Antioxidative enzymes in seedlings of Nelumbo nucifera germinated under water

Dry seeds of anoxia-tolerant lotus ( Nelumbo nucifera Gaertn= Nelumbium speciosum Willd.) have green shoots with plastids containing chlorophyll, so photosynthesis starts even in seedlings germinated under water, namely hypoxia. Here we investigated antioxidative enzyme changes in N. nucifera seedlings responding to oxygen deficiency. The activity of superoxide dismutase (SOD; EC 1.15.1.1), dehydroascorbate reductase (DHAR; EC 1.8.5.1) and glutathione reductase (GR; EC 1.6.4.2) were lower in seedlings germinated under water (submerged condition) in darkness (SD seedlings) than those found in seedlings germinated in air and darkness (AD seedlings). In contrast, ascorbate peroxidase (APX; EC 1.11.1.11) activity was higher in SD seedlings and the activity of catalase (EC 1.11.1.6) and monodehydroascorbate reductase (MDAR; EC 1.6.5.4) in SD seedlings was nearly the same as in AD seedlings. When SD seedlings were exposed to air, the activity of SOD, DHAR and GR increased, while the activity of catalase and MDAR decreased. Seven electrophoretically distinct SOD isozymes were detectable in N. nucifera . The levels of plastidic Cu,Zn-SODs and Fe-SOD in SD seedlings were comparable with those found in AD seedlings, which may reflect the maintenance of green plastids in SD seedlings as well as in AD seedlings. These results were substantially different from those previously found in rice seedlings germinated under water.     

DnrD cyclase involved in the biosynthesis of doxorubicin: purification and characterization of the recombinant enzyme

Mutations in the Streptomyces peucetius dnrD gene block the ring cyclization leading from aklanonic acid methyl ester (AAME) to aklaviketone (AK), an intermediate in the biosynthetic pathway to daunorubicin (DNR) and doxorubicin. To investigate the role of DnrD in this transformation, its gene was overexpressed in Escherichia coli and the DnrD protein was purified to homogeneity and characterized. The enzyme was shown to catalyze the conversion of AAME to AK presumably via an intramolecular aldol condensation mechanism. In contrast to the analogous intramolecular aldol cyclization catalyzed by the TcmI protein from the tetracenomycin (TCM) C pathway in Streptomyces glaucescens, where a tricyclic anthraquinol carboxylic acid is converted to its fully aromatic tetracyclic form, the conversion catalyzed by DnrD occurs after anthraquinone formation and requires activation of a carboxylic acid group by esterification of aklanonic acid, the AAME precursor. Also, the cyclization is not coupled with a subsequent dehydration step that would result in an aromatic ring. As the substrates for the DnrD and TcmI enzymes are among the earliest isolable intermediates of aromatic polyketide biosynthesis, an understanding of the mechanism and active site topology of these proteins will allow one to determine the substrate and mechanistic parameters that are important for aromatic ring formation. In the future, these parameters may be able to be applied to some of the earlier polyketide cyclization processes that currently are difficult to study in vitro.     

A Clostridium perfringens hem gene cluster contains a cysG(B) homologue that is involved in cobalamin biosynthesis

The hem gene cluster, which consists of hemA, cysG(B), hemC, hemD, hemB, and hemL genes, and encodes enzymes involved in the biosynthetic pathway from glutamyl-tRNA to uroporphyrinogen III, has been identified by the cloning and sequencing of two overlapping DNA fragments from Clostridium perfringens NCTC8237. The deduced amino acid sequence of the N-terminal region of C. perfringens HemD is homologous to those reported for the C-terminal region of Salmonella typhimurium CysG and Clostridium josui HemD. C. perfringens CysG(B) is a predicted 220-residue protein which shows homology to the N-terminal region of S. typhimurium CysG. Disruption of the cysG(B) gene in C. perfringens strain 13 by homologous recombination reduced cobalamin (vitamin B12) levels by a factor of 200. When grown in vitamin B12-deficient medium, the mutant strain showed a four-fold increase in its doubling time compared with that of the wild-type strain, and this effect was counteracted by supplementing the medium with vitamin B12. These results suggest that C. perfringens CysG(B) is involved in the chelation of cobalt to precorrin II as suggested for the CysG(B) domain of S. typhimurium CysG, enabling the synthesis of cobalamin.     

