Rupam Biswas | Indian Institute of Technology Kharagpur (original) (raw)

Papers by Rupam Biswas

FabG belongs to the short-chain dehydrogenase/reductase (SDR) family. As a part of fatty acid elo... more FabG belongs to the short-chain dehydrogenase/reductase (SDR) family. As a part of fatty acid elongation cycle, the
enzyme executes the 2nd step of the cycle by reducing β-ketoacyl-acyl carrier protein using NADPH cofactor. The insolution
oligomeric organisations of FabGs tend to differ in various organisms although the crystal structures are
mainly tetrameric. Importantly, this oligomerization propensity is crucial because it directs enzymatic activation upon
cofactor binding and substrate cooperativity. Here, the aim of our work is to find out the underlying molecular basis that
switches the different oligomeric conformations of FabGs as well as how it is associated with the presence of cofactor
and substrate. To establish our proposition a chimeric FabG has been designed by using domain swapping strategy.
Our data suggest that the substitution of a modified P-interface can alter the dimer-tetramer equilibrium in FabGs.
Overall, the study provides insights into the structural dynamics of FabGs and allows us to understand the key
structural features that regulate oligomeric conformation as well as the substrate, and cofactor binding.

FabGs, or β-oxoacyl reductases, are involved in fatty acid synthesis. The reaction entails NADPH/... more FabGs, or β-oxoacyl reductases, are involved in fatty acid synthesis. The reaction entails NADPH/NADH-mediated conversion of β-oxoacyl-ACP (acyl-carrier protein) into βhydroxyacyl-ACP. HMwFabGs (high-molecular-weight FabG) form a phylogenetically separate group of FabG enzymes. FabG4, an HMwFabG from Mycobacterium tuberculosis, contains two distinct domains, an N-terminal 'flavodoxintype' domain and a C-terminal oxoreductase domain. The catalytically active C-terminal domain utilizes NADH to reduce β-oxoacyl-CoA to β-hydroxyacyl-CoA. In the present study the crystal structures of the FabG4-NADH binary complex and the FabG4-NAD + -hexanoyl-CoA ternary complex have been determined to understand the substrate specificity and catalytic mechanism of FabG4. This is the first report to demonstrate how FabG4 interacts with its coenzyme NADH and hexanoyl-CoA that mimics an elongating fattyacyl chain covalently linked with CoA. Structural analysis shows that the binding of hexanoyl-CoA within the active site cavity of FabG significantly differs from that of the C 16 fattyacyl substrate bound to mycobacterial FabI [InhA (enoyl-ACP reductase)]. The ternary complex reveals that both loop I and loop II interact with the phosphopantetheine moiety of CoA or ACP to align the covalently linked fattyacyl substrate near the active site. Structural data ACP inhibition studies indicate that FabG4 can accept both CoA-and ACP-based fattyacyl substrates. We have also shown that in the FabG4 dimer Arg 146 and Arg 445 of one monomer interact with the C-terminus of the second monomer to play pivotal role in substrate association and catalysis.

Secretary proteins of Mycobacterium tuberculosis are key players of the mycobacterial infection p... more Secretary proteins of Mycobacterium tuberculosis are key players of the mycobacterial infection pathway. MTC28 is a 28-kDa proline-rich secretary antigen of Mycobacterium tuberculosis and is only conserved in pathogenic strains of mycobacteria. Here we report the crystal structure of MTC28 at 2.8-and 2.15-Å resolutions for the structure-based epitope design. MTC28 shares a "mog1p"-fold consisting of seven antiparallel ␤ strands stacked between ␣ helices. Five probable epitopes have been located on a solvent-accessible flexible region by computational analysis of the structure of MTC28. Simultaneously, the protein is digested with trypsin and the resulting fragments are purified by HPLC. Such 10 purified peptide fragments are screened against sera from patients infected with pulmonary tuberculosis (PTB). Two of these 10 fragments, namely 127 ALDITLPMPPR 137 and 138 WTQVPDPNVPDAFVVIADR 156 , are found to be major immunogenic epitopes that are localized on the outer surface of the protein molecule and are part of a single continuous epitope that have been predicted in silico. Mutagenesis and antibody inhibition studies are in accordance with the results obtained from epitope mapping.

Herein, we present dual inhibitors of new targets FabG4 and HtdX for the first time. In this work... more Herein, we present dual inhibitors of new targets FabG4 and HtdX for the first time. In this work, eight compounds have been designed, synthesized, characterized and evaluated for bio-activities. Amongst them, six compounds have shown inhibitory activities. Three of them (12e14) demonstrate dual inhibition of both FabG4 and HtdX at low micromolar concentration. In addition, the dual inhibitors show good anti-mycobacterial properties against both planktonic growth and biofilm culture of Mycobacterium species. This study is an important addition to tuberculosis drug discovery because it explores two new enzymes as drug targets and presents their dual inhibitors as good candidates for pre-clinical trials.

