Biochemical and Biophysical Research Communications 313 (2004) 907–914

BBRCwww.elsevier.com/locate/ybbrc

Structures of human purine nucleoside phosphorylase complexedwith inosine and ddI

Fernanda Canduri,a,b Denis Marangoni dos Santos,a,b Rafael Guimar~aaes Silva,c

Maria Anita Mendes,b,d Luiz Augusto Basso,c M�aario S�eergio Palma,b,d

Walter Filgueira de Azevedo Jr.,a,b,* and Di�oogenes Santiago Santosc,e,*

a Departamento de F�ıısica, UNESP, S~aao Jos�ee do Rio Preto, SP 15054-000, Brazilb Center for Applied Toxinology, Instituto Butantan, S~aao Paulo, SP 05503-900, Brazil

c Rede Brasileira de Pesquisas em Tuberculose, Departamento de Biologia Molecular e Biotecnologia, UFRGS, Porto Alegre, RS 91501-970, Brazild Laboratory of Structural Biology and Zoochemistry-CEIS/Department of Biology, Institute of Biosciences, UNESP, Rio Claro, SP 13506-900, Brazil

e Faculdade de Farm�aacia/Instituto de Pesquisas Biom�eedicas, Pontif�ııcia Universidade Cat�oolica do Rio Grande do Sul, Porto Alegre, RS, Brazil

Received 5 November 2003

Abstract

Human purine nucleoside phosphorylase (PNP) is a ubiquitous enzyme which plays a key role in the purine salvage pathway, and

PNP deficiency in humans leads to an impairment of T-cell function, usually with no apparent effect on B-cell function. PNP is

highly specific for 6-oxopurine nucleosides and exhibits negligible activity for 6-aminopurine nucleosides. The catalytic efficiency for

inosine is 350,000-fold greater than for adenosine. Adenine nucleosides and nucleotides are deaminated by adenosine deaminase and

AMP deaminase to their corresponding inosine derivatives which, in turn, may be further degraded. Here we report the crystal

structures of human PNP in complex with inosine and 20,30-dideoxyinosine, refined to 2.8�AA resolution using synchrotron radiation.

The present structures provide explanation for ligand binding, refine the purine-binding site, and can be used for future inhibitor

design.

� 2003 Elsevier Inc. All rights reserved.

Keywords: PNP; Synchrotron radiation; Structure; Drug design

Purine nucleoside phosphorylase (PNP, E.C. 2.4.2.1.)

catalyzes the cleavage of the glycosidic bond of ribo-

and deoxyribonucleosides of guanine, hypoxanthine,

and a number of related nucleoside congeners [1], in the

presence of inorganic orthophosphate (Pi) as a secondsubstrate, to generate the purine base and ribose(de-

oxyribose)-1-phosphate. PNP is a ubiquitous enzyme of

purine metabolism that functions in the salvage path-

way, thus enabling the cells to utilize purine bases

recovered from metabolized purine ribo- and deoxy-

ribonucleosides to synthesize purine nucleotides [2]. The

salvage enzymes involved in the process allow circum-

vention of the respective de novo pathways when pre-cursors are provided [3]. Adenosine and deoxyadenosine

* Corresponding authors. Fax: +55-17-221-2247.

E-mail addresses: walterfa@df.ibilce.unesp.br (W.F. de Azevedo

Jr.), diogenes@pucrs.br (D.S. Santos).

0006-291X/$ - see front matter � 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.bbrc.2003.11.179

are not degraded by mammalian PNP. Rather, adenine

nucleosides and nucleotides are deaminated by adeno-

sine deaminase and AMP deaminase to their corre-

sponding inosine derivatives which, in turn, may be

further degraded. Human PNP is an attractive target fordrug design and it has been submitted to extensive

structure-based design. More recently, the three-di-

mensional structure of human PNP has been refined to

2.3�AA resolution, using synchrotron radiation and cryo-

crystallography techniques [4], which allowed a redefi-

nition of the residues involved in the substrate binding

providing a more reliable model for structure-based

design of inhibitors. The crystallographic structure is atrimer and analysis of human PNP in solution, using the

integration of geometric docking and small-angle X-ray

scattering (SAXS), confirmed that the crystallographic

trimer is conserved even in solution [5]. Furthermore,

the crystallographic structure of human PNP complexed

Fig. 1. Catabolic pathway of inosine and ddI.

