the direction of the ribosyl moiety to avoid steric clashes with Gly 92C α, Val 205C γ2, and Asp 203O δ1 atoms.

This base displacement induces the ribosyl group to adopt an unusual C3′- exo conformation. The influence of Cl 6 and Br 8 modifications in catalysis and nucleoside binding. The C3′- exo pucker was already observed in the nucleoside 9-β-D-xylofuranosyladenine bound to EcPNP (PDB code 1PR6, ) and it is considered incompatible with the sugar conformation required for PNP catalysis. Moreover, structural comparisons with the EcPNP-Ado-PO 4 complex (PDB code 1PK7, ) showed that the chlorine atom may prevent the Asp 203 side chain to approach to the N 7 atom to donate a proton during catalysis (). Thus, these findings suggest that Cl-Guo as well as other NAs with 6-substituents heavier than chlorine cannot be cleaved by BsPNP233 and other hexameric PNPs. Corroboration of the Competitive Inhibition Mechanism of Hexameric PNP by Tubercidin Tubercidin (7-deazaadenosine) is an adenosine analogue which presents antiviral, antischistosomal and antifungal properties as well as antitumor activity –.

Furthermore, tubercidin and other 7-deazapurine nucleosides are inhibitors of EcPNP,. TBN presented an interaction mode very similar to that seen for the natural substrate adenosine (). Slightly differences were observed in its ribosyl moiety that assumed an O4′– exo pucker instead of the C3′- exo conformation of Ado in complex with BsPNP233 (). The C 7 substituent in TBN makes hydrophobic and van der Waals interactions with residues Cys 91 and Ser 202.

The ribosyl moiety is stabilized by a conserved network of hydrogen bonds involving His 4*, Glu 180 and Arg 87 side chains and by hydrophobic interactions with Glu 178 (C α and C β atoms) and Met 179C γ atom (). Our structural data corroborate the competitive inhibition mechanism of hexameric PNP by TBN defined by in vitro studies. The substitution of N 7 by a carbon prevents the protonation step of the N 7 atom required for catalysis, making TBN a non-cleavable adenosine analogue by EcPNP and probably by other PNPs. Ganciclovir Inhibits Both Trimeric and Hexameric PNPs Ganciclovir (GCV) is an acyclic NA used to treat cytomegalovirus infections. It is also used together with herpes simplex virus thymidine kinase in a suicide gene therapy system that has been studied for the treatment of hepatocellular carcinoma.

GCV is an inhibitor of the human PNP (trimeric) and probably has inhibitory effects on hexameric PNPs as well. Our structural data support this hypothesis revealing that GCV binds to the nucleoside binding site of BsPNP233 ().

The binding mode of acyclic nucleosides. The guanine moiety of GCV conserves the position observed for the 2′-deoxyguanosine base but it is rotated by about 10° to accommodate the acyclic chain in the ribose-binding site (). A water molecule mediates hydrogen bonds between the ligand O 6 atom and the side chains of Ser 202 and Asp 203.

The N 7 atom interacts with Ser 202O γ and Gly 92N atoms, and the base is stabilized by hydrophobic contacts with Ser 90, Cys 91, Ser 202 and Phe 159 (). Interestingly, the three oxygens of the acyclic radical occupy similar positions to those observed for the three oxygens of dGuo ribosyl group, mimetizing its binding mode ().

From the three hydrogen bonds observed for dGuo ribosyl moiety, the GCV acyclic radical conserves two, involving the His 4* and Glu 180 side chains. Moreover, the C 4′ atom of the GCV acyclic moiety preserves the hydrophobic interactions with Met 179C β and Met 179C γ atoms performed by the dGuo C 3′ atom (). Therefore our data indicate that GCV is also a competitive inhibitor for hexameric PNPs. Aciclovir Acyclic Chain Adopts Two Conformations in the BsPNP233 Ribosyl Binding Site Aciclovir (ACV) is an antiviral drug used to treat herpes virus infections and has modest inhibitory effects on human PNP. Here, we present for the first time the crystal structure of a hexameric PNP with ACV. This structure revealed differences in the aciclovir binding mode, which can be explored for drug design targeting hexameric PNPs from pathogens such as P. Falciparum and T.

Aciclovir binds to the BsPNP233 nucleoside binding site and is stabilized by hydrophobic interactions and a hydrogen-bonding network mediated by solvent molecules (). Interestingly, the acyclic tail assumes two alternative conformations that, seen simultaneously, resemble the conformation observed for the ganciclovir acyclic radical ().

In one of these conformations, the 3′ hydroxyl group of ACV is attached to the carboxyl group of Glu 180 side chain while the carbon atoms make hydrophobic contacts with the main chain of Glu 178 and with the Met 179C β and Met 179C γ atoms (). A phosphate ion, modeled with half occupancy based on difference maps, also makes a hydrogen bond with the ligand 3′ hydroxyl group (). The other conformation is stabilized by a hydrogen bond between the 3′-OH group of ACV and the His 4* side chain (). The ACV guanine moiety assumes a different position and orientation from that observed for GCV (), getting closer to the Phe 159 side chain. The main chain of Cys 91 and the side chain of Val 177 also contribute with hydrophobic interactions to the base ().