Herein we report six novel triazole linked polyphenol-aminobenzene hybrids (3-8) as inhibitors of... more Herein we report six novel triazole linked polyphenol-aminobenzene hybrids (3-8) as inhibitors of Mycobacterium tuberculosis FabG4 (Rv0242c), a less explored b-ketoacyl CoA reductase that has immense potential to be the future anti-tuberculosis drug target due to its possible involvement in drug resistance and latent infection. Novel triazole linked polyphenol-aminobenzene hybrids have been synthesized, characterized and evaluated for their inhibitory activity against FabG4. All of them inhibit FabG4 at low micromolar concentrations. In silico docking study has been carried out to explain the experimental findings. A comparative study of these new inhibitors with previously reported gallate counterparts leads to structure-activity relations (SAR) of substituent linked to N-1 of triazole ring.

Fatty acid biosynthesis type II in mycobacteria delivers the fatty acids required for mycolic aci... more Fatty acid biosynthesis type II in mycobacteria delivers the fatty acids required for mycolic acid synthesis.

Rv0241c (HtdX) is a putative (3R)-hydroxyacyl-CoA dehydratase of Mycobacterium tuberculosis. The ... more Rv0241c (HtdX) is a putative (3R)-hydroxyacyl-CoA dehydratase of Mycobacterium tuberculosis. The htdX gene belongs to a conserved operon and is expressed in mycobacteria in the presence of several fatty-acid synthase II drugs. To elucidate the structure of HtdX, the protein was cloned, overexpressed, purified to homogeneity and crystallized. The protein was crystallized from two conditions: (i) 3 M sodium chloride, 0.1 M Na HEPES pH 8.0 and (ii) 2.5 M sodium chloride, 0.1 M Tris-HCl pH 8.5. A complete diffraction data set was collected from crystals from both conditions. The crystal from the first condition diffracted to 2.3 Å resolution and belonged to space group I4 1 , with unit-cell parameters a = b = 61.51, c = 143.81 Å . Crystals from the second condition diffracted to 3.

FabG belongs to the short-chain dehydrogenase/reductase (SDR) family. As a part of fatty acid elo... more FabG belongs to the short-chain dehydrogenase/reductase (SDR) family. As a part of fatty acid elongation cycle, the
enzyme executes the 2nd step of the cycle by reducing β-ketoacyl-acyl carrier protein using NADPH cofactor. The insolution
oligomeric organisations of FabGs tend to differ in various organisms although the crystal structures are
mainly tetrameric. Importantly, this oligomerization propensity is crucial because it directs enzymatic activation upon
cofactor binding and substrate cooperativity. Here, the aim of our work is to find out the underlying molecular basis that
switches the different oligomeric conformations of FabGs as well as how it is associated with the presence of cofactor
and substrate. To establish our proposition a chimeric FabG has been designed by using domain swapping strategy.
Our data suggest that the substitution of a modified P-interface can alter the dimer-tetramer equilibrium in FabGs.
Overall, the study provides insights into the structural dynamics of FabGs and allows us to understand the key
structural features that regulate oligomeric conformation as well as the substrate, and cofactor binding.

FabGs, or β-oxoacyl reductases, are involved in fatty acid synthesis. The reaction entails NADPH/... more FabGs, or β-oxoacyl reductases, are involved in fatty acid synthesis. The reaction entails NADPH/NADH-mediated conversion of β-oxoacyl-ACP (acyl-carrier protein) into βhydroxyacyl-ACP. HMwFabGs (high-molecular-weight FabG) form a phylogenetically separate group of FabG enzymes. FabG4, an HMwFabG from Mycobacterium tuberculosis, contains two distinct domains, an N-terminal 'flavodoxintype' domain and a C-terminal oxoreductase domain. The catalytically active C-terminal domain utilizes NADH to reduce β-oxoacyl-CoA to β-hydroxyacyl-CoA. In the present study the crystal structures of the FabG4-NADH binary complex and the FabG4-NAD + -hexanoyl-CoA ternary complex have been determined to understand the substrate specificity and catalytic mechanism of FabG4. This is the first report to demonstrate how FabG4 interacts with its coenzyme NADH and hexanoyl-CoA that mimics an elongating fattyacyl chain covalently linked with CoA. Structural analysis shows that the binding of hexanoyl-CoA within the active site cavity of FabG significantly differs from that of the C 16 fattyacyl substrate bound to mycobacterial FabI [InhA (enoyl-ACP reductase)]. The ternary complex reveals that both loop I and loop II interact with the phosphopantetheine moiety of CoA or ACP to align the covalently linked fattyacyl substrate near the active site. Structural data ACP inhibition studies indicate that FabG4 can accept both CoA-and ACP-based fattyacyl substrates. We have also shown that in the FabG4 dimer Arg 146 and Arg 445 of one monomer interact with the C-terminus of the second monomer to play pivotal role in substrate association and catalysis.

