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 Zn2+ and Mg2+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.
The protein sequence (320 residues long) for GLK (E.C.18.104.22.168, 320aa) from S. pneumonaie was retrieved from GenBank
FASTA format. The basic local alignment search tool (BLAST) A search at PDB for S. pneumonaie GLK identified a template
structure (PDB code: 2QM1 wih 2.02 Å resolution) E. faecalis with 65% identity
(). Sequence alignment () revealed that the
active site amino acid residue Asp114 (represented in pink) in the template was conserved in S. pneumonaie Glk (Asp115).
The ligand binding residues in the template Val76, Asp77, Glu102, Pro108, H155 and Gly159 (represented in green i) were also conserved
both in target and template (Val77, Asp78, Glu103, Pro109, His 156 and Gly161. The metal ion interacting conserved residues are Asn113,
Ala115, Asn116, Gly145, His166, Cys176, Cys178 and Cys183.
Figure 1 (a) Pairwise alignment of GLK from S. pneumoniae and the template PDB ID: 2QM1 from E. feacalis. Dash
represents insertion and deletion: conserved residues which are not involved in active site, ligand binding and metal binding
are shaded in blue. Conserved (more ...)
Homology modeling and structure refinement
The homology model of S. pneumonaie GLK was built using MODELLER. We then incorporated the Mg2+ ion
and Zn2+ ion from the template structure into the modeled structure. The atomic charges of the amino acid side chains
involved in Mg2+ ion and Zn2+ ion coordination system is similar to those of the template. Structural refinement
through energy minimization model was performed using spdbv.
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
(). 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.
Accessible surface area (ASA) analysis of the predicted GLK model showed the active site amino acid Asp115 with zero ASA value
() 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.