Three-dimensional structure of a mutant E.coli aspartate aminotransferase with increased enzymic activity

The aspartate and tyrosine aminotransferases from Escherichia coli have 43% sequence identity and nearly identical active sites. Both are equally good enzymes for dicarboxylate substrates, but the latter transaminates aromatic amino acids 1000 times faster. In an attempt to discover the critical residues for this differential substrate specificity, the aspartate aminotransferase mutant V39L has recently been prepared. It showed improved kcal/Km values for aspartate, glutamate and tyrosine and the corresponding oxo acids, mainly due to two to ten times lower Km values. For example, the Km values of V39L (wild type) for Asp and Glu are 0.12 (1.0) and 0.85 (2.7) mM respectively. The mutant was co-crystallized with 30 mM maleate from both polyethylene glycol and ammonium sulfate. Both structures were solved and refined to R-factors of 0.22 and 0.20 at 2.85 and 2.5 Å resolution respectively. They bear strong resemblance to the closed structure of the wild type enzyme complexed with maleate. The unexpected feature is that, for the first time, the closed form was produced in crystals grown from ammonium sulfate. It is concluded that the mutation has shifted the conformational equilibrium towards the closed form, which leads to generally reduced substrate Km

The structure of aliphatic amine adducts of uranyl acetylacetonate. II. Dioxobis(2,4-pentanedionato)mono (2-N,N-dimethylaminopentan-4-one)uranium(VI)

Introduction: In a previous analysis of a compound of this type, we have established that the adduct molecule is bonded through O and that the geometry about U is pentagonal bipyramidal (Haigh, Nassimbeni, Pauptit, Rodgers & Sheldrick, 1976). We have carried out the present analysis to study the conformational effects on the ligand brought about by substitution at N

The structure of aliphatic amine adducts of uranyl acetylacetonate. IV. Dioxobis(2,4-pentanedionato) mono(2-aminopentan-4-one)uranium(VI)

Introduction: We have shown in three earlier determinations of aliphatic amine adducts of U02(AA)2 (part I: Haigh, Nassimbeni, Pauptit, Rodgers & Sheldrick, 1976; part II: Nassimbeni, Orpen, Pauptit, Rodgers & Haigh, 1977; part III: Rodgers, Nassimbeni & Haigh, 1977) that the conformation of the adduct is dependent on its ability to form hydrogen bonds. The present compound has two H atoms available for hydrogen bonding and may be regarded as the parent of the series

The structure of aliphatic amine adducts of uranyl acetylacetonate. I. Dioxobis(2,4-pentanedionato)mono (2-N-methylaminopentan-4-one)uranium(VI)

Crystals of the title compound are monoclinic with a= 8.314 (5), b= 22.723 (9), c= 12.589 (6) A, /3= 123.0 (2t, Z=4, space group P2dc. The structure was determined by Patterson and Fourier methods and refined by full-matrix least squares to a final R of 0.030 for 2043 independent reflexions. The U atom has pentagonal bipyramidal coordination and the N-methylacetylacetoneamine is bonded to U via O. There are two intramolecular N-H. . .0 hydrogen bonds which govern the geometry of the adduct molecule

Targeting RNA transcription and translation in ovarian cancer cells with pharmacological inhibitor CDKI-73

Dysregulation of cellular transcription and translation is a fundamental hallmark of cancer. As CDK9 and Mnks play pivotal roles in the regulation of RNA transcription and protein synthesis, respectively, they are important targets for drug development. We herein report the cellular mechanism of a novel CDK9 inhibitor CDKI-73 in an ovarian cancer cell line (A2780). We also used shRNA-mediated CDK9 knockdown to investigate the importance of CDK9 in the maintenance of A2780 cells. This study revealed that CDKI-73 rapidly inhibited cellular CDK9 kinase activity and downregulated the RNAPII phosphorylation. This subsequently caused a decrease in the eIF4E phosphorylation by blocking Mnk1 kinase activity. Consistently, CDK9 shRNA was also found to down-regulate the Mnk1 expression. Both CDKI-73 and CDK9 shRNA decreased anti-apoptotic proteins Mcl-1 and Bcl-2 and induced apoptosis. The study confirmed that CDK9 is required for cell survival and that ovarian cancer may be susceptible to CDK9 inhibition strategy. The data also implied a role of CDK9 in eIF4Emediated translational control, suggesting that CDK9 may have important implication in the Mnk-eIF4E axis, the key determinants of PI3K/Akt/mTOR- and Ras/Raf/MAPKmediated tumorigenic activity. As such, CDK9 inhibitor drug candidate CDKI-73 should have a major impact on these pathways in human cancers

