Insights from Streptococcus pneumoniae glucose kinase structural model.

Streptococcus pneumonia is the common cause of sepsis and meningitis. Emergence of multiple antibiotic resistant strains in the community‐acquired bacterium is catastrophic. Glucose kinase (GLK) is a regulatory enzyme capable of adding phosphate group to glucose in the first step of streptomycin biosynthesis. The activity of glucose kinase was regulated by the Carbon Catabolite Repression (CCR) system. Therefore, it is important to establish the structure‐function relation of GLK in S. pneumoniae. However, a solved structure for S. pneumoniae GLK is not available at the protein data bank (PDB). Therefore, we created a model of GLK from S. pnemoniae using the X‐ray structure of Glk from E. faecalis as template with MODELLER (a comparative modeling program). The model was validated using protein structure checking tools such as PROCHECK, WHAT IF and ProSA for reliability. The active site amino acid Asp114 in the template is retained in S. pneumoniae GLK model (Asp115). Solvent accessible surface area (ASA) analysis of the GLK model showed that known key residues playing important role in active site for ligand binding and metal ion binding are buried and hence not accessible to solvent. The information thus discussed provides insight to the molecular understanding of glucose kinase in S. pneumoniae.


Background:
The continuing battles in the hospitals against drugresistant bacteria highlight the need for new antibiotics. The world largest causes of mortality and morbidity of pneumonia is caused by Streptococcus pneumoniae (S. pneumonia). S. pneumoniae is the most common cause of fatal community-acquired pneumonia, middle ear infection and meningitis. The disease rate is especially high in young children, the elderly and immuno-compromised individuals with predisposing conditions such as asplenia or acquired immunodeficiency syndrome (AIDS) [1, 2, 3]. More than 90 different pneumococcal serotypes have been identified by their difference in polysaccharide capsules. However, about 90% of clinical episodes of invasive pneumococcal infections in humans are caused by 23 pneumococcal serotypes [4]. The mortality in the United Staes alone is about 40,000 per yea which is, higher than that for other bacterial diseases [5,6]. The efficacy of available vaccines is limited. The use of antibiotics results in either capsular type shifting or in the rapid appearance and spread of antibiotic resistance determinants [7,8,9]. Thus prevention and treatment of the infection is a top priority for the scientific community [10]. This requires a better understanding of the S. pneumoniae proteins using structural and functional data for target definition and validation. Here, we describe the modeled structure of glucose kinase (GLK) from S. pneumonia towards establishing its molecular function. GLK (E.C.2.7.1.2) is an important enzyme in the biosynthesis of streptomycin. Glk catalyzes the conversion of glucose to glucose-6phosphate and is catabolically repressed by higher concentrations of glucose (carbon catabolite repression (CCR)). However, a 3-dimenisonal structure for S. pneumonia GLK is yet not available. Hence, we constructed the model structure for S. pneumonia GLK using known structural templates and describe its structural features to understand molecular function.

Methodology: Tools and systems
The study was performed on an AMD 64 bits dual processor with Linux operating system. Sequence alignment was performed with ClustalX and homology modeling was carried out using MODELLER (comparative modeling software). Energy minimization of the developed model was performed using Swiss PDB Viewer spdbv [11]. Protein structure checks were conducted using the ADIT validation server (http://deposit.pdb.org/validate/); WHAT IF web interface (http://swift.cmbi.ru.nl) and ProSA-web (https://prosa.services.came.sbg.ac.at). Figures were developed using Pymol 1.0 (http://pymol.sourceforge.net/).
The study was performed on an AMD 64 bits dual processor with Linux operating system. Sequence alignment was performed with ClustalX [11] and homology modeling was carried out using modeller (comparative modeling software). Energy minimization of the developed model was performed using Swiss PDB Viewer spdbv [12]. Protein structure checks were conducted using the ADIT validation server

Discussion:
Streptococcus pneumoniae infction is the major cause of morbidity and mortality elderly and young children. GLK is the enzyme involved in streptomycin biosynthesis and converts glucose to glucose-6-phosphate. Hence, it is important to establish the structure-function relationship for Streptococcus pneumoniae GLK. We developed a structural model using 2QMI (PDB ID) as a template. The sequence similarity is 65% with the template and reliability of the predicted model thus generated using MODELLER is high. Kinases are Zn 2+ and Mg 2+ dependent enzymes. The ions play an important role in the catalytic process of the enzyme by reducing the overall entropy by coordinating to substrates and water molecules. Here, we describe the consequence of model development towards establishing the specific function of S. pneumoniae GLK using predicted model.

Homology modeling and structure refinement
The homology model of S. pneumonaie GLK was built using MODELLER. We then incorporated the Mg 2+ ion and Zn 2+ ion from the template structure into the modeled structure. The atomic charges of the amino acid side chains involved in Mg 2+ ion and Zn 2+ ion coordination system is similar to those of the template. Structural refinement through energy minimization model was performed using spdbv.

Model validation
The constructed model of Glk from S. pneumoniae was examined for validation using different criteria. The RMSD analysis of the developed model was evaluated by means of deviation from its template using SUPERPOSE (Figure 1b). The Cα RMSD and the backbone RMSD deviations for the model and the template crystal structure are 0.36 Å and 0.42 Å respectively. The stereo chemical quality of the predicted model was evaluated using PROCHECK in ADIT. The Ramachandran plot of phi/psi distribution in the model is developed using PROCHECK for checking non-GLY residues at the disallowed regions. Standard bond lengths and bond angles of the model were determined using WHAT IF. The analysis revealed RMS Z-scores for bond lengths and bond angles to be 0.885 and 0.926, respectively. The values are almost equal to 1 suggesting high model quality. ProSA-Web analysis of the model revealed a Z-score value of -9.49 and it is in the range of native conformations of the template.

ASA analysis
Accessible surface area (ASA) analysis of the predicted GLK model showed the active site amino acid Asp115 with zero ASA value ( Figure 1c) and is buried inside the catalytic cleft. Some of the ligand binding residues and metal ion binding residues were found to have high ASA values (Pro109, His156, Gly161, Cys179, Cys181, Cys186) and some others were found to have low ASA values (Val77, Asp78, Glu103, Asn114, Ala116, Asn117, Gly146, His168). Residues with high ASA values are on the surface of the cleft and those with low values are buried inside.

Conclusion
The structure of S. pneumoniae GLK is important for establishing its molecular function. However, a three dimensional structure is not available as yet at PDB. We developed a homology model for S. pneumoniae GLK using MODELLER. The model was further analyzed for residue solvent accessibility in establishing its molecular function. Solvent accessible surface area (ASA) analysis of the GLK model showed that known key residues playing important role in active site for ligand binding and metal ion binding are buried and not accessible to solvent. The analysis highlights the importance of solvent exposed catalytic residues in molecular function.