K. J. Pampa

Crystal structure studies of NADP+ dependent isocitrate dehydrogenase from Thermus thermophilus exhibiting a novel terminal domain.

NADP(+) dependent isocitrate dehydrogenase (IDH) is an enzyme catalyzing oxidative decarboxylation of isocitrate into oxalosuccinate (intermediate) and finally the product α-ketoglutarate. The crystal structure of Thermus thermophilus isocitrate dehydrogenase (TtIDH) ternary complex with citrate and cofactor NADP(+) was determined using X-ray diffraction method to a resolution of 1.80 Å. The overall fold of this protein was resolved into large domain, small domain and a clasp domain. The monomeric structure reveals a novel terminal domain involved in dimerization, very unique and novel domain when compared to other IDHs. And, small domain and clasp domain showing significant differences when compared to other IDHs of the same sub-family. The structure of TtIDH reveals the absence of helix at the clasp domain, which is mainly involved in oligomerization in other IDHs. Also, helices/beta sheets are absent in the small domain, when compared to other IDHs of the same sub family. The overall TtIDH structure exhibits closed conformation with catalytic triad residues, Tyr144-Asp248-Lys191 are conserved. Oligomerization of the protein is quantized using interface area and subunit-subunit interactions between protomers. Overall, the TtIDH structure with novel terminal domain may be categorized as a first structure of subfamily of type IV.

2-Chloro-N-(4-methoxybenzoyl)benzene-sulfonamide

In the title compound, C14H12ClNO4S, the dihedral angle between the aromatic rings is 82.07 (1)° and the dihedral angle between the planes defined by the S—N—C=O fragment and the sulfonyl benzene ring is 82.46 (3)°. In the crystal, the molecules are linked into C(4) chains running along [001] by strong N—H⋯O hydrogen bonds. A C—H⋯O interaction reinforces the [001] chains: its graph-set symbol is C(7). The chains are cross-linked into (100) sheets by further C—H⋯O interactions as C(6) chains along [001]. The structure also features weak π–π stacking interactions [centroid–centroid distances = 3.577 (1) and 3.8016 (1) Å].

Purification, crystallization and preliminary X-ray diffraction studies of a putative UDP-N-acetyl-D-mannosamine dehydrogenase from Pyrococcus horikoshii OT3.

A putative UDP-N-acetyl-D-mannosamine dehydrogenase from Pyrococcus horikoshii OT3, an essential enzyme for polysaccharide biosynthesis, has been overexpressed in Escherichia coli and purified. Crystals were obtained using the oil-microbatch method at 291 K. A native data set extending to 1.8 A resolution has been collected and processed in space group P2(1). Assuming the presence of a dimer in the asymmetric unit, the V(M) value is calculated to be 2.3 A3 Da(-1), which is consistent with the result of a dynamic light-scattering experiment that shows a dimeric state of the protein in solution.

Synthesis, spectral, biological activity, and crystal structure evaluation of novel pyrazoline derivatives having sulfonamide moiety

In the present study, synthesis and characterization of pyrazoline derivatives integrated with sulfonamide scaffold have been performed. The characterization of the molecules was done by elemental analysis, ultraviolet–visible, infrared, nuclear magnetic resonance (NMR), and mass spectra. Crystal structure of compounds 2e and 2g were determined by single crystal X-ray diffraction. In the compounds 2e and 2g, the intra molecular hydrogen bonds N15--H15…O13 and N14--H14…N1 were closed to form a S(6) ring motif, whereas the N14--H14…O17 hydrogen bond links, pairs of molecules related by inversion, forming the familiar R22(10) ring motif. The Hirshfeld surface analysis comprising of the dnorm surface plots, electrostatic potentials and two-dimensional fingerprint plots were generated in order to give visual confirmation of the intermolecular interactions. The molecules were screened for their in vitro antitubercular and antimicrobial activity. The molecules 2n and 2m have shown high potent against M. tuberculosis and most of the molecules have shown good potential against different bacteria and fungi.

Synthesis, Characterization, Crystal Structure, and Hirshfeld Surface Analysis of 2–(2–Hydroxy–3–methoxyphenyl)–1–(4–methylphenyl)–4, 5–diphenyl–1H–imidazole

The substituted imidazole C29H24N2O2 was prepared via multicomponent reactions and the product crystallized using dimethylformamide. The structure of the compound was established by elemental analysis, FT-IR, thermogravimetric analysis, UV-Visible, and single-crystal X-ray diffraction. The molecule is crystallized in the tetragonal crystal system with the space group P43212 and with unit cell parameters a = 12.226(1) Å, b = 12.226(1) Å, c = 31.807(3) Å, and Z = 8. The molecular and crystal structure of the title molecule is stabilized by intramolecular interactions, O‒H···N and C‒H···N, and intermolecular interaction, C‒H···O.

Synthesis, Crystal Structure and Characterization of (Z)-2-N′-hydroxyisonicotinamidine

The compound (Z)-2-N′-hydroxyisonicotinamidine, (2) was synthesized and characterized by 1H NMR, FT-IR, FAB-Mass, UV-Visible Spectra, and elemental Analysis. Its molecular structure was solved by single crystal X-ray diffraction method. The title molecule, C6H7N3O is crystallized in the orthorhombic crystal system with the space group Pna21 and with unit cell parameters a = 12.5664(8) Å, b = 8.8622(6) Å, c = 5.7953(4) Å, α = 90°, β = 90°, γ = 90°, and Z = 4. The molecular and crystal structure of the title molecule is stabilized by an intramolecular interaction of the type N—H···O, and the intermolecular interactions of types N—H···N and O—H···N.