X-ray crystallographic studies of eight organic compounds

ABSTRACT Part one of this thesis contains the x-ray crystal structure analyses of six compounds related to natural product chemistry. The first three analyses were performed in order to identify two isomers, separated by chromatography, that were potential intermediates in the syntheses, of stemodin. and aphidicolin, and differed only in the orientation of a cyclobutyl moiety. The first eluted isomer was shown to be a β-cyclobutyl tricyclic enone (C₂₂H₃₂O₃ monoclinic, space group P2₁/n, a = 11.832(1), b = 11.423(1), c = 14.637(1) Å, β = 98.71(2)°, Z = 4, solved by direct methods and refined to R = 0.034 for 2052 observed reflections). The second eluted isomer was the c-cyclobutyl species (C₂₂H₃₂O₃, monoclinic, space group P2₁/n, a = 15.722(4), b = 7.463(2), c = 17.213(6) Å, β =- 104.67(1) ⁰, Z = 4, solved by direct methods- and- refined to R = 0.040 for 702 observed reflections). The third analysis was of the p-bromobenzoate derivative of the second eluted isomer, and confirmed the c-cyclobutyl structure (C₂₉H₃₇BrO₄, triclinic, space group PT, a = 11.023(2), b = 11.877(1), c = 10.900(1) Å, α = 90.461(8), β = 111.57(1), ƴ = 80.51(1)°, Z = 2, solved by Patterson methods and refined to R = 0.032 for 2715 observed reflect ions). The fourth structure was also a p-bromobenzoate derivative of a system involving a four-membered ring, and was undertaken to verify the 1,4-homoenol structure of camphor-1,4-homoenol p-bromobenzoate (C ₁₇H₁₉BrO₂, orthorhombic, space group P2₁2₁2₁, a = 6.875(1), b = 8.522(2), c = 26.658(6) Å, Z = 4, solved by both direct and Patterson methods and refined to R =0.045 for 697 observed reflections). The last two structures of this part proved to be crystallographically difficult. One was the previously unknown structure of raucubaine, an indole alkaloid isolated from the plant Rauwolfia salicifolia griseb. (C₂₀H2₂₄N₂0₃, monoclinic, space group P2₁, a = 7.2179(3), b = 12.8169(7), c = 9.1996(2) Å, β = 93.040(3)°, Z = 2, solved by direct methods (with great difficulty) and refined to R = 0.046 for 1700 observed reflections). The other was a sugar that had remained unsolved for fourteen years (C₂₄H₂₄Cl₂O₈, monoclinic, space group P2₁, a = 5.752(3), b = 15.436(3), c = 13.698(3) Å, β = 93.74(3)°, Z = 2, solved by direct methods (with great difficulty) and refined to R = 0.042 for 898 observed reflections). Part two contains two optically active structures as part of a project concerning spontaneous resolution in binaphthyl systems: the first being naphthidine (C₂₀H₁₆N₂, tetragonal, space group P4₁2₁2 or P4₃2₁2, a = 7.945(1), c = 24.264(5) Å, Z = 4, solved by direct methods and refined to R = 0.068 for 548 reflections) and the other 1, 1'-binaphthyl (C₂₀H₁₄, tetragonal, space group P4₁2₁2 or P4₃2, a = 7.164(2), c = 27.70(1) Å, Z = 4, solved by direct methods and refined to R = 0.030 for 562 observed reflections). These structures are compared to those of several related compounds.Science, Faculty ofChemistry, Department ofGraduat