Quinolone synthase from Aegle marmelos (AmQNS) is a type III polyketide synthase that yields therapeutically effective quinolone and acridone compounds. Addressing the structural and molecular underpinnings of AmQNS and its substrate interaction in terms of its high selectivity and specificity can aid in the development of numerous novel compounds. This paper presents a high-resolution AmQNS crystal structure and explains its mechanistic role in synthetic selectivity. Additionally, we provide a model framework to comprehend structural constraints on ketide insertion and postulate that AmQNS's steric and electrostatic selectivity plays a role in its ability to bind to various core substrates, resulting in its synthetic diversity. AmQNS prefers quinolone synthesis and can accommodate large substrates because of its wide active site entrance. However, our research suggests that acridone is exclusively synthesized in the presence of high malonyl-CoA concentrations. Potential implications of functionally relevant residue mutations were also investigated, which will assist in harnessing the benefits of mutations for targeted polyketide production. The pharmaceutical industry stands to gain from these findings as they expand the pool of potential drug candidates, and these methodologies can also be applied to additional promising enzymes. A high-resolution structure provides a mechanistic explanation to the synthetic selectivity of quinolone synthase (AmQNS) and possible consequences of functionally relevant residue mutations.

Quinolone synthase from Aegle marmelos (AmQNS) is a type III polyketide synthase that yields therapeutically effective quinolone and acridone compounds. Based on the high-resolution protein structure of AmQNS, this study provided a mechanistic explanation of the structure to synthetic selectivity. Additionally, it displays the comparatively wide active site entry that allows the catalytic pocket to accommodate bulky substrates, which affects the enzyme catalysis. We also develop a model framework for comprehending the structural constraints on ketide insertion, and postulate that AmQNS synthetic diversity is owing to its steric and electrostatic selectivity, which allows it to bind to a variety of core substrates. We further establish that AmQNS is structurally biased toward quinolone synthesis and only synthesizes acridone when malonyl-CoA concentrations are significantly high. In a nutshell, we anticipate that addressing the structural and molecular underpinnings of AmQNS–substrate interaction in terms of its high selectivity and specificity can aid in the development of numerous novel compounds. Besides, the approaches can also be expanded to other potential enzymes, which will help the pharmaceutical sector by expanding the pool of potential medication leads.

Crystal structures of jacalin in complex with GlcNAc beta-(1,3) Gal-beta-OMe and Gal beta-(1,3) Gal-beta-OMe have been determined. The binding of the ligands to jacalin is similar to that of analogous -substituted disaccharides. However, the beta-substituted beta-(1,3) linked disaccharides get distorted at the anomeric center and the glycosidic linkage. The distortion results in higher internal energies of the ligands leading to lower affinity to the lectin. This confirms the possibility of using ligand distortion as a strategy for modulating binding affinity. Unlike in the case of alpha-substituted monosaccharides bound to jacalin, where a larger distortion at the anomeric center was observed, smaller distortions are distributed among two centers in the structures of the two beta-substituted beta-(1,3) linked disaccharides presented here. These disaccharides, like the unsubstituted and alpha-substituted counterparts, bind jacalin with the reducing Gal at the primary binding site, indicating that the lower binding affinity of beta-substituted disaccharides is not enough to overcome the intrinsic propensity of Gal -(1,3) Gal-based disaccharides to bind jacalin with the reducing sugar at the primary site. (C) 2017 IUBMB Life, 69(2): 72-78, 2017.

A lectin from Methanococcus voltae A3 has been cloned, expressed, purified and characterized. The lectin appears to be specific for complex sugars. The protein crystallized in a tetragonal space group, with around 16 subunits in the asymmetric unit. Sequence comparisons indicate the lectin to have a β-prism I fold, with poor homology to lectins of known three-dimensional structure.