Effect of a glycine residue insertion into crustacean hyperglycemic hormone on hormonal activity

Crustacean hyperglycemic hormone (CHH) and molt-inhibiting hormone (MIH) have similar amino acid sequences and therefore comprise a peptide family referred to as the CHH family. All MIHs unexceptionally have an additional glycine residue at position 12, which is lacking in all CHHs. In order to understand the relevance of the absence of the glycine residue for hyperglycemic activity, a mutant CHH having a glycine residue insertion was prepared, and its hyperglycemic activity was assessed. This mutant CHH had the same disulfide bond arrangement as the recombinant CHH produced in Escherichia coli cells, and exhibited a similar circular dichroism spectrum to the recombinant CHH, indicating that the two CHHs possessed similar conformations. The mutant CHH showed a hyperglycemic effect weaker than the recombinant CHH by about one order of magnitude. These results suggest that the insertion of a glycine residue is one of the indices for structural and functional divergence of the CHH family peptides.     

Carboxy terminus of glucose transporter 3 contains an apical membrane targeting domain

We previously demonstrated that distinct facilitative glucose transporter isoforms display differential sorting in polarized epithelial cells. In Madin-Darby canine kidney (MDCK) cells, glucose transporter 1 and 2 (GLUT1 and GLUT2) are localized to the basolateral cell surface whereas GLUTs 3 and 5 are targeted to the apical membrane. To explore the molecular mechanisms underlying this asymmetric distribution, we analyzed the targeting of chimeric glucose transporter proteins in MDCK cells. Replacement of the carboxy-terminal cytosolic tail of GLUT1, GLUT2, or GLUT4 with that from GLUT3 resulted in apical targeting. Conversely, a GLUT3 chimera containing the cytosolic carboxy terminus of GLUT2 was sorted to the basolateral membrane. These findings are not attributable to the presence of a basolateral signal in the tails of GLUTs 1, 2, and 4 because the basolateral targeting of GLUT1 was retained in a GLUT1 chimera containing the carboxy terminus of GLUT5. In addition, we were unable to demonstrate the presence of an autonomous basolateral sorting signal in the GLUT1 tail using the low-density lipoprotein receptor as a reporter. By examining the targeting of a series of more defined GLUT1/3 chimeras, we found evidence of an apical targeting signal involving residues 473-484 (DRSGKDGVMEMN) in the carboxy tail. We conclude that the targeting of GLUT3 to the apical cell surface in MDCK cells is regulated by a unique cytosolic sorting motif.     

Cyanobacterial phytochrome-like PixJ1 holoprotein shows novel reversible photoconversion between blue- and green-absorbing forms

The gene, pixJ1 (formerly pisJ1), is predicted to encode a phytochrome-like photoreceptor that is essential for positive phototaxis in the unicellular cyanobacterium Synechocystis sp. PCC 6803 [Yoshihara et al. (2000) Plant Cell Physiol. 41: 1299]. The PixJ1 protein was overexpressed as a fusion with a poly-histidine tag (His-PixJ1) and isolated from Synechocystis cells. A zinc-fluorescence assay suggested that a linear tetrapyrrole was covalently attached to the His-PixJ1 protein as a chromophore. His-PixJ1 showed novel photoreversible conversion between a blue light-absorbing form (Pb, lambdaAmax=425-435 nm) and a green light-absorbing form (Pg, lambdaAmax=535 nm). Dark incubation led Pg to revert to Pb, indicative of stability of the Pb form in darkness. Red or far-red light irradiation, which is effective for photochemical conversion of the known phytochromes, produced no change in the spectra of Pb and Pg forms. Site-directed mutagenesis revealed that a Cys-His motif in the second GAF domain of PixJ1 is responsible for binding of the chromophore. Possible chromophore species are discussed with regard to the novel photoconversion spectrum.