Molecular and Structural Characterization of Guanosine

Monophosphate Reductase of Streptococcus pneumonia - a potential drug

target for pneumonia

Rajasekhar Chikati2 Richa Gulati

3 Sireesha Garimella

3 Sphoorthi Sree Chalumuri

1

Prasanthi Chittineedi1 Santhi Latha Pandrangi

1

1 Onco-Stem Cell Research Laboratory Dept of Biochemistry and Bioinformatics

GITAM Institute of Science GITAM Deemed to be University Visakhapatnam-

530045 India

2 Dept of Biochemistry University College of Science Osmania University

Hyderabad-500007 India

3 Dept of Biotechnology GITAM Institute of Science GITAM Deemed to be

University Visakhapatnam-530045 India

Abstract

In developed and developing countries Streptococcus pneumoniae remains a major pathogen

responsible for high morbidity and mortality rates S pneumoniae is estimated to kill about one

million children under the age of five worldwide per year Today antibiotic resistance is

widespread and growing in S pneumonia Guanosine monophosphate reductase (GMPR)

catalyses and plays a critical role in the re-utilization of free intracellular bases and purine

nucleosides by irreversible and NADPH-dependent reductive deamination of Guanosine

Monophosphate (GMP) to Inosine Mono Phosphate (IMP) The 3D structure of GMP reductase

of Streptococcus pneumonia-R6 was established in this paper by comparing the crystal structure

of human guanosine monophosphate reductase 2 (hGMPr2 2A7R) The 3D Structure was

constructed using the alignment file information built in ClustalW 183 in Modeler 9v8 The

developed GMP reductase S-pneumonia-R6 model was submitted to the PROCHECK and

WHATIF programs for stereochemical consistency validation and structure analysis ProSA site

review calculated the Z-score The model developed was deposited in PMDB and accepted with

stereochemical failures of lt3 percent This research will provide useful insight into the quest and

rational drug design of a new generation of broad-spectrum pneumonia drugs through a clear

understanding of GMP reductase structural characterization

Key Words Streptococcus pneumonia GMP reductase homology modeling Modeller 9v8

Potential drug targets for Pneumonia

Abbreviations

GMP Guanosine Mono Phosphate

GMPR Guanosine Mono Phosphate Reductase

hGMPR2 Human Guanosine Mono Phosphate Reductase

2

IMP Inosine Mono Phosphate

PBPs Penicillin-binding Proteins

PMDB Protein Data Model Base

RMSD Root Mean Square Deviation

VMD Visual Molecular Dynamics

WHO World Health Organization

Introduction

Pneumonia is a serious infection andor inflammation of our lungs Pneumonia and

influenza combined together were ranked as the seventh leading cause of death Pneumonia was

caused by a variety of bacteria including Streptococcus Staphylococcus Pseudomonas

Haemophilus Chlamydia Mycoplasma several viruses certain fungi and protozoans Based on

the available data Streptococcus pneumoniae (S pneumoniae) a major human bacterial

pathogen has elevated rates of morbidity and mortality It is estimated to kill annually close to

one million children of less than five years of age worldwide [WHO 2012] S pneumoniae is a

common spherical gram-positive bacterium normally found in the nasopharynx of 5-10 of

healthy adults and 20-40 of healthy children [Whitney et al 2003] Pneumonia can cause up to

24 million deaths annually mainly in the World Health Organizations African Southeast Asian

and Eastern Mediterranean regions (WHO) This constitutes 15-2 times more child deaths in

Key Words Streptococcus pneumonia GMP reductase homology modeling Modeller 9v8

Potential drug targets for Pneumonia

Abbreviations

GMP Guanosine Mono Phosphate

GMPR Guanosine Mono Phosphate Reductase

hGMPR2 Human Guanosine Mono Phosphate Reductase

2

IMP Inosine Mono Phosphate

PBPs Penicillin-binding Proteins

PMDB Protein Data Model Base

RMSD Root Mean Square Deviation

VMD Visual Molecular Dynamics

WHO World Health Organization

Introduction

Pneumonia is a serious infection andor inflammation of our lungs Pneumonia and

influenza combined together were ranked as the seventh leading cause of death Pneumonia was

caused by a variety of bacteria including Streptococcus Staphylococcus Pseudomonas

