ABSTRACT
Aminotransferases of subfamily Iß, which include histidinol phosphate aminotransferases (HspATs) and aromatic amino acid aminotransferases (ArATs), are structurally similar but possess distinct substrate specificities. This study, encompassing structural and biochemical characterisation of HspAT and ArAT from Mycobacterium tuberculosis demonstrates that the residues lining the substrate binding pocket and N-terminal lid are the primary determinants of their substrate specificities. In mHspAT, hydrophilic residues in the substrate binding pocket and N-terminal lid allow the entry and binding of its preferential substrate, Hsp. On the other hand, the hydrophobic nature of both the substrate binding pocket and the N-terminal lid of mArAT is responsible for the discrimination of a polar substrate such as Hsp, while facilitating the binding of Phe and other aromatic residues such as Tyr and Trp. In addition, the present study delineates the ligand induced conformational rearrangements, providing insights into the plasticity of aminotransferases. Furthermore, the study also demonstrates that the adventitiously bound ligand 2-(N-morpholino)ethanesulfonic acid (MES) is indeed a specific inhibitor of HspAT. These results suggest that previously untapped morpholine-ring scaffold compounds could be explored for the design of new anti-TB agents.
Subject(s)
Bacterial Proteins/chemistry , Mycobacterium tuberculosis/enzymology , Transaminases/chemistry , Alkanesulfonic Acids/chemistry , Amino Acid Sequence , Bacterial Proteins/antagonists & inhibitors , Catalytic Domain , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Kinetics , Models, Molecular , Morpholines/chemistry , Phenylalanine/chemistry , Protein Binding , Protein Conformation, alpha-Helical , Structural Homology, Protein , Substrate Specificity , Succinic Acid/chemistry , Transaminases/antagonists & inhibitorsABSTRACT
A recombinant version of a putative aspartate aminotransferase, AspB (encoded by the ORF Rv3565), from Mycobacterium tuberculosis (Mtb) was overexpressed in M. smegmatis and purified to homogeneity using liquid chromatography. Crystals of AspB were grown in a condition consisting of 0.2â M ammonium phosphate monobasic, 0.1â M calcium chloride dihydrate employing the hanging-drop vapour-diffusion method at 298â K. The crystals diffracted to a limit of 2.50â Å resolution and belonged to the orthorhombic space group P212121, with unit-cell parameters a=93.27, b=98.19, c=198.70â Å. The structure of AspB was solved by the molecular-replacement method using a putative aminotransferase from Silicibacter pomeroyi (PDB entry 3h14) as the search model. The template shares 46% amino-acid sequence identity with Mtb AspB. The crystal asymmetric unit contains four AspB molecules (the Mr of each is 42,035â Da).
Subject(s)
Aspartate Aminotransferases/chemistry , Bacterial Proteins/chemistry , Mycobacterium tuberculosis/chemistry , Amino Acid Sequence , Aspartate Aminotransferases/genetics , Aspartate Aminotransferases/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cloning, Molecular , Crystallization , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Molecular Sequence Data , Mycobacterium smegmatis/genetics , Mycobacterium smegmatis/metabolism , Mycobacterium tuberculosis/enzymology , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Structural Homology, ProteinABSTRACT
In the present study, comparative genome analysis between Clostridium perfringens and the human genome was carried out to identify genes that are essential for the pathogen's survival, and non-homologous to the genes of human host, that can be used as potential drug targets. The study resulted in the identification of 426 such genes. The number of these potential drug targets thus identified is significantly lower than the genome's protein coding capacity (2558 protein coding genes). The 426 genes of C. perfringens were further analyzed for overall similarities with the essential genes of 14 different bacterial species present in Database of Essential Genes (DEG). Our results show that there are only 5 essential genes of C. perfringens that exhibit similarity with 12 species of the 14 different bacterial species present in DEG database. Of these, 1 gene was similar in 12 species and 4 genes were similar in 11 species. Thus, the study opens a new avenue for the development of potential drugs against the highly pathogenic bacterium. Further, by selecting these essential genes of C. perfringens, which are common and essential for other pathogenic microbial species, a broad spectrum anti-microbial drug can be developed. As a case study, we have built a homology model of one of the potential drug targets, ABC transporter-ATP binding protein, which can be employed for in silico docking studies by suitable inhibitors.
ABSTRACT
Phosphoglucose isomerase (PGI) plays a key role in both glycolysis and gluconeogenesis inside the cell, whereas outside the cell it exhibits cytokine properties. PGI is also known to act as an autocrine motility factor, a neuroleukin agent and a differentiation and maturation mediator. Here, the first crystal structure of PGI from Mycobacterium tuberculosis H37Rv (Mtb) is reported. The structure was refined at 2.25 A resolution and revealed the presence of one molecule in the asymmetric unit with two globular domains. As known previously, the active site of Mtb PGI contains conserved residues including Glu356, Glu216 and His387 (where His387 is from the neighbouring molecule). The crystal structure of Mtb PGI was observed to be rather more similar to human PGI than other nonbacterial PGIs, with only a few differences being detected in the loops, arm and hook regions of the human and Mtb PGIs, suggesting that the M. tuberculosis enzyme uses the same enzyme mechanism.