Crystal structure of product-bound complex of UDP-N-acetyl-d-mannosamine dehydrogenase from Pyrococcus horikoshii OT3.

UDP-N-acetyl-d-mannosamine dehydrogenase (UDP-d-ManNAcDH) belongs to UDP-glucose/GDP-mannose dehydrogenase family and catalyzes Uridine-diphospho-N-acetyl-d-mannosamine (UDP-d-ManNAc) to Uridine-diphospho-N-acetyl-d-mannosaminuronic acid (UDP-d-ManNAcA) through twofold oxidation of NAD(+). In order to reveal the structural features of the Pyrococcus horikoshii UDP-d-ManNAcADH, we have determined the crystal structure of the product-bound enzyme by X-ray diffraction to resolution of 1.55Å. The protomer folds into three distinct domains; nucleotide binding domain (NBD), substrate binding domain (SBD) and oligomerization domain (OD, involved in the dimerization). The clear electron density of the UDP-d-ManNAcA is observed and the residues binding are identified for the first time. Crystal structures reveal a tight dimeric polymer chains with product-bound in all the structures. The catalytic residues Cys258 and Lys204 are conserved. The Cys258 acts as catalytic nucleophile and Lys204 as acid/base catalyst. The product is directly interacts with residues Arg211, Thr249, Arg244, Gly255, Arg289, Lys319 and Arg398. In addition, the structural parameters responsible for thermostability and oligomerization of the three dimensional structure are analyzed.

2-(6-Hy-droxy-1-benzo-furan-3-yl)acetamide.

In the title compound, C10H9NO3, the dihedral angle between the benzofuran ring system (r.m.s. deviation for the non-H atoms = 0.009 Å) and the –C—C(O)—N– segment is 83.76 (1)°. In the crystal, molecules are linked by N—H⋯O and O—H⋯O hydrogen bonds, generating (001) sheets, which feature C(4) and C(10) chains.

The first crystal structure of NAD-dependent 3-dehydro-2-deoxy-D-gluconate dehydrogenase from Thermus thermophilus HB8

2-Keto-3-deoxygluconate (KDG) is one of the important intermediates in pectin metabolism. An enzyme involved in this pathway, 3-dehydro-3-deoxy-D-gluconate 5-dehydrogenase (DDGDH), has been identified which converts 2,5-diketo-3-deoxygluconate to KDG. The enzyme is a member of the short-chain dehydrogenase (SDR) family. To gain insight into the function of this enzyme at the molecular level, the first crystal structure of DDGDH from Thermus thermophilus HB8 has been determined in the apo form, as well as in complexes with the cofactor and with citrate, by X-ray diffraction methods. The crystal structures reveal a tight tetrameric oligomerization. The secondary-structural elements and catalytically important residues of the enzyme were highly conserved amongst the proteins of the NAD(P)-dependent SDR family. The DDGDH protomer contains a dinucleotide-binding fold which binds the coenzyme NAD(+) in an intersubunit cleft; hence, the observed oligomeric state might be important for the catalytic function. This enzyme prefers NAD(H) rather than NADP(H) as the physiological cofactor. A structural comparison of DDGDH with mouse lung carbonyl reductase suggests that a significant difference in the α-loop-α region of this enzyme is associated with the coenzyme specificity. The structural data allow a detailed understanding of the functional role of the conserved catalytic triad (Ser129-Tyr144-Lys148) in cofactor and substrate recognition, thus providing substantial insights into DDGDH catalysis. From analysis of the three-dimensional structure, intersubunit hydrophobic interactions were found to be important for enzyme oligomerization and thermostability.

Purification, crystallization and preliminary X-ray diffraction studies of the ATP-binding subunit of an ABC transporter from Geobacillus kaustophilus

ATP-binding cassette (ABC) transporters, also known as traffic ATPases, form a large family of integral membrane proteins responsible for the translocation of a variety of chemically diverse substrates across the lipid bilayers of cellular membranes of both prokaryotes and eukaryotes by the hydrolysis of ATP. The ATP-binding subunit of an ABC transporter from Geobacillus kaustophilus, a homodimeric enzyme, was overexpressed in Escherichia coli and purified. Crystals were obtained using the microbatch-under-oil method at 291 K. X-ray diffraction data to 1.6 Å resolution were collected on SPring-8 beamline BL26B1. The crystals belonged to the orthorhombic space group I222, with unit-cell parameters a=54.94, b=78.63, c=112.96 Å. Assuming the presence of a dimer in the asymmetric unit gave a crystal volume per protein weight (VM) of 2.32 Å3 Da(-1) and a solvent content of 47%; this was consistent with the results of a dynamic light-scattering experiment, which showed a dimeric state of the protein in solution. Molecular-replacement trials using the crystal structure of HisP from the Salmonella typhimurium ATP-binding subunit of an ABC transporter as a search model did not provide a satisfactory solution, indicating that the two ATP-binding subunits of ABC transporters have substantially different structures.