908 F. Canduri et al. / Biochemical and Biophysical Research Communications 313 (2004) 907–914

with guanine revealed a new phosphate site, which may

be the second regulatory phosphate-binding site [6].

We have obtained the crystallographic structures of

the complexes between HsPNP and inosine (HsPNP:I-

no), and HsPNP and 20,30-dideoxyinosine (HsPNP:ddI).

The ddI is an analogue of the naturally occurring purinenucleoside inosine. This nucleoside is converted within

target cells to its active form ddA-triphosphate. In ad-

dition to the intracellular formation of ddA-TP, ddI can

be broken down to hypoxanthine which can either re-

enter the purine metabolic pool or be degraded further

to uric acid, the enzymes involved are PNP and xanthine

oxidase, respectively [7]. Fig. 1 shows this catabolic

pathway. Figs. 2A–E show the molecular structures of

Fig. 2. Molecular structures of PNP ligands. (A) Inosine, (B)

ligands, inosine, ddI, and guanine, and of inhibitors

immucillin-H and acyclovir. Our analyses of the

HsPNP:Ino and HsPNP:ddI structural data and struc-

tural differences between the PNP apoenzyme and

complexes provide explanation for substrate binding,

identify water molecules, and can be used for futureinhibitor design.

Materials and methods

Crystallization and data collection. Recombinant human PNP was

expressed and purified as previously described [8]. HsPNP:Ino and

HsPNP:ddI were crystallized using the experimental conditions

described elsewhere [9,10]. In brief, a PNP solution was concentrated

ddI, (C) guanine, (D) acyclovir, and (E) immucillin-H.

F. Canduri et al. / Biochemical and Biophysical Research Communications 313 (2004) 907–914 909

to 12mgml�1 against 10mM potassium phosphate buffer (pH 7.1) and

incubated in the presence of 0.6mM ligand (inosine and ddI) (Sigma),

in the molar ratio between protein and ligand of 1:2. Hanging drops

were equilibrated by vapor diffusion at 25 �C against reservoir

containing 19% (v/v) saturated ammonium sulfate solution in 0.05M

citrate buffer (pH 5.3).

In order to increase the resolution of the HsPNP:Ino and

HsPNP:ddI crystal, we collected data from a flash-cooled crystal at

104K. Prior to flash cooling, glycerol was added, up to 50% by vol-

ume, to the crystallization drop. X-ray diffraction data were collected

at a wavelength of 1.4310�AA using the Synchrotron Radiation Source

(Station PCr, Laborat�oorio Nacional de Luz S�iincrotron, LNLS,

Campinas, Brazil) and a CCD detector (MARCCD) with an exposure

time of 30 s per image at a crystal to detector distance of 120mm, for

the two data sets. X-ray diffraction data were processed to 2.8�AA res-

olution using the program MOSFLM and scaled with the program

SCALA [11].

