Cloning and structure-function analyses of quinolone- and acridone-producing novel type III polyketide synthases from Citrus microcarpa.

Two novel type III polyketide synthases, quinolone synthase (QNS) and acridone synthase (ACS), were cloned from Citrus microcarpa (Rutaceae). The deduced amino acid sequence of C. microcarpa QNS is unique, and it shared only 56-60% identities with C. microcarpa ACS, Medicago sativa chalcone synthase (CHS), and the previously reported Aegle marmelos QNS. In contrast to the quinolone- and acridone-producing A. marmelos QNS, C. microcarpa QNS produces 4-hydroxy-N-methylquinolone as the "single product" by the one-step condensation of N-methylanthraniloyl-CoA and malonyl-CoA. However, C. microcarpa ACS shows broad substrate specificities and produces not only acridone and quinolone but also chalcone, benzophenone, and phloroglucinol from 4-coumaroyl-CoA, benzoyl-CoA, and hexanoyl-CoA, respectively. Furthermore, the x-ray crystal structures of C. microcarpa QNS and ACS, solved at 2.47- and 2.35-Å resolutions, respectively, revealed wide active site entrances in both enzymes. The wide active site entrances thus provide sufficient space to facilitate the binding of the bulky N-methylanthraniloyl-CoA within the catalytic centers. However, the active site cavity volume of C. microcarpa ACS (760 Å(3)) is almost as large as that of M. sativa CHS (750 Å(3)), and ACS produces acridone by employing an active site cavity and catalytic machinery similar to those of CHS. In contrast, the cavity of C. microcarpa QNS (290 Å(3)) is significantly smaller, which makes this enzyme produce the diketide quinolone. These results as well as mutagenesis analyses provided the first structural bases for the anthranilate-derived production of the quinolone and acridone alkaloid by type III polyketide synthases.

Benzophenone synthase from Garcinia mangostana L. pericarps.

The cDNA of a benzophenone synthase (BPS), a type III polyketide synthase (PKS), was cloned and the recombinant protein expressed from the fruit pericarps of Garcinia mangostana L., which contains mainly prenylated xanthones. The obtained GmBPS showed an amino acid sequence identity of 77-78% with other plant BPSs belonging to the same family (Clusiaceae). The recombinant enzyme produced 2,4,6-trihydroxybenzophenone as the predominant product with benzoyl CoA as substrate. It also accepted other substrates, such as other plant PKSs, and used 1-3 molecules of malonyl CoA to form various phloroglucinol-type and polyketide lactone-type compounds. Thus, providing GmBPS with various substrates in vivo might redirect the xanthone biosynthetic pathway.

Magnesium deprivation inhibits a MEK-ERK cascade and cell proliferation in renal epithelial Madin-Darby canine kidney cells.

AIMS: Loss of magnesium (Mg(2+)) inhibits cell proliferation and augments nephrotoxicant-induced renal injury, but the role of Mg(2+) has not been clarified in detail. We examined the effect of extracellular Mg(2+) deprivation on a MEK-ERK cascade and cell proliferation using a renal epithelial cell line, Madin-Darby canine kidney (MDCK) cells. MAIN METHODS: MDCK cells were cultured in Mg(2+)-containing or Mg(2+)-free media. A HA-tagged constitutively active (CA)-MEK1 and a dominant negative (DN)-MEK1 were transfected into MDCK cells. The level of protein was examined by Western blotting. The intracellular free Mg(2+) concentration ([Mg(2+)](i)) was measured using a fluorescent dye, mag-fura 2. Cell proliferation was determined by WST-1 assay. Dead cells were identified by staining with annexin V-FITC and propidium iodide. KEY FINDINGS: In the presence of fetal calf serum (FCS), Mg(2+) deprivation decreased phosphorylated-ERK1/2 (p-ERK1/2) levels and [Mg(2+)](i). Re-addition of Mg(2+) increased p-ERK1/2 levels, which were inhibited by U0126, a specific inhibitor of a MEK-ERK cascade. Glutathione-S-transferase pull-down and coimmunoprecipitation assays showed that CA-MEK1 and DN-MEK1 binds with ERK1/2 in the presence of Mg(2+). In contrast, neither CA-MEK1 nor DN-MEK1 bound to ERK1/2 in the absence of Mg(2+). These results indicate that the MEK-ERK cascade is regulated by [Mg(2+)](i). Cell proliferation was increased by the treatment with FCS or the expression of CA-MEK1 in the presence of Mg(2+), but was inhibited by Mg(2+) deprivation. Mg(2+) deprivation did not increase the number of dead cells. SIGNIFICANCE: Mg(2+) is involved in the regulation of the MEK-ERK cascade and cell proliferation in MDCK cells.

Extracellular Mg(2+) regulates the tight junctional localization of claudin-16 mediated by ERK-dependent phosphorylation.

Claudin-16 is involved in the paracellular reabsorption of Mg(2+) in the thick ascending limb of Henle. Little is known about the mechanism regulating the tight junctional localization of claudin-16. Here, we examined the effect of Mg(2+) deprivation on the distribution and function of claudin-16 using Madin-Darby canine kidney (MDCK) cells expressing FLAG-tagged claudin-16. Mg(2+) deprivation inhibited the localization of claudin-16 at tight junctions, but did not affect the localization of other claudins. Re-addition of Mg(2+) induced the tight junctional localization of claudin-16, which was inhibited by U0126, a MEK inhibitor. Transepithelial permeability to Mg(2+) was also inhibited by U0126. The phosphorylation of ERK was reduced by Mg(2+) deprivation, and recovered by re-addition of Mg(2+). These results suggest that the MEK/ERK-dependent phosphorylation of claudin-16 affects the tight junctional localization and function of claudin-16. Mg(2+) deprivation decreased the phosphothreonine levels of claudin-16. The phosphothreonine levels of T225A and T233A claudin-16 were decreased in the presence of Mg(2+) and these mutants were widely distributed in the plasma membrane. Furthermore, TER and transepithelial Mg(2+) permeability were decreased in the mutants. We suggest that the tight junctional localization of claudin-16 requires a physiological Mg(2+) concentration and the phosphorylation of threonine residues via a MEK/ERK-dependent pathway.