ABSTRACT
Fsa2 and Phm7 are a unique pair of pericyclases catalyzing [4 + 2] cycloaddition reactions with reverse stereoselectivities in the biosynthesis of equisetin and phomasetin, both of which are potent HIV-1 integrase inhibitors. We here solve the crystal structures of Fsa2 and Phm7, both of which possess unusual "two-ß barrel" folds. Different residues are evident between the active sites of Fsa2 and Phm7, and modeling experiments provide key structural information determining the reverse stereoselectivities. These results provide a better understanding of how natural pericyclases control the catalytic stereoselectivities and benefit the protein engineering in future.
ABSTRACT
Pimarane-type diterpenoids are widely distributed in all domains of life, but no structures or catalytic mechanisms of pimarane-type diterpene synthases (DTSs) have been characterized. Here, we report that two class I DTSs, Sat1646 and Stt4548, each accept copalyl diphosphate (CPP) as the substrate to produce isopimara-8,15-diene (1). Sat1646 can also accept syn-CPP and produce syn-isopimaradiene/pimaradiene analogues (2-7), among which 2 possesses a previously unreported "6/6/7" ring skeleton. We solve the crystal structures of Sat1646, Sat1646 complexed with magnesium ions, and Stt4548, thereby revealing the active sites of these pimarane-type DTSs. Substrate modeling and subsequent site-directed mutagenesis experiments demonstrate different structural bases of Sat1646 and Stt4548 for 1 production. Comparisons with previously reported DTSs reveal their distinct carbocation intermediate stabilization mechanisms, which control the conversion of a single substrate CPP into structurally diverse diterpene products. These results illustrate the structural bases for enzymatic catalyses of pimarane-type DTSs, potentially facilitating future DTS engineering and combinatorial biosynthesis.
ABSTRACT
Chemical examination of the bright yellow sponge Leucetta chagosensis resulted in the isolation of three new imidazole-based alkaloid complexes namely chagosendines A - C (1 - 3), together with known analogues pyronaamidine, naamidine J, and naamine C. Their structures were determined on the basis of extensive spectroscopic (IR, MS, NMR, and single-crystal X-ray diffraction) analysis in association with the chemical conversion. The isolated alkaloids together with three synthesized new homodimer complexes were evaluated for the cytotoxic activities against a panel of tumor cell lines. The copper complexes of imidazole alkaloids 2 and 3, as found from nature for the first time, exerted selective and remarkable activities against the tumor cell lines K562, HepG2, and HeLa.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Coordination Complexes/pharmacology , Imidazoles/pharmacology , Porifera/chemistry , Animals , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/isolation & purification , Cell Proliferation/drug effects , Cell Survival/drug effects , Coordination Complexes/chemistry , Coordination Complexes/isolation & purification , Crystallography, X-Ray , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , HeLa Cells , Hep G2 Cells , Humans , Imidazoles/chemistry , Imidazoles/isolation & purification , K562 Cells , Models, Molecular , Molecular Structure , Structure-Activity RelationshipABSTRACT
Chemical examination of the gorgonian coral Junceella fragilis resulted in the isolation of four pairs of acetyl isomers belonging to briarane diterpenoids, including five new compounds. Their structures were determined on the basis of extensive spectroscopic (IR, MS, NMR, and single-crystal X-ray diffraction) analysis in association with the chemical conversion. Each pair of isomers featured by dynamical interconversion through as acetyl migration in 1,2-diol, which was postulated to be generated under the formation of a cyclic orthoacetate intermediate. All compounds exerted the inhibitory activities against the nitric oxide production in RAW264.7 macrophage cells.
Subject(s)
Anthozoa/chemistry , Diterpenes/chemistry , Animals , Diterpenes/pharmacology , Halogenation , Isomerism , Macrophages/metabolism , Mice , Molecular Structure , Nitric Oxide/antagonists & inhibitors , Nitric Oxide/biosynthesis , RAW 264.7 CellsABSTRACT
Rat calvarial osteoblasts were treated with lanthanum chloride (LaCl3) to explore its effect on the mineral crystalline phase during the process of osteoblast calcification in vitro. The results confirmed that La was readily deposited in the mineral component of the matrix. Employing high-resolution transmission electron microscopy and Fourier transform infrared microspectroscopy techniques, we demonstrated that features comparable to dicalcium phosphate dihydrate (DCPD) and octacalcium phosphate, and hydroxyapatite (HAP) were detected in the mineral phases in vitro. Particularly, LaCl3 treatment retarded conversion from DCPD-like phase into HAP during mineralization. In addition, La was introduced in DCPD powder during wet chemical synthesis. When compared with that of La-free DCPD, the dissolution rate of La-incorporated DCPD was lower, thereby leading to a delayed DCPD-to-HAP phase transformation. Thus, it can be concluded that LaCl3 treatment influences the kinetics of inorganic phase transition by decreasing the dissolution rate of DCPD.