Upon cooling the cell parameters shrank from a ¼ b ¼ 142:90�AA,

c ¼ 165:20�AA to a ¼ b ¼ 140:99�AA, and c ¼ 161:13�AA for HsPNP:Ino

Table 1

Data collection and refinement statistics

Statistics

Cell parameters

a (�AA)

b (�AA)

c (�AA)

a (�)b (�)c (�)

Space group

Number of measurements with I > 2rðIÞAverage I=sðIÞ valueNumber of independent reflections

Multiplicity

Completeness in the range from 56.80 to 2.80�AA (%)

Rsyma (%)

Highest resolution shell (�AA)

Completeness in the highest resolution shell (%)

Rsyma in the highest resolution shell (%)

Resolution range used in the refinement (�AA)

Rfactorb (%)

Rfreec (%)

Observed r.m.s.d from ideal geometry

Bond lengths (�AA)

Bond angles (�)Dihedrals (�)

B valuesd (�AA2)

Main chain

Side chains

Ligand

Waters

Sulfate groups

Residues in most favored regions of the Ramachandran plot (%)

Residues in additionally allowed regions of the Ramachandran plot (%)

Residues in generously allowed regions of the Ramachandran plot (%)

Residues in disallowed regions of the Ramachandran plot (%)

No. of water molecules

No. of sulfate groups

aRsym ¼ 100P

jIðhÞ � hIðhÞi=P

IðhÞ with IðhÞ, observed intensity and hIbRfactor ¼ 100�

PjFobs � Fcalcj=

PðFobs), the sums being taken over all re

cRfree ¼ Rfactor for 10% of the data, which were not included during crystdB values¼ average B values for all non-hydrogen atoms.

and a ¼ b ¼ 141:33�AA, and c ¼ 161:45�AA for HsPNP:ddI. The volume

of the unit cell for complexes is 2.793� 106 �AA3 compatible with one

monomer in the asymmetric unit with a Vm value of 4.8�AA3/Da. As-

suming a value of 0.25 cm3 g�1 for the protein partial specific volume,

the calculated solvent content in the crystal is 75% and the calculated

crystal density 1.1 g cm�3.

Crystal structure. The crystal structures of the HsPNP:Ino and

HsPNP:ddI were determined by standard molecular replacement

methods using the program AMoRe [12]. For the structure HsPNP:Ino

was used as search model the structure of HsPNP:acyclovir (PDB

access code: 1PWY) [9]. The ligand and water molecules were removed

from search model HsPNP:acyclovir. For the structure HsPNP:ddI

was used as search model the structure of HsPNP (PDB access code:

1M73) [4]. Structure refinement was performed using X-PLOR [13].

The atomic positions obtained from molecular replacement were used

to initiate the crystallographic refinement. The overall stereochemical

quality of the final models for HsPNP:Ino and HsPNP:ddI complexes

was assessed by the program PROCHECK [14]. Atomic models were

superposed using the program LSQKAB from CCP4 [11].

PNP:ddI PNP:Ino

141.33 140.99

141.33 140.99

161.45 161.13

90.00 90.00

90.00 90.00

120.00 120.00

R32 R32

94,304 109,144

7.5 6.6

12,886 15,363

5.5 5.5

84.3 93.3

5.7 7.2

2.95–2.80 2.95–2.80

87.9 95.7

29.2 26.8

8.0–2.8 8.0–2.8

21.4 20.8

30.6 29.0

0.013 0.015

1.83 2.00

24.63 25.25

37.02 41.61

38.32 44.54

36.68 51.02

27.65 34.08

32.27 33.92

78.7 83.6

16.8 13.1

3.3 0.8

1.2 2.5

34 45

3 3

ðhÞi, mean intensity of reflection h over all measurement of IðhÞ.flections with F =rðF Þ > 2 cutoff.

allographic refinement.

910 F. Canduri et al. / Biochemical and Biophysical Research Communications 313 (2004) 907–914

Results and discussion

Molecular replacement and crystallographic refinement

The standard procedure of molecular replacement

using AMoRe [12] was used to solve both structures.