Haemophilus Chlamydia Mycoplasma several viruses certain fungi and protozoans Based on

the available data Streptococcus pneumoniae (S pneumoniae) a major human bacterial

pathogen has elevated rates of morbidity and mortality It is estimated to kill annually close to

one million children of less than five years of age worldwide [WHO 2012] S pneumoniae is a

common spherical gram-positive bacterium normally found in the nasopharynx of 5-10 of

healthy adults and 20-40 of healthy children [Whitney et al 2003] Pneumonia can cause up to

24 million deaths annually mainly in the World Health Organizations African Southeast Asian

and Eastern Mediterranean regions (WHO) This constitutes 15-2 times more child deaths in

these regions than those from malaria and HIV infection combined HIV has resulted in a rapid

rise in the incidence of morbidity and mortality of pneumonia in sub-Saharan Africa [WHO] S

pneumoniae infections cause 100000-135000 hospitalizations for pneumonia around 6 million

cases of otitis media and more than 60000 invasive disease cases including 3300 meningitis

cases per year according to the CDC Division of Bacterial and Mycotic Diseases

India accounts for almost 40 percent of the [Center for Disease Control and Prevention] cases of

childhood pneumonia worldwide Pneumococcal disease is more prevalent in the winter months

and in the transmission of respiratory viruses such as influenza Most deaths from pneumococcal

disease occur in older people in the United States although several children die of pneumococcal

pneumonia in developing nations

S pneumoniae is responsible for a wide variety of diseases such as those triggered by

upper respiratory infections (sinusitis acute otitis media tracheobronchitis) lower respiratory

infections (bronchopneumonia pneumonia with or without bacteremia or empyema) invasive

infections (primary childhood bacteremia meningitis spontaneous bacterial peritonitis tissue

seeding sepsis (septic arthritis myositis tissue seeding) otitis media and acute sinusitis (main

causes are infection with pneumococci species and Haemophilus influenzae) and pneumonia It

also causes infections in other areas of the body as well such as the bloodstream (bacteremia)

brain and spinal cord lining (meningitis) bones (osteomyelitis) joints (arthritis) ears (otitis

media) and sinuses (sinusitis) It spreads through auto-inoculation in people carrying bacteria in

their upper respiratory tracts and via direct in-person contact through respiratory droplets

The irreversible and NADPH-dependent reductive deamination of GMP to IMP is

catalysed by guanosine monophosphate reductase (GMPR) and plays a crucial role in the re-use

of free intracellular bases and purine nucleosides It transforms nucleobase nucleoside and

nucleotide derivatives of G to A nucleosides and maintains the intracellular equilibrium of

nucleotides of A and G In Fig1 the essential and simple reaction is shown

Fig1 The reaction carried out by the enzyme involvement of GMP reductase catalyzes the

irreversible and NADPH- dependent reductive deamination of GMP to IMP

At one stage S pneumoniae-caused diseases could be treated reliably with antibiotics

The various antibiotics used for the treatment of diseases caused by S pneumoniae are given in

Table 1 Today however antibiotic resistance in the S Pneumoniae is prevalent and growing

[GVDoern et al 1999] Streptococcus pneumoniae remained overwhelmingly susceptible to all

groups of antibiotics that were active against pathogens from the beginning of the antibiotic

period to the mid-1970s In Streptococcus pneumoniae mutations in the target enzymes for β-

lactam antibiotics the PBPs have been identified as a significant resistance mechanism

[Hakenbeck R 2006] Resistance in S pneumoniae has started to increase to other non-szlig-lactam

products such as macrolides clindamycin tetracycline (132) chloramphenicol (72) and

trimethoprimsulfamethoxazole (311) [Jones et al 1998] In patients with pneumococcal

infections treated with previously successful medications therapeutic failures have been

identified [Kaplan SL Mason EO Jr 1998]

