Actinorhodin biosynthesis: structural requirements for post-PKS tailoring intermediates revealed by functional analysis of ActVI-ORF1 reductase

Actinorhodin (ACT) produced by Streptomyces coelicolor A3(2) is an aromatic polyketide antibiotic, whose basic carbon skeleton is derived from type II polyketide synthase (PKS). Although an acyl carrier protein (ACP) serves as an anchor of nascent intermediates during chain elongation in the type II PKS complex, it generally remains unknown when an ACP-free intermediate is released from the complex to post-PKS modification ("tailoring") steps. In ACT biosynthesis, a stereospecific ketoreductase (RED1) encoded by actVI-ORF1 reduces the 3beta-keto group of a proposed bicyclic intermediate to an (S) secondary alcohol. The bicyclic intermediate is formed from the steps of PKS and its closely associated enzymes and lies at the interface toward ACT-tailoring steps. To clarify whether RED1 recognizes the ACP-bound bicyclic intermediate or the ACP-free bicyclic intermediate, recombinant RED1 was purified for enzymatic characterization. RED1 was heterologously expressed in Escherichia coli and purified using Ni-chelate and gel filtration column chromatographies to homogeneity in soluble form. Enzymatic studies in vitro on RED1 with synthetic analogues, in place of an unstable bicyclic intermediate, showed that RED1 recognizes 3-oxo-4-naphthylbutyric acid (ONBA) as a preferred substrate and not its N-acetylcysteamine thioester. This strongly suggests that RED1 recognizes ACP-free bicyclic beta-keto acid as the first committed intermediate of tailoring steps. Kinetic studies of RED1 showed high affinity with ONBA, consistent with the requirement for an efficient reduction of a labile beta-keto carboxylic acid. Interestingly, the methyl ester of ONBA acted as a competitive inhibitor of RED1, indicating the presence of strict substrate recognition toward the terminal acid functionality.     

Analysis of membrane stereochemistry with homology modeling of sn-glycerol-1-phosphate dehydrogenase

Different enantiomeric isomers, sn-glycerol-1-phosphate and sn-glycerol-3-phosphate, are used as the glycerophosphate backbones of phospholipids in the cellular membranes of Archaea and the remaining two kingdoms, respectively. In Archaea, sn-glycerol-1-phosphate dehydrogenase is involved in the generation of sn-glycerol-1-phosphate, while sn-glycerol-3-phosphate dehydrogenase synthesizes the enantiomer in Eukarya and Bacteria. The coordinates of sn-glycerol-3-phosphate dehydrogenase are available, although neither the tertiary structure nor the reaction mechanism of sn-glycerol-1-phosphate dehydrogenase is known. Database searching revealed that the archaeal enzyme shows sequence similarity to glycerol dehydrogenase, dehydroquinate synthase and alcohol dehydrogenase IV. The glycerol dehydrogenase, with coordinates that are available today, is closely related to the archaeal enzyme. Using the structure of glycerol dehydrogenase as the template, we built a model structure of the Methanothermobacter thermautotrophicus sn-glycerol-1-phosphate dehydrogenase, which could explain the chirality of the product. Based on the model structure, we determined the following: (1) the enzyme requires a Zn(2+) ion for its activity; (2) the enzyme selectively uses the pro-R hydrogen of the NAD(P)H; (3) the putative active site and the reaction mechanism were predicted; and (4) the archaeal enzyme does not share its evolutionary origin with sn-glycerol-3-phosphate dehydrogenase.     

Functional studies on a ketoreductase gene from Streptomyces sp. AM-7161 to control the stereochemistry in medermycin biosynthesis

Medermycin shows the same trans (3S,15R) configuration as actinorhodin in the pyran ring crucial for its bioactivity. One medermycin biosynthetic gene, med-ORF12, is assumed to be involved in the stereochemical control at C-3. Functional complementation suggested that it plays a similar role as actVI-ORF1 previously proved to determine the stereospecificity at C-3 in actinorhodin biosynthesis. Co-expression of med-ORF12 with actinorhodin early biosynthetic genes further demonstrated that med-ORF12 encodes a ketoreductase responsible for the enantioselective reduction at C-3 in the formation of the pyran ring.     