After translation function computation the correlation

was of 75.1% and the Rfactor of 30.8% for the structure

Fig. 3. Ribbon diagrams of (A) PNP apoenzyme, (B) HsPNP:Ino, and

HsPNP:Ino and 74.1% and the Rfactor of 31.1% forthe structure HsPNP:ddI. The highest magnitude of

the correlation coefficient function was obtained for the

Euler angles a ¼ 117:07�; b ¼ 59:42�, and c ¼ 153:27�for HsPNP:Ino and a ¼ 113:65�; b ¼ 57:46�, and

c ¼ 158:07� for HsPNP:ddI. The fractional coordinates

are Tx ¼ 0.8315, Ty ¼ 0.9584, Tz ¼ 0.3649, and Tx ¼0.1640, Ty ¼ 0.6251, Tz ¼ 0.0318 for HsPNP:Ino and

(C) HsPNP:ddI generated by Molscript [28] and Raster3d [29].

F. Canduri et al. / Biochemical and Biophysical Research Communications 313 (2004) 907–914 911

HsPNP:ddI, respectively. At this stage 2Fobs )Fcalc omitmaps were calculated. These maps showed clear electron

density for the inosine and ddI in the complexes. Fur-

ther refinement in X-PLOR continued with simulated

annealing using the slow-cooling protocol, followed by

alternate cycles of positional refinement and manual

rebuilding using XtalView [15]. Initial models of ligands

were generated using Sybyl (Tripos). Finally, the posi-

tions of ligands, water, and sulfate molecules werechecked and corrected in Fobs )Fcalc maps. The final

model for the HsPNP:Ino has an Rfactor 20.8% and an

Rfree of 29.0% with 45 water molecules, 3 sulfate ions,

and the inosine. HsPNP:ddI has an Rfactor of 21.4% and

an Rfree of 30.6%, with 34 water molecules, 3 sulfate

ions, and the ddI (Table 1).

Ignoring low-resolution data, a Luzzati plot [16] gives

the best correlation between the observed and calculateddata for a predicted mean coordinate error of 0.33�AA for

HsPNP:Ino and 0.35�AA for HsPNP:ddI. The average B

factor for main chain and side chain atoms, and analysis

of the Ramachandran plot are given in Table 1. It is

interesting to observe that Thr221 occupies disallowed

regions in the two complex structures, which was also

observed in the structures of HsPNP previously solved

[17], although it is well positioned in the electron-densitymap (2Fobs )Fcalc).

Overall description

Analysis of the crystallographic structures ofHsPNP:Ino and HsPNP:ddI complexes indicates a tri-

meric structure. The core of one PNP monomer consists

of an extended b-sheet. This sheet is surrounded by ahelices. The structure contains an eight-stranded mixed

b-sheet and a five-stranded mixed b-sheet, which join to

form a distorted b-barrel. These secondary structural

elements are linked by extended loops, a characteristic

feature of all PNP molecules [2] (Fig. 3). The ligand islocked between the monomers, as also observed for the

structures of PNP complexed with immucillin-H and

acyclovir [9,10].

Fig. 4. Gate movement after binding of inosine and ddI

Ligand-binding conformational changes

There is a conformational change in the PNP struc-

tures when ligands bind in the active site. The largest

movement was observed for Ala263 in the present

structures. The residues 241–260 act as a gate that opens

during substrate binding. The r.m.s. deviation difference

of the superimposition of HsPNP complex on the PNP

apoenzyme, in the coordinates of Ca is 0.34�AA, disre-garding the gate (Fig. 4). The r.m.s.d. in the coordinates

of all Ca is 1.16�AA upon superimposition of HsPNP:Ino

on the PNP apoenzyme, and 1.28�AA upon superimpo-

sition of HsPNP:ddI on the PNP apoenzyme. The gate

is anchored near the central b-sheet at one end and near

the C-terminal helix at the other end and it is respon-

sible for controlling access to the active site. The gate

movement involves a transition from coil to helix ofresidues 257–265 in the change of the apoenzyme-

complex (Figs. 3A–C).

