S-Adenosyl-L-methionine:3-hydroxy-N-methylcoclaurine 4-O-methyltransferase (4-OMT) catalyzes the conversion of 3-hydroxy-N-methylcoclaurine to reticuline, an important intermediate in synthesizing isoquinoline alkaloids. In an earlier step in the biosynthetic pathway to reticuline, another O-methyltransferase, S-adenosyl-L-methionine:norcoclaurine 6-O-methyltransferase (6-OMT), catalyzes methylation of the 6-hydroxyl group of norcoclaurine. We isolated two kinds of cDNA clones that correspond to the internal amino acid sequences of a 6-OMT/4-OMT preparation from cultured Coptis japonica cells. Heterologously expressed proteins had 6-OMT or 4-OMT activities, indicative that each cDNA encodes a different enzyme. 4-OMT was purified using recombinant protein, and its enzymological properties were characterized. It had enzymological characteristics similar to those of 6-OMT; the active enzyme was the dimer of the subunit, no divalent cations were required for activity, and there was inhibition by Fe 2؉ , Cu 2؉ , Co 2؉ , Zn 2؉ , or Ni 2؉ , but none by the SH reagent. 4-OMT clearly had different substrate specificity. It methylated (R,S)-6-O-methylnorlaudanosoline, as well as (R,S)-laudanosoline and (R,S)-norlaudanosoline. Laudanosoline, an N-methylated substrate, was a much better substrate for 4-OMT than norlaudanosoline. 6-OMT methylated norlaudanosoline and laudanosoline equally. Further characterization of the substrate saturation and product inhibition kinetics indicated that 4-OMT follows an ordered Bi Bi mechanism, whereas 6-OMT follows a Ping-Pong Bi Bi mechanism. The molecular evolution of these two related O-methyltransferases is discussed.
Six GEX1 compounds, GEX1A/herboxidiene and its related 5 novel compounds, were isolated from a culture broth of Streptomyces sp. GEX1 compounds induced both G1 and G2/M arrest in a human normal fibroblast cell line, WI-38. All six compounds up-regulated luciferase reporter gene expression directed by enhancer/promoter of various genes, such as cdc2, IL-2 and SV40 early genes. All GEX1 compounds showed cytotoxic activities in the same order of the up-regulating activities on gene expression, suggesting that these two activities are related. Despite the up-regulating activities on the reporter gene expression, GEX1A/herboxidiene did not enhance the expression of any endogenous genes involved in the cell cycle, proliferation and apoptosis. Although the unique effects of GEX1 compounds on cell cycle and the reporter gene expression were similar to those of trichostatin A (TSA), an inhibitor of histone deacetylase (HDAC), GEX1A/herboxidiene did not affect histone acetylation in cells. In addition, GEX1A/herboxidiene treatment gave rise to the shorter sized transcripts of the cdc25A and cdc2 genes as well as the normal sized ones. These results suggest that GEX1 compounds modulate gene expression by an unknown mechanism. New generation of anti-tumor agents exert inhibitory activities on cell proliferation by modulating the cell cycle and gene expression, e.g. histone deacetylase (HDAC) inhibitors1) and all-trans-retinoic acid2). Thus these pathways are attractive targets for anti-cancer drug discovery. We recently isolated six structurally related antitumor antibiotics, GEX1 compounds, from the culture broth of Streptomyces sp.3). A major compound GEX1A was identified as a herbicide, herboxidiene4), and GEX1 Q1-Q5 were novel compounds (Fig. 1). GEX1 compounds had cytotoxic activity, but the mechanism of action was unknown. In this paper, we report the effects of these compounds on the cell cycle and gene expression analyzed by flow cytometry, luciferase reporter assay and RT-PCR methods, and discuss the mode of action of GEX1 compounds. Materials and Methods Drugs GEX1 compounds were isolated from the culture broth of Streptomyces sp. GEX13). Trichostatin A was purchased from Wako Pure Chemical Industry, LTD. Test samples were dissolved in dimethyl sulfoxide (DMSO), diluted
The aim of this study was to investigate the mechanism of inhibition of Eg5 (kinesin spindle protein), a mitotic kinesin that plays an essential role in establishing mitotic spindle bipolarity, by the novