Synthesis of R-GABA Derivatives via Pd(II) Catalyzed Enantioselective C(sp3)-H Arylation and Virtual Validation with GABAB1 Receptor for Potential leads.

GABA (γ-amino butyric acid) analogues like baclofen, tolibut, phenibut, etc., are well-known GABAB1 inhibitors and pharmaceutically important drugs. However, there is a huge demand for more chiral GABA aryl analogues with promising pharmacological actions. Here, we demonstrate the chiral ligand acetyl-protected amino quinoline (APAQ) mediated enantioselective synthesis of GABAB1 inhibitor drug scaffolds from easily accessible GABA via Pd-catalyzed C(sp3)-H activation. The synthetic methodology shows moderate to good yields, up to 74% of ee. We have successfully demonstrated the deprotection and removal of the directing group to synthesize R-tolibut in 86% yield. Further, we employed computation to probe the binding of R-GABA analogues to the extracellular domain of the human GABAB1 receptor. Our Rosetta-based molecular docking calculations show better binding for four R-enantiomers of GABA analogues than R-baclofen and R-phenibut. In addition, we employed GROMACS MD simulations and MMPB(GB)SA calculations to identify per-residue contribution to binding free energy. Our computational results suggest analogues (3R)-4-amino-3-(3,4-dimethylphenyl) butanoic acid, (3R)-4-amino-3-(3-fluorophenyl) butanoic acid, (3R)-3-(4-acetylphenyl)-4-aminobutanoic acid, (3R)-4-amino-3-(4-methoxyphenyl) butanoic acid, and (3R)-4-amino-3-phenylbutanoic acid are potential leads which could be synthesized from our methodology reported here.

Structural Analyses of Substrate-pH Activity Pairing Observed across Diverse Polysaccharide Lyases.

Anionic polysaccharides found in nature are functionally and structurally diverse, and so are the polysaccharide lyases (PLs) that catalyze their degradation. Atomic superposition of various PL folds according to their cleavable substrate structure confirms the occurrence of structural convergence at PL active sites. This suggests that various PL folds have emerged to cleave a particular class of anionic polysaccharide during the course of evolution. Whereas the structural and mechanistic similarity of PL active site has been highlighted in earlier studies, a detailed understanding regarding functional properties of this catalytic convergence remains an open question, especially the role of extrinsic factors such as pH in the context of substrate binding and catalysis. Our earlier structural and functional work on pH directed multisubstrate specificity of Smlt1473 inspired us to regroup PLs according to substrate type to analyze the pH dependence of their catalytic activity. Interestingly, we find that particular groups of substrates are cleaved in a particular pH range (acidic/neutral/basic) irrespective of PL fold, boosting the idea of functional convergence as well. On the basis of this observation, we set out to define structurally and computationally the key constituents of an active site among PL families. This study delineates the structural determinants of conserved "substrate-pH activity pairing" within and between PL families.

Structural insights into the mechanism of pH-selective substrate specificity of the polysaccharide lyase Smlt1473.

Polysaccharide lyases (PLs) are a broad class of microbial enzymes that degrade anionic polysaccharides. Equally broad diversity in their polysaccharide substrates has attracted interest in biotechnological applications such as biomass conversion to value-added chemicals and microbial biofilm removal. Unlike other PLs, Smlt1473 present in the clinically relevant Stenotrophomonas maltophilia strain K279a demonstrates a wide range of pH-dependent substrate specificities toward multiple, diverse polysaccharides: hyaluronic acid (pH 5.0), poly-ß-D-glucuronic (celluronic) acid (pH 7.0), poly-ß-D-mannuronic acid, and poly-α-L-guluronate (pH 9.0). To decode the pH-driven multiple substrate specificities and selectivity in this single enzyme, we present the X-ray structures of Smlt1473 determined at multiple pH values in apo and mannuronate-bound states as well as the tetra-hyaluronate-docked structure. Our results indicate that structural flexibility in the binding site and N-terminal loop coupled with specific substrate stereochemistry facilitates distinct modes of entry for substrates having diverse charge densities and chemical structures. Our structural analyses of wild-type apo structures solved at different pH values (5.0-9.0) and pH-trapped (5.0 and 7.0) catalytically relevant wild-type mannuronate complexes (1) indicate that pH modulates the catalytic microenvironment for guiding structurally and chemically diverse polysaccharide substrates, (2) further establish that molecular-level fluctuation in the enzyme catalytic tunnel is preconfigured, and (3) suggest that pH modulates fluctuations resulting in optimal substrate binding and cleavage. Furthermore, our results provide key insight into how strategies to reengineer both flexible loop and regions distal to the active site could be developed to target new and diverse substrates in a wide range of applications.

Leishmania major biotin protein ligase forms a unique cross-handshake dimer.

Biotin protein ligase catalyses the post-translational modification of biotin carboxyl carrier protein (BCCP) domains, a modification that is crucial for the function of several carboxylases. It is a two-step process that results in the covalent attachment of biotin to the ϵ-amino group of a conserved lysine of the BCCP domain of a carboxylase in an ATP-dependent manner. In Leishmania, three mitochondrial enzymes, acetyl-CoA carboxylase, methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase, depend on biotinylation for activity. In view of the indispensable role of the biotinylating enzyme in the activation of these carboxylases, crystal structures of L. major biotin protein ligase complexed with biotin and with biotinyl-5'-AMP have been solved. L. major biotin protein ligase crystallizes as a unique dimer formed by cross-handshake interactions of the hinge region of the two monomers formed by partial unfolding of the C-terminal domain. Interestingly, the substrate (BCCP domain)-binding site of each monomer is occupied by its own C-terminal domain in the dimer structure. This was observed in all of the crystals that were obtained, suggesting a closed/inactive conformation of the enzyme. Size-exclusion chromatography studies carried out using high protein concentrations (0.5 mM) suggest the formation of a concentration-dependent dimer that exists in equilibrium with the monomer.

Spectroscopic Evidences for Strong Hydrogen Bonds with Selenomethionine in Proteins.

Careful protein structure analysis unravels many unknown and unappreciated noncovalent interactions that control protein structure; one such unrecognized interaction in protein is selenium centered hydrogen bonds (SeCHBs). We report, for the first time, SeCHBs involving the amide proton and selenium of selenomethionine (Mse), i.e., amide-N-H···Se H-bonds discerned in proteins. Using mass selective and conformer specific high resolution vibrational spectroscopy, gold standard quantum chemical calculations at CCSD(T), and in-depth protein structure analysis, we establish that amide-N-H···Se and amide-N-H···Te H-bonds are as strong as conventional amide-NH···O and amide-NH···O═C H-bonds despite smaller electronegativity of selenium and tellurium than oxygen. It is in fact, electronegativity, atomic charge, and polarizability of the H-bond acceptor atoms are at play in deciding the strength of H-bonds. The amide-N-H···Se and amide-N-H···Te H-bonds presented here are not only new additions to the ever expanding world of noncovalent interactions, but also are of central importance to design new force-fields for better biomolecular structure simulations.