In this paper we predicted the 3D structure of GMP reductase of Streptococcus

pneumonia based on the Crystal Structure of Human Guanosine Monophosphate Reductase 2

(Gmpr2) The template (2A7R) sequence taken from PDB and the model was developed in

modeller 9v8

Table 1 Antibiotics used for the treatment of diseases caused by S pneumoniae

Drug Mechanism of action Major side-effects

Fig1 The reaction carried out by the enzyme involvement of GMP reductase catalyzes the

irreversible and NADPH- dependent reductive deamination of GMP to IMP

At one stage S pneumoniae-caused diseases could be treated reliably with antibiotics

The various antibiotics used for the treatment of diseases caused by S pneumoniae are given in

Table 1 Today however antibiotic resistance in the S Pneumoniae is prevalent and growing

[GVDoern et al 1999] Streptococcus pneumoniae remained overwhelmingly susceptible to all

groups of antibiotics that were active against pathogens from the beginning of the antibiotic

period to the mid-1970s In Streptococcus pneumoniae mutations in the target enzymes for β-

lactam antibiotics the PBPs have been identified as a significant resistance mechanism

[Hakenbeck R 2006] Resistance in S pneumoniae has started to increase to other non-szlig-lactam

products such as macrolides clindamycin tetracycline (132) chloramphenicol (72) and

trimethoprimsulfamethoxazole (311) [Jones et al 1998] In patients with pneumococcal

infections treated with previously successful medications therapeutic failures have been

identified [Kaplan SL Mason EO Jr 1998]

In this paper we predicted the 3D structure of GMP reductase of Streptococcus

pneumonia based on the Crystal Structure of Human Guanosine Monophosphate Reductase 2

(Gmpr2) The template (2A7R) sequence taken from PDB and the model was developed in

modeller 9v8

Table 1 Antibiotics used for the treatment of diseases caused by S pneumoniae

Drug Mechanism of action Major side-effects

Ceftaroline Transpeptidase activity inhibition and

PbPs binding

Diarrhea nausea rash

infusion-site reactions

Omadacycline Action on efflux pumps and ribosomal

protection associated with chemical

structure modifications

Gastrointestinal side

effects

Solithromycin Bacterial translation inhibition by

binding to the 23S ribosomal RNA

Hepatotoxicity

Delafloxacin Bacterial DNA topoisomerase IV and

DNA gyrase inhibition

Chest pain

transaminase

elevations nausea

Materials and Methods

Fasta sequence of GMP reductase of S Pneumoniae- R6 is extracted from the NCBI BLASTp

software and the sequence is compared to structurally related sequences in NCBI for good

template selection The human guanosine monophosphate reductase 2 (hGmpr2) crystal structure

(PDB ID 2A7R) was taken as a pneumonia example [Jixi Li et al 2006] By using the clustalW

online server the GMP reductase sequences were matched with the template [Thompson JD et

al 1994] Model was built using modeller 9v8 (httpwwwsalilaborgmodeller 9v8) Hundred

models were generated and inspected SWISS-PDB Viewer (spdbv) [Guex N and Peitsch MC

1997] was used for verification of RMS values all the models

The Ramachandran plot carried out the stereochemical quality control of the formed structure

For the prediction of the secondary structure of target and template sequences online server PDB

Sum was used [Laskowski et al 2005] By submitting PDB ID 2A7R to PDB Sum the active

site of the template structure was obtained In order to assess the stereochemical quality and

structural analysis of the constructed GMP reductase structure PROCHECK and WHATIF

programs were used [Morris et al 1992 Vriend G 1990] Z-score of the GMP reductase

formed was obtained by submitting its PDB structure (target) to ProSa (Protein Structure

Analysis) [Sippl MJ 1993] To reflect the existing structure Pymol and VMD software were

used [httppymolsourceforgenet Humphrey et al 1996]