The crystal structures of jacalin complexed with Gal alpha-(1,4) Gal and Gal alpha-(1,3) Gal beta-(1,4) Gal have been determined with the primary objective of exploring the effect of linkage on the location of reducing and non-reducing sugars in the extended binding site of the lectin, an issue which has not been studied thoroughly. Contrary to the earlier surmise based on simple steric considerations, the two structures demonstrate that alpha-linked sugars can bind to jacalin with nonreducing sugar at the primary binding site. This is made possible substantially on account of the hitherto underestimated plasticity of a non-polar region of the extended binding site. Modeling studies involving conformational search and energy minimization, along with available crystallographic and thermodynamic data, indicate a strong preference for complexation with Gal beta-(1,3) Gal with the reducing Gal at the primary site, followed by that with Gal alpha-(1,3) Gal, with the reducing or non-reducing Gal located at the primary binding site. This observation is in consonance with the facility of jacalin to bind mucin type O-glycans containing T-antigen core. (C) 2016 IUBMB Life, 68(12): 971-979, 2016

This thesis consists of two parts. The first part is concerned with further structural and related studies of jacalin, one of the two lectins found in jack fruit seeds. The second part deals with the search of mycobacterial and archeal genomes for lectins. The β-prism I fold was identified as a lectin fold through the X-ray analysis of jacalin way back in 1996. Subsequent structural studies on jacalin are described in the first chapter in context of the overall efforts on lectins with particular reference to those on lectins with β-prism I fold. The structure of jacalin has been thoroughly characterized through the analysis of several crystals. The extended binding site of the lectin, made up of the primary binding site and secondary sites A and B, has also been characterized through studies on different jacalin-sugar complexes. However, nuances of jacalin-carbohydrate interactions remain underexplored with respect to two specific issues. The first issue is concerned with the structural basis for the lower affinity of jacalin for β-substituted sugars. The second has to do with the influence of the anomeric nature of the glycosidic linkage on the location of the reducing and non-reducing sugars in disaccharides when interacting with jacalin. Part of the work described in the thesis addresses these two issues. It was surmised that the lower affinity of β-galactosides to jacalin as compared to α-galactosides, is caused by steric interactions of the substituents in the former with the protein. This issue is explored both energetically and structurally in Chapter 2 using appropriately derivatized monosaccharide complexes of jacalin. It turns out that the earlier surmise is not correct. The interactions of the substituent with the binding site remain essentially the same irrespective of the anomeric nature of the substitution. This is achieved through a distortion of the sugar ring in β-galactosides. The difference in energy, and therefore affinity, is caused by the distortion of the sugar ring in β-galactosides. The elucidation of this unprecedented distortion of the ligand as a strategy for modulating affinity is of general interest. The crystal structures also provide a rationale for the relative affinities of the different carbohydrate ligands to jacalin. The crystal structures of jacalin complexed with α-linked oligosaccharides Gal α-(1,4) Gal and Gal α-(1,3) Gal β-(1,4) Gal, as described in Chapter 3, have been determined with the primary objective of exploring the effect of linkage on the location of reducing and non-reducing sugars in the extended binding site of the lectin, an issue which has not been studied thoroughly. Contrary to the earlier surmise based on simple steric considerations, the two structures demonstrate that α-linked sugars can bind to jacalin with non-reducing sugar at the primary binding site. This is made possible substantially on account of the hitherto underestimated plasticity of a non-polar region of the extended binding site. Modelling studies involving conformational search and…

Jacalin is among the most thoroughly studied lectins. Its carbohydrate-binding site has also been well characterized. It has been postulated that the lower affinity of β-galactosides for jacalin compared with α-galactosides is caused by steric interactions of the substituents in the former with the protein. This issue has been explored energetically and structurally using different appropriate carbohydrate complexes of jacalin. It turns out that the earlier postulation is not correct. The interactions of the substituent with the binding site remain essentially the same irrespective of the anomeric nature of the substitution. This is achieved through a distortion of the sugar ring in β-galactosides. The difference in energy, and therefore in affinity, is caused by a distortion of the sugar ring in β-galactosides. The elucidation of this unprecedented distortion of the ligand as a strategy for modulating affinity is of general interest. The crystal structures also provide a rationale for the relative affinities of the different carbohydrate ligands for jacalin.