Secretary proteins of Mycobacterium tuberculosis are key players of the mycobacterial infection p... more Secretary proteins of Mycobacterium tuberculosis are key players of the mycobacterial infection pathway. MTC28 is a 28-kDa proline-rich secretary antigen of Mycobacterium tuberculosis and is only conserved in pathogenic strains of mycobacteria. Here we report the crystal structure of MTC28 at 2.8-and 2.15-Å resolutions for the structure-based epitope design. MTC28 shares a "mog1p"-fold consisting of seven antiparallel ␤ strands stacked between ␣ helices. Five probable epitopes have been located on a solvent-accessible flexible region by computational analysis of the structure of MTC28. Simultaneously, the protein is digested with trypsin and the resulting fragments are purified by HPLC. Such 10 purified peptide fragments are screened against sera from patients infected with pulmonary tuberculosis (PTB). Two of these 10 fragments, namely 127 ALDITLPMPPR 137 and 138 WTQVPDPNVPDAFVVIADR 156 , are found to be major immunogenic epitopes that are localized on the outer surface of the protein molecule and are part of a single continuous epitope that have been predicted in silico. Mutagenesis and antibody inhibition studies are in accordance with the results obtained from epitope mapping.

Herein, we present dual inhibitors of new targets FabG4 and HtdX for the first time. In this work... more Herein, we present dual inhibitors of new targets FabG4 and HtdX for the first time. In this work, eight compounds have been designed, synthesized, characterized and evaluated for bio-activities. Amongst them, six compounds have shown inhibitory activities. Three of them (12e14) demonstrate dual inhibition of both FabG4 and HtdX at low micromolar concentration. In addition, the dual inhibitors show good anti-mycobacterial properties against both planktonic growth and biofilm culture of Mycobacterium species. This study is an important addition to tuberculosis drug discovery because it explores two new enzymes as drug targets and presents their dual inhibitors as good candidates for pre-clinical trials.

Herein we report six novel triazole linked polyphenol-aminobenzene hybrids (3-8) as inhibitors of... more Herein we report six novel triazole linked polyphenol-aminobenzene hybrids (3-8) as inhibitors of Mycobacterium tuberculosis FabG4 (Rv0242c), a less explored b-ketoacyl CoA reductase that has immense potential to be the future anti-tuberculosis drug target due to its possible involvement in drug resistance and latent infection. Novel triazole linked polyphenol-aminobenzene hybrids have been synthesized, characterized and evaluated for their inhibitory activity against FabG4. All of them inhibit FabG4 at low micromolar concentrations. In silico docking study has been carried out to explain the experimental findings. A comparative study of these new inhibitors with previously reported gallate counterparts leads to structure-activity relations (SAR) of substituent linked to N-1 of triazole ring.

Fatty acid biosynthesis type II in mycobacteria delivers the fatty acids required for mycolic aci... more Fatty acid biosynthesis type II in mycobacteria delivers the fatty acids required for mycolic acid synthesis.

Rv0241c (HtdX) is a putative (3R)-hydroxyacyl-CoA dehydratase of Mycobacterium tuberculosis. The ... more Rv0241c (HtdX) is a putative (3R)-hydroxyacyl-CoA dehydratase of Mycobacterium tuberculosis. The htdX gene belongs to a conserved operon and is expressed in mycobacteria in the presence of several fatty-acid synthase II drugs. To elucidate the structure of HtdX, the protein was cloned, overexpressed, purified to homogeneity and crystallized. The protein was crystallized from two conditions: (i) 3 M sodium chloride, 0.1 M Na HEPES pH 8.0 and (ii) 2.5 M sodium chloride, 0.1 M Tris-HCl pH 8.5. A complete diffraction data set was collected from crystals from both conditions. The crystal from the first condition diffracted to 2.3 Å resolution and belonged to space group I4 1 , with unit-cell parameters a = b = 61.51, c = 143.81 Å . Crystals from the second condition diffracted to 3.