Interactions with ligands

The specificity and affinity between enzyme and its

ligand depend on directional hydrogen bonds and ionic

interactions, as well as on shape complementarity of the

contact surfaces of both partners [18–25]. The electro-

static potential surface of the ligands complexed with

HsPNP was calculated with GRASP [26] (figure not

shown). The analysis of the charge distribution of the

binding pocket indicates the presence of some chargecomplementarity between inhibitor and enzyme (purine

binding site), though most of the binding pocket is hy-

drophobic (ribose binding site). The previously de-

scribed participation of Lys244 [27] in ligand binding

was not identified in the present study and in the

structures of human PNP complexed with inhibitors

[7,8].

Comparison of the present structureswith humanPNPcomplexed with guanine (HsPNP:Gua) [6], acyclovir

(HsPNP:Acy) [9], and immucillin-H (HsPNP:ImmH) [10]

indicates that human PNP presents multiple modes of

to human PNP, compared with PNP apoenzyme.

Fig. 5. Multiple modes of binding to human PNP. (A) HsPNP:inosine, (B) HsPNP:ddI, (C) HsPNP:guanine, (D) HsPNP:acyclovir, and

(E) HsPNP:immucillin-H.

912 F. Canduri et al. / Biochemical and Biophysical Research Communications 313 (2004) 907–914

binding to the active site. Figs. 5A–E show the interaction

between ligands and PNP. The main residues involved in

binding in all PNP complexes are Glu201 and Asn243.Analysis of the hydrogen bonds between inosine and PNP

reveals seven hydrogen bonds, involving the residues

Tyr88, Glu201, Met219, Asn243, and His257 (Table 2).

There are four hydrogen bonds between ddI and human

PNP, involving the residues Glu201, Asn243, and His257

(Table 3).Analysis of the complexes indicates thatGlu201

occupies approximately the same position in all the

complexes of human PNP studied so far. However, theside chain of Asn243 shows some flexibility, which causes

differences in the hydrogen bond pattern of this residue.

The complexes HsPNP:Ino, HsPNP:ddI, HsPNP:ImmH

[10], and HsPNP:Gua [6] show intermolecular hydrogenbonds involving the following atom pairs: Asn243 ND2-

O6 and Asn243 OD1-N7. The participation of Asn243

OD1 is not observed in the HsPNP:Acy complex [9]. The

precise definition of the modes of binding to human PNP

may help in future structure-based design of inhibitors.

The atomic coordinates and the structure factors for

the complexes HsPNP:Ino and HsPNP:ddI have been

deposited in the PDB with accession codes: 1RCT and1V3Q, respectively.

Fig. 5. (continued).

Table 2

Hydrogen bonds between HsPNP and inosine

Inosine PNP Distance (�AA)

N1 Glu201 OE1 3.15

N1 OE2 2.50

O6 Asn243 ND2 2.87

N7 OD1 2.57

O5� His257 ND1 2.99

O2� Met219 N 3.05

O3� Tyr88 OH 3.03

Table 3

Hydrogen bonds between HsPNP and 20; 30-dideoxyinosine

20; 30-Dideoxyinosine PNP Distance (�AA)

N1 Glu201 OE2 2.45

O6 Asn243 ND2 3.33

N7 OD1 2.97

O5� His257 ND1 2.77

F. Canduri et al. / Biochemical and Biophysical Research Communications 313 (2004) 907–914 913

Acknowledgments

We acknowledge the expertise of Denise Cantarelli Machado for

the expansion of the cDNA library and Deise Potrich for the DNA

sequencing. This work was supported by grants from FAPESP

(SMOLBNet, Proc.01/07532-0, and 02/04383-7), CNPq, CAPES and

Instituto do Mileenio (CNPq-MCT). W.F.A. (CNPq, 300851/98-7),

M.S.P. (CNPq, 300337/2003-5), and L.A.B. (CNPq, 520182/99-5) are

researchers for the Brazilian Council for Scientific and Technological

Development, F.C. is post-doctoral fellow under FAPESP fellowship.

References

[1] J.D. Stoeckler, in: R.I. Glazer (Ed.), Developments in Cancer

Chemotherapy, CRC, Boca Raton, FL, 1984, pp. 35–60.