Results and Discussion

GMP reductase (EC 1717) (spr1128 CDS) is involved in the Purine metabolism of S

pneumoniae- R6 The fasta sequence of target sequence was obtained from NCBI and this

sequence was subjected to BLASTp analysis The protein with the highest bit score was selected

as the template ie 2A7R hGMPR2 from the BLASTp data The PDB ID 2A7R template

sequence is downloaded from RCSB PDB as compared to the fasta sequence in the NCBI

BLASTp program hGMPr2 shows 34 identities bit score and expected e-value 175 2e-44

respectively The template has the following parameters such as 300 Ao resolutions 0228(obs)

R-value and 0276 R-free value Multiple sequence alignment between the GMPR of S

pneumoniae- R6 and GMPR2 of human using the online platform ClustalW 183 (Fig2a) was

obtained In red color boxes the conserved regions were seen Hundred models of GMP

reductase were produced using modeller 9v8 the best one taken from hundred models developed

based on procheck analysis Pymol and VMD softwares were used for representation of

developed structure of GMP reductase of S pneumoniae_R6 (Fig2b)

Fig2a) GMP reductase of Spneumoniae-R6 aligned with template human GMPr2 (PDB

ID 2A7R) conserved regions showed in red color boxes and catalytic residue

showed in thick red color box 2b) The Developed 3D structure of GMP

reductase of Spneumoniae_R6 in which α-helicals showed in Red color β-turns

showed in Yellow color and loops showed in Green color

Results and Discussion

GMP reductase (EC 1717) (spr1128 CDS) is involved in the Purine metabolism of S

pneumoniae- R6 The fasta sequence of target sequence was obtained from NCBI and this

sequence was subjected to BLASTp analysis The protein with the highest bit score was selected

as the template ie 2A7R hGMPR2 from the BLASTp data The PDB ID 2A7R template

sequence is downloaded from RCSB PDB as compared to the fasta sequence in the NCBI

BLASTp program hGMPr2 shows 34 identities bit score and expected e-value 175 2e-44

respectively The template has the following parameters such as 300 Ao resolutions 0228(obs)

R-value and 0276 R-free value Multiple sequence alignment between the GMPR of S

pneumoniae- R6 and GMPR2 of human using the online platform ClustalW 183 (Fig2a) was

obtained In red color boxes the conserved regions were seen Hundred models of GMP

reductase were produced using modeller 9v8 the best one taken from hundred models developed

based on procheck analysis Pymol and VMD softwares were used for representation of

developed structure of GMP reductase of S pneumoniae_R6 (Fig2b)

Fig2a) GMP reductase of Spneumoniae-R6 aligned with template human GMPr2 (PDB

ID 2A7R) conserved regions showed in red color boxes and catalytic residue

showed in thick red color box 2b) The Developed 3D structure of GMP

reductase of Spneumoniae_R6 in which α-helicals showed in Red color β-turns

showed in Yellow color and loops showed in Green color

The formed reductase GMP of S Pneumoniae- R6 was sent for stereochemical quality

analysis to PROCHECK SWISS-PDB Viewer of spdbv WHAT IF and ProSA According to

Ramachandran Plot 949 percent of residues in the most favored region 44 percent of residues

in the additionally permitted region and just 03 percent of residues in generous and disallowed

regions are statistics of the established model (Fig3a) The key statistics on chain and side chain

parameters also indicate that there are no considerable bad contacts It explains that the

suggested model is of high quality

SWISS-PDB viewer (spdbv) version 37 was used for determining the RMSD of the

experimentally determined structure of GMPR of S pneumoniae- R6 The structural

superimposition of C crystal structure (2A7R) structures on GMPR of S pneumoniae- R6

model revealed the RMSD value of 032A0 and 1336 number of atoms involved which is

characterized as good theoretical model The data of WHATIF explains the evaluation

parameters (quality conformation) of the structural model of GMPR of S pneumoniae- R6 It

shows average package quality 1168 for target and 1707 for template rotamer normality 0778

for target and 2618 for template backbone conformation 0954 for target and 0850 for template

bond length 0924 for target and 0366 for template and bond angles 1244 for target and0660

for template Z-score of GMPR of S pneumoniae- R6 was calculated by using of online software