The O 6 atom makes water mediated hydrogen bonds with the Asp 203 side chain and with the Phe 159 carbonyl oxygen (). The same is observed for N 1 and N 2 atoms, which interact through a water molecule with the Gln 158 carbonyl oxygen; for N 7 atom, which makes water mediated hydrogen bonds with Asp 203 side chain, and; for N 9 atom, whose interaction with both Ser 90 and Ser 202 hydroxyl groups is also mediated by a solvent molecule (). Structural comparison between BsPNP233-ACV and human PNP (HsPNP)-ACV (PDB code 1PWY, ) complexes showed differences in the binding mode. In the HsPNP-ACV complex, the base N 1, N 2, N 7 and O 6 atoms interact directly with active-site residues through hydrogen bonds.

In addition, the acyclic chain adopts a different conformation, which is stabilized by hydrophobic interactions with Phe 200 side chain and Ala 116/Ala 117 main chains (). To investigate if differences in the interaction mode of aciclovir with BsPNP233 and HsPNP may result in different binding affinities, we estimated the strength of protein–ligand interactions using the rerank score function of MOLEGRO. According to this analysis, ACV presented similar predicted binding affinities in both complexes, which was slightly higher (lower rerank score value) for the BsPNP233 complex (). The same analysis was performed for GCV whose predicted binding affinity was considerable higher than that observed for ACV (). This result indicates that GCV is a better inhibitor for hexameric PNPs than ACV. Structural Basis of Distinct Kinetic Models for Phosphate Binding in Hexameric PNPs The asymmetric unit of the BsPNP233 crystal structure belonging to the H32 space group presented a catalytic dimer whose protomers adopt an open and a closed conformation, respectively ().

The electron density map clearly showed a tetrahedral molecule in the active site of both subunits (). As the crystallization condition was phosphate free and contained high concentrations of ammonium sulfate, we modeled sulfate ions in both sites. Structural basis of distinct kinetic models for phosphate binding in hexameric PNPs. The open and closed conformations of BsPNP233-sulfate complex were already observed in EcPNP-sulfate/phosphate structures and have been associated with two dissociation constants that characterize phosphate binding to EcPNP,. The closed conformation is defined by a disruption of helix α7 and subsequent displacement of its N-terminal portion and the precedent loop towards the active site ().

This conformation seems to be triggered by the interaction of Arg 24 side chain with phosphate and results in an approximation of Arg 216 to the catalytic residue Asp 203 (). As BsPNP233 protomers are able to adopt open and closed conformations like EcPNP subunits, this suggests that the negative cooperativity of phosphate binding demonstrated for EcPNP is also applied for BsPNP233. Comparison between BsPNP233-sulfate and Bacillus cereus adenosine phosphorylase (BcAdoP)-sulfate complexes (PDB codes 3UAV, 3UAW, 3UAX, 3UAY, 3UAZ, ) showed that BcAdoP assumes an intermediate conformation where only the first turn of helix α7 is disrupted (). In the BcAdoP-sulfate complex structure (PDB code 3UAW, ), Arg 217 (corresponding to BsPNP233-Arg 216) points to the active site but it is not able to approach Asp 204 (BsPNP233-Asp 203) such as BsPNP233-Arg 216 (). The apparent inability of BcAdoP to adopt the closed conformation seems to be caused by a steric hindrance imposed by Thr 91 to the conformational change that Phe 221 (BsPNP233-Phe 220) undergoes for the closed conformation being achieved (). In BsPNP233 and EcPNP this threonine residue is replaced by a serine, which allows Phe 220 side chain to adopt the rotamer observed in the closed conformation (). These analyses suggest that the negative cooperativity model of phosphate binding displayed by EcPNP cannot be applied for BcAdoP, as BcAdoP apparently presents only one conformational state.

This hypothesis is supported by functional studies, which showed that BcAdoP obeys Michaelis–Menten kinetics. A previous work reported that BsPNP233 is specific for 6-aminopurine nucleosides. However, Xie and coworkers recently showed that BsPNP233 (named PNP 702) exhibits a broad substrate specificity and present comparable activity towards both guanosine (6-oxopurine nucleoside) and adenosine (6-aminopurine nucleoside). Our structural data is in agreement with Xie and coworkers data indicating that BsPNP233 conserves the same catalytic mechanism proposed for EcPNP, where catalysis occurs in the closed conformation (). Conclusion This report provided a broad description of how the hexameric PNP from B. Subtilis interacts with natural substrates and the impact of modifications in such substrates on binding and catalysis.

The structural analysis reported here can be instrumental for studies aiming to optimize BsPNP233 or other hexameric PNPs for biotechnological applications such as industrial synthesis of nucleoside analogues or gene therapy against solid tumors. An initiative of this sort has been taken for E. Coli PNP to optimize the cleavage of the prodrug Me( talo)-MeP-R with great success. The crystal structure of six ligands (adenine, 2′deoxyguanosine, aciclovir, ganciclovir, 8-bromoguanosine and 6-chloroguanosine) in complex with a hexameric PNP are presented for the first time.

The information extracted from these structures can be extended to homologous hexameric PNPs to help the development of new inhibitors against pathogens such as T. Vaginalis and P. Falciparum as well as new prodrugs for gene therapies against tumors,.

In addition, our results and comparative analyses shed light on distinct kinetic models for phosphate binding in hexameric PNPs. According to our model the substitution of the conserved residue Ser 90 by a threonine disrupts the open/close mechanism of hexameric PNPs subunits, which results in the loss of the negative cooperativity of phosphate binding.