[2] A. Bzowska, E. Kulikowska, D. Shugar, Purine nucleoside

phosphorylases: properties, functions, and clinical aspects, Phar-

macol. Therapeut. 88 (2000) 349–425.

[3] C. Mao, W.J. Cook, M. Zhou, A.A. Federov, S.C. Almo, S.E.

Ealick, Calf spleen purine nucleoside phosphorylase complexed

with substrates and substrate analogues, Biochemistry 37 (1998)

7135–7146.

[4] W.F. De Azevedo, F. Canduri, D.M. dos Santos, R.G. Silva, J.S.

Oliveira, L.P.S. Carvalho, L.A. Basso, M.A. Mendes, M.S. Palma,

D.S. Santos, Crystal structure of human purine nucleoside

phosphorylase at 2.3�AA resolution, Biochem. Biophys. Res. Com-

mun. 308 (3) (2003) 545–552.

[5] W.F. De Azevedo, G.C. Santos, D.M. dos Santos, J.R. Olivieri, F.

Canduri, R.G. Silva, L.A. Basso, M.A. Mendes, M.S. Palma, D.S.

Santos, Docking and small angle X-ray scattering studies of

purine nucleoside phosphorylase, Biochem. Biophys. Res. Com-

mun. 309 (2003) 928–933.

[6] W.F. De Azevedo, F. Canduri, D.M. dos Santos, J.H. Pereira,

M.V.B. Dias, R.G. Silva, M.A. Mendes, L.A. Basso, M.S. Palma,

D.S. Santos, Crystal structure of human PNP complexed with

guanine, Biochem. Biophys. Res. Commun. 312 (2003) 767–772.

[7] D.J. Back, S. Ormesher, J.F. Tjia, R. Macleod, Metabolism of

20,30-dideoxyinosine (ddI) in human blood, Br. J. Clin. Pharmacol.

33 (1992) 319–322.

914 F. Canduri et al. / Biochemical and Biophysical Research Communications 313 (2004) 907–914

[8] R.G. Silva, L.P. Carvalho, J.S. Oliveira, C.A. Pinto, M.A.

Mendes, M.S. Palma, L.A. Basso, D.S. Santos, Cloning, overex-

pression, and purification of functional human purine nucleoside

phosphorylase, Protein Expr. Purif. 27 (1) (2003) 158–164.

[9] D.M. dos Santos, F. Canduri, J.H. Pereira, M.V.B. Dias, R.G.

Silva, M.A. Mendes, M.S. Palma, L.A. Basso, W.F. de Azevedo,

D.S. Santos, Crystal structure of human purine nucleoside

phosphorylase complexed with acyclovir, Biochem. Biophys.

Res. Commun. 308 (3) (2003) 553–559.

[10] W.F. De Azevedo, F. Canduri, D.M. dos Santos, J.H. Pereira,

M.V.B. Dias, R.G. Silva, M.A. Mendes, L.A. Basso, M.S. Palma,

D.S. Santos, Structural basis for inhibition of human PNP by

immucillin-H, Biochem. Biophys. Res. Commun. 309 (2003) 922–

927.

[11] Collaborative Computational Project, Acta Crystallogr. D 50 (4)

(1994) 760–763.

[12] J. Navaza, AMoRe: an automated package for molecular

replacement, Acta Crystallogr. A 50 (1994) 157–163.

[13] A.T. Br€uunger, X-PLOR Version 3.1: A System for Crystallogra-

phy and NMR, Yale University Press, New Haven, 1992.

[14] R.A. Laskowski, M.W. MacArthur, D.K. Smith, D.T. Jones,

E.G. Hutchinson, A.L. Morris, D. Naylor, D.S. Moss, J.M.

Thorton, PROCHECK v.3.0—program to check the stereochem-

istry quality of protein structures—operating instructions, 1994.