ProSA web analysis The constructed model shows the overall value of model quality-849

which is well within the range of a standard native structure (Fig3b) and the residue energies are

negative for local model quality All these studies show that the evolved GMPR model of S

Pneumoniae- R6 is of decent quality

Online server PDB sum was used for the generation of secondary structure of GMPR of

S pneumoniae- R6 and structure of hGMPr2 (PDB ID 2A7R) A single disulphide bond

between 68-95 residues and catalytic residue ASN is shown in the template structure (PDB ID

2A7R) Developed structure of GMPR of S pneumoniae- R6 shows highly conserved regions in

between 176 to 188 it contain 66 strands (190) alpha helix 111 (320) 3-10 helix11 (32)

others 159 (458) and total 347 residues Based on the alignment of the templates catalytic

residue (active site) are determined in built model of GMPR of S pneumoniae- R6 The template

structure (hGMPr2) (PDB ID 2A7R) was submitted to online server PDBSUM and it showed

that the catalytic residue (active site) ASN -158 was conserved in hGMPR2 In the thick red

color box of Multiple Sequence Alignment the active site area is shown (Fig2a) Catalytic

residues (active amino acids) are seen in red and yellow colors and are also involved in

secondary structural conformation (Fig3c) The developed GMPR of S pneumoniae- R6 model

was submitted to PMDB with a model ID as PM-0075465 The built model was accepted as

less than 3 stereochemical check failures [Tiziana et al 2006]

Fig3a) PROCHEK plot statistics of GMP reductase of Spneumoniae-R6 shows 949

residues in most favored region 44 residues in additionally allowed region and

only 03 of residues in generously and disallowed regions 3b) The generated

model GMP reductase of Spneumoniae-R6 Z-score value -849 well inside the

range of a typical native structure 3c) Developed 3D structure of GMP reductase

of Spneumoniae-R6 catalytic residue (active site region) ASPARGINE-158

showed in Red color

Conclusion

It is very difficult to generate the 3D structure of GMP reductase S-pneumoniae-R6 using X-ray

diffraction information and NMR data Computational studies are widely accepted for the

prediction of S-pneumoniae-3D R6s GMPR structure and may help structure-based drug design

The developed structure GMPR of S pneumoniae- R6 shows 949 residues in most favored

region Our study was focused on gaining valuable information on research research and rational

drug design of new generation of wide spectrum pneumonia drugs

Conflict of interest None declared

Author Contributions Performed the experiments RSC and RG Analyzed the data RG SG

PCSSC and SLP Helped in performing the studies GS PC SSC Overall supervision of the

study SLP Contributed materials analysis tools SLP Conceptualized the study SLP and RSC

Wrote the manuscript SLP RSC and RG All authors read and approved the final manuscript

Acknowledgements

SLP gratefully acknowledges UGC (Ref No NoF30-4562018 (BSR) for the financial support

RSC gratefully acknowledges University Grant Commission (UGC) New Delhi (Lr No F 11-

482008(BSR) Dated 12-02-2010) for financial support and also to thanks DS Kothari Post

Doctoral Fellowship (No F 4-206 (BSR)BL13- 140228 Dated 021213)

References

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orally administered antimicrobial agents against four species of bacterial respiratory

pathogens from US medical centers in 1992 and 1993rsquo Antimicrob Agents Chemother

38 pp2419-25

2 Guex N and Peitsch MC (1997) lsquoSWISS MODEL and the Swiss pdb viewer an

environment for comparative protein modelingrsquo Electrophoresis 18 pp2714-2723

3 Hakenbeck R Bruumlckner R Denapaite D and Maurer P (2012)

lsquoMolecular mechanisms of β-lactam resistance in Streptococcus pneumoniaersquo Fut