Metabolic changes in Arabidopsis thaliana expressing the feedback-resistant anthranilate synthase alpha subunit gene OASA1D

Anthranilate synthase (AS) is a key enzyme in tryptophan (Trp) biosynthesis. Metabolic changes in transgenic Arabidopsis plants expressing the feedback-resistant anthranilate synthase alpha subunit gene OASA1D were investigated with respect to Trp synthesis and effects on secondary metabolism. The Trp content varied depending on the transgenic line, with some lines showing an approximately 200-fold increase. The levels of AS activity in crude extracts from the transgenic lines were comparable to those in the wild type. On the other hand, the enzyme prepared from the lines accumulating high levels of Trp showed a relaxed feedback sensitivity. The AS activity, determined in the presence of 50 microM L-Trp, correlated well with the amount of free Trp in the transgenic lines, indicating the important role of feedback inhibition in control of Trp pool size. In Arabidopsis, Trp is a precursor of multiple secondary metabolites, including indole glucosinolates and camalexin. The amount of indol-3-ylmethyl glucosinolate (I3 M) in rosette leaves of the high-Trp accumulating lines was 1.5- to 2.1-fold greater than that in wild type. The treatment of the leaves with jasmonic acid resulted in a more pronounced accumulation of I3 M in the high-Trp accumulating lines than in wild type. The induction of camalexin formation after the inoculation of Alternaria brassicicola was not affected by the accumulation of a large amount of Trp. The accumulation of constitutive phenylpropanoids and flavonoids was suppressed in high-Trp accumulating lines, while the amounts of Phe and Tyr increased, thereby indicating an interaction between the Trp branch and the Phe and Tyr branch in the shikimate pathway.     

Protein kinase Cβ isoform down-regulates the expression of MDR3 P-glycoprotein in human Chang liver cells

The MDR3 protein is a transporter of phosphatidylcholine on the canalicular membrane of human hepatocytes. Previously we showed that the expression of MDR3 mRNA was down-regulated by phorbol 12-myristate 13-acetate (PMA) in human Chang liver cells. In the present study, to elucidate the isoform of protein kinase C (PKC), which influences the level of MDR3 protein, we investigated the effects of PKC-specific inhibitors and antisense oligonucleotides. The level of protein decreased around 50% after treatment for 3–5 days using the dosage of PMA effective against the mRNA expression. The half-life of the MDR3 protein was estimated to be about 5 days. This decrease was antagonized by GF109203X, a non-selective inhibitor of PKCs, and Gö6976, a selective inhibitor for PKCα/β. These inhibitors also suppressed the reduction in MDR3 protein. To specify the isoform of PKC, the cells were treated with antisense oligonucleotide of PKCα or PKCβ. The suppressive effects on MDR3 mRNA of PMA were attenuated in antisense PKCβ-treated cells, but those in antisense PKCα-treated cells were not attenuated. These suggested that PKCβ plays a regulatory role in the expression of MDR3.     

Molecular and Biochemical Characterization of Three Anthocyanin Synthetic Enzymes from Gentiana triflora

Full length cDNA clones of flavonoid 3',5'-hydroxylase, dihydroflavonol4-reductase and flavonoid 3-glucosyltransferase were clonedfrom petals of Gentiana triflora. Their sequences were homologousto counterparts from other plants. Flavonoid 3',5'-hydroxylaseand flavonoid 3-glucosyltransferase were enzymatically characterizedby expressing cDNAs in heterologous expression systems. (Received May 21, 1996; Accepted June 4, 1996)     

A proposal on a contribution of space biological sciences to sexual health in the human space life.

Sexuality is a basic property of the human being and provides an important background for the human life and society. The present situation in the world strongly suggests that the human being will extensively and intensively spread out in space and the population in space, particularly on the space stations in orbit, moon and possibly Mars, will rapidly increase during this century. Therefore sexual health will become a serious issue for the development of space and a better life of the human in the space environment. It is not too early to consider sexuality in the space life and to discuss how to maintain sexual health, how to control it in the space society, or whether it should be controlled. Space biological sciences should be the key and play an important role in studying scientifically sexuality especially the sex, sexual behavior and reproduction in the space environment.