[15] D.E. McRee, XtalView/Xfit—a versatile program for manipulat-

ing atomic coordinates and electron density, J. Struct. Biol. 125

(1999) 156–165.

[16] P.V. Luzzati, Traitement statistique des erreurs dans la determina-

tion des structures cristallines, Acta Crystallogr. 5 (1952) 802–810.

[17] S.E. Ealick, S.A. Rule, D.C. Carter, T.J. Greenhough, Y.S. Babu,

W.J. Cook, J. Habash, J.R. Helliwell, J.D. Stoeckler, R.E. Parks

Jr., S.-F. Chen, C.E. Bugg, Three-dimensional structure of human

erythrocytic purine nucleoside phosphorylase at 3.2�AA resolution,

J. Biol. Chem. 265 (3) (1990) 1812–1820.

[18] W.F. De Azevedo, H.J. MuellerDieckmann, U. SchulzeGahmen,

P.J. Worland, E. Sausville, S.H. Kim, Structural basis for

specificity and potency of a flavonoid inhibitor of human

CDK2, a cell cycle kinase, Proc. Natl. Acad. Sci. USA 93 (7)

(1996) 2735–2740.

[19] W.F. De Azevedo, S. Leclerc, L. Meijer, L. Havlicek, M, Strnad,

S.H. Kim, Inhibition of cyclin-dependent kinases by purine

analogues—crystal structure of human cdk2 complexed with

roscovitine, Eur. J. Biochem. 243 (1–2) (1997) 518–526.

[20] S.H. Kim, U. Schulze-Gahmen, J. Brandsen, W.F. de Azevedo,

Structural basis for chemical inhibition of CDK2, Prog. Cell Cycle

Res. 2 (1996) 137–145.

[21] W.F. De Azevedo, F. Canduri, N.J.F. da Silveira, Structural basis

for inhibition of cyclin-dependent kinase 9 by flavopiridol,

Biochem Biophys. Res. Commun. 293 (2002) 566–571.

[22] W.F. De Azevedo, R.T. Gaspar, F. Canduri, J.C. Camera, N.J.F.

da Silveira, Molecular model of cyclin-dependent kinase 5

complexed with roscovitine, Biochem Biophys. Res. Commun.

297 (2002) 1154–1158.

[23] W.F. De Azevedo, J.S. de Oliveira, L.A. Basso, M.S. Palma, J.H.

Pereira, F. Canduri, D.S. Santos, Molecular model of shikimate

kinase from Mycobacterium tuberculosis, Biochem. Biophys. Res.

Commun. 295 (1) (2002) 142–148.

[24] F. Canduri, N.J.F. Silveira, J.C. Camera, W.F. de Azevedo,

Structural bioinformatics study of cyclin-dependent kinases com-

plexed with inhibitors, Ecletica Quim. 28 (2003) 45–53.

[25] J.H. Pereira, F. Canduri, J.S. Oliveira, N.J.F. Silveira, L.A. Basso,

M.S. Palma, W.F. De Azevedo, D.S. Santos, Structural bioinfor-

matics study of EPSP synthase from Mycobacterium tuberculosis,

Biochem. Biophys. Res. Commun. 312 (2003) 608–614.

[26] A. Nicholls, K. Sharp, B. Honig, Protein folding and association:

insights from the interfacial and thermodynamic properties of

hydrocarbons, Proteins Struct. Funct. Genet. 11 (1991) 281–296.

[27] J.A. Montgomery, Purine nucleoside phosphorylase: a target for

drug design, Med. Res. Rev. 13 (3) (1993) 209–228.

[28] P.J. Kraulis, MOLSCRIPT: a program to produce both detailed

and schematic plots of proteins, J. Appl. Cryst. 24 (1991) 946–

950.

[29] E.A. Merritt, D.J. Bacon, Raster3D: photorealistic molecular

graphics, Methods Enzymol. 277 (1997) 505–524.