Microbio Volume 7 No 3

4 Humphrey W dalke A and Schulten K (1996) lsquoVMD-Visual Molecular Dynamicsrsquo

Journal of molecular Graphics 141 pp33-38

5 Jixi Li Zhiyi Wei Mei Zheng Xing Gu Yingfeng Deng Rui Qiu Fei Chen

Chaoneng Ji Weimin Gong Yi Xie and Yumin Mao (2006) lsquoCrystal structure of

human guanosine monophosphate reductase 2 (hGMPR2) in Complex with GMPrsquo

Journal of Molecular Biology 355 5 pp980-988

6 Jones RN Pfaller MA and Doern GV (1998) lsquoComparative antimicrobial activity

of trovafloxacin tested against 3049 Streptococcus pneumoniae isolates from the 1997-

1998 respiratory infection seasonrsquo Diagn Microbiol Infect Dis 32 pp119-26

7 Jorgensen JH Doern GV Maher LA Howell AW and Redding JS (1990)

lsquoAntimicrobial resistance among respiratory isolates of Haemophilus influenza

Moraxella catarrhalis and Streptococcus pneumonia in the United Statesrsquo Antimicrob

Agents Chemother 34 pp2075- 80

8 Kaplan SL Mason EO Jr (1998) lsquoManagement of infections due to antibiotic-

resistant Streptococcus pneumoniaersquo Clin Microbiol Rev 11 pp628-44

9 Laskowski RA Watson JD and Thornton JM (2005) lsquoProtein function prediction

using local 3D templatesrsquo Journal of Molecular Biology 351 pp614- 626

10 Laskowski RA Watson JD and Thornton JM (2005) lsquoProFunc a server for

predicting protein function from 3D structurersquo Nucleic Acids Research 33 W pp89-

93

11 Morris AL MacArthur MW Hutchinson EG and Thornton JM (1992)

lsquoStereochemical quality of protein structure coordinatesrsquo Proteins 12 pp345ndash364

12 Robinson KA Baughman W Rothrock G Barrett NL Pass M and Lexau C

(2001) lsquoEpidemiology of invasive Streptococcus pneumoniae infections in the United

States 1995-1998 Opportunities for prevention in the conjugate vaccine erarsquo JAMA

285 (13) pp1729-35

13 Sippl MJ (1993) lsquoRecognition of errors in three-dimensional structures of Proteinsrsquo

Proteins 17 pp355-362

14 Spika JS Acklam RR Plikaytis BD and Oxtoby MG (1991) lsquoThe Pneumococcal

Surveillance Working Group Antimicrobial resistance of Streptococcus pneumoniae in

the United States 1979-1987rsquo J Infect Dis 163 pp1273-78

15 Thompson JD Higgins DG and Gibson TJ (1994) lsquoClustalW improving the

sensitivity of progressive multiple sequence alignment through sequence weighting

position-specific gap penalties and weight matrix choicersquo Nucleic Acids Research 22

pp4673-4680

16 Thornsberry C Brown SD Yee C Bouchillon SK Marler JK and Rich T

(1993) lsquoIncreasing penicillin resistance in Streptococcus pneumoniae in the USrsquo

Infections in Medicine Supplement 93 pp15-24

17 Tiziana Castrignano Paolo D Onorio De Meo Domenico ozzetto Ivan Guiseppe

Talamo and Anna Tramontano (2006) lsquoThe PMBD Protein Model Database Nuclic

Acids Research 34 306-309

18 Vriend G (1990) lsquoWHATIF a molecular modeling and drug design programrsquo Journal

of Molecular Graphics 8 pp52ndash56

19 Whitney CG Farley MM Hadler J Lee HHN Bennett M Ruth Lynfield

Arthur Reingold Cieslak Paul R Tamara Pilishvili Delois Jackson Richard R

Facklam James H and Anne Schuchat (2003) lsquoDecline in invasive pneumococcal

disease after the introduction of protein-polysaccharide conjugate vaccinersquo N Engl J

Med 348 pp1737-1746

20 World Health Organization (2012) Pneumococcal vaccines WHO position paperndash

2012 Wkly Epidemiol Rec 87 129ndash44

Websites

httpwwwcdcgovncidoddbmddiseaseinfostreppneum_thtml (CDC)

httpwwwsalilaborgmodeller 9v8 (Modeller )

httppymolsourceforgenet (Pymol)

httpwwwwhointen (World Health Organization)