Forty-six lectin domains which have homologues among well established eukaryotic and bacterial lectins of known three-dimensional structure, have been identified through a search of 165 archeal genomes using a multipronged approach involving domain recognition, sequence search and analysis of binding sites. Twenty-one of them have the 7-bladed β-propeller lectin fold while 16 have the β-trefoil fold and 7 the legume lectin fold. The remainder assumes the C-type lectin, the β-prism I and the tachylectin folds. Acceptable models of almost all of them could be generated using the appropriate lectins of known three-dimensional structure as templates, with binding sites at one or more expected locations. The work represents the first comprehensive bioinformatic study of archeal lectins. The presence of lectins with the same fold in all domains of life indicates their ancient origin well before the divergence of the three branches. Further work is necessary to identify archeal lectins which have no homologues among eukaryotic and bacterial species. Proteins 2016; 84:21–30. © 2015 Wiley Periodicals, Inc.

AbstractStructural studies in this laboratory encompass four of the five major classes of plant lectins, including the one discovered by us. In addition to addressing issues specific to individual lectins, the work provided insights into protein folding, quaternary association and generation of ligand specificity. Legume and β-prism fold lectins constitute families of proteins in which small alterations in essentially the same tertiary structure lead to large variations in quaternary structure, including that involving an open structure. Strategies for generating ligand specificity include water bridges, variation in loop length, post translational modification and oligomerization. Three of the structural classes investigated have subunits with three-fold symmetry. The symmetry in the structure is reflected in the sequence to different extents in different sub-classes. The evolutionary implications of this observation have been explored. The work on lectins has now been extended to those from mycobacteria.

Sixty-four sequences containing lectin domains with homologs of known three-dimensional structure were identified through a search of mycobacterial genomes. They appear to belong to the β-prism II, the C-type, the Microcystis virdis (MV), and the β-trefoil lectin folds. The first three always occur in conjunction with the LysM, the PI-PLC, and the β-grasp domains, respectively while mycobacterial β-trefoil lectins are unaccompanied by any other domain. Thirty heparin binding hemagglutinins (HBHA), already annotated, have also been included in the study although they have no homologs of known three-dimensional structure. The biological role of HBHA has been well characterized. A comparison between the sequences of the lectin from pathogenic and nonpathogenic mycobacteria provides insights into the carbohydrate binding region of the molecule, but the structure of the molecule is yet to be determined. A reasonable picture of the structural features of other mycobacterial proteins containing one or the other of the four lectin domains can be gleaned through the examination of homologs proteins, although the structure of none of them is available. Their biological role is also yet to be elucidated. The work presented here is among the first steps towards exploring the almost unexplored area of the structural biology of mycobacterial lectins. Proteins 2013. © 2012 Wiley Periodicals, Inc.

Previous studies of complexes of Mycobacterium tuberculosis PanK (MtPanK) with nucleotide diphosphates and nonhydrolysable analogues of nucleoside triphosphates in the presence or the absence of pantothenate established that the enzyme has dual specificity for ATP and GTP, revealed the unusual movement of ligands during enzyme action and provided information on the effect of pantothenate on the location and conformation of the nucleotides at the beginning and the end of enzyme action. The X-ray analyses of the binary complexes of MtPanK with pantothenate, pantothenol and N-nonylpantothenamide reported here demonstrate that in the absence of nucleotide these ligands occupy, with a somewhat open conformation, a location similar to that occupied by phosphopantothenate in the `end' complexes, which differs distinctly from the location of pantothenate in the closed conformation in the ternary `initiation' complexes. The conformation and the location of the nucleotide were also different in the initiation and end complexes. An invariant arginine appears to play a critical role in the movement of ligands that takes place during enzyme action. The work presented here completes the description of the locations and conformations of nucleoside diphosphates and triphosphates and pantothenate in different binary and ternary complexes, and suggests a structural rationale for the movement of ligands during enzyme action. The present investigation also suggests that N-alkylpantothenamides could be phosphorylated by the enzyme in the same manner as pantothenate.