Crystal structure of an aromatic ring opening dioxygenase LigAB, a protocatechuate 4,5-dioxygenase, under aerobic conditions

AbstractBackground: Sphingomonas paucimobilis SYK-6 utilizes an extradiol-type catecholic dioxygenase, the LigAB enzyme (a protocatechuate 4,5-dioxygenase), to oxidize protocatechuate (or 3,4-dihydroxybenzoic acid, PCA). The enzyme belongs to the family of class III extradiol-type catecholic dioxygenases catalyzing the ring-opening reaction of protocatechuate and related compounds. The primary structure of LigAB suggests that the enzyme has no evolutionary relationship with the family of class II extradiol-type catecholic dioxygenases. Both the class II and class III enzymes utilize a non-heme ferrous center for adding dioxygen to the substrate. By elucidating the structure of LigAB, we aimed to provide a structural basis for discussing the function of class III enzymes.Results: The crystal structure of substrate-free LigAB was solved at 2.2 Å resolution. The molecule is an α2β2 tetramer. The active site contains a non-heme iron coordinated by His12, His61, Glu242, and a water molecule located in a deep cleft of the β subunit, which is covered by the α subunit. Because of the apparent oxidation of the Fe ion into the nonphysiological Fe(III) state, we could also solve the structure of LigAB complexed with a substrate, PCA. The iron coordination sphere in this complex is a distorted tetragonal bipyramid with one ligand missing, which is presumed to be the O2-binding site.Conclusions: The structure of LigAB is completely different from those of the class II extradiol-type dioxygenases exemplified by the BphC enzyme, a 2,3-dihydroxybiphenyl 1,2-dioxygenase from a Pseudomonas species. Thus, as already implicated by the primary structures, no evolutionary relationship exists between the class II and III enzymes. However, the two classes of enzymes share many geometrical characteristics with respect to the nature of the iron coordination sphere and the position of a putative catalytic base, strongly suggesting a common catalytic mechanism

Effects of vasoactive intestinal contractor (VIC) and endothelin on intracellular calcium level in neuroblastoma NG108-15 cells

AbstractEffects on [Ca2+]i levels of endothelin-1 (ET) and vasoactive intestinal contractor peptide (VIC), which is a novel member of the endothelin family, were examined in fura 2-loaded neuroblastoma NG108-15 cells. VIC was found to be a very effective stimulus for intracellular Ca2+ mobilization and to be more potent than ET. Intracellular calcium response to sequential addition of two stimulants exhibited the homologous desensitization of either ET or VIC, but no heterologous desensitization between ET and VIC. This indicates evidence suggesting that these two peptides act through distinct receptors

PcpA, which is involved in the degradation of pentachlorophenol in Sphingomonas chlorophenolica ATCC39723, is a novel type of ring-cleavage dioxygenase

AbstractThe pentachlorophenol (PCP) mineralizing bacterium Sphingomonas chlorophenolica ATCC39723 degrades PCP via 2,6-dichlorohydroquinone (2,6-DCHQ). The pathway converting PCP to 2,6-DCHQ has been established previously; however, the pathway beyond 2,6-DCHQ is not clear, although it has been suggested that a PcpA plays a role in 2,6-DCHQ conversion. In this study, PcpA expressed in Escherichia coli was purified to homogeneity and shown to have novel ring-cleavage dioxygenase activity in conjunction with hydroquinone derivatives, and converting 2,6-DCHQ to 2-chloromaleylacetate

Effects of Abietane diterpenes from Rosmarinus officinalis on guinea pig hearts

A plant in the Lamiaceae family, rosemary ( Rosmarinus officinalis ) is a perennial shrub native to the Mediterranean region. The current study used right atrial specimens from guinea pigs to assess the biological activity of 3 catechol diterpenes with an abietane skeleton―carnosic acid (CA ― 1), demethylsalvicanol (DS ― 1), and carnosol (CN ― 1)―contained in rosemary. Administration of CA ― 1 had a positive inotropic effect (PIE) while administration of CN ― 1 conversely had a negative inotropic effect (NIE). Administration of DS ― 1 resulted in no changes in myocardial contractility. Administration of CA ― 1 or DS ― 1 did not result in significant changes in heart rate. However, administration of CN ― 1 had a negative chronotropic effect (NCE), and administration of CN ― 1 at a final concentration of 10 － 4 M caused cardiac arrest. In addition, the PIE of CA ― 1 was inhibited by prior administration of the phosphodiesterase (PDE) inhibitor IBMX (5 μM)

Effects of ent -Kaurene diterpenes from Rabdosia excisa on the cardiac function of guinea pigs

The medicinal plant Rabdosia excisa in the Lamiaceae family is found in Northeastern China and is used to treat a fever due to a cold, mastitis, arthralgia, and bruises[1]. A previous study reported that an aqueous extract from that plant yielded ent -kaurene diterpenes with antitumor action. In addition, diterpenes have been used to synthesize analogs in relatively large quantities, and their structureactivity relationship in terms of cytotoxic activity[2] and inhibition of the activation of the intracellular transcription factor NF-κB[3] has been examined. Various studies have also examined ent -kaurene diterpenes[4] ― [7]. Three diterpenes―kamebakaurin (1), kamebanin (2), and excisanin A (3)―can be obtained in relatively large quantities from R. excisa . The current study examined the effects of those diterpenes on cardiac function in guinea pigs. Some of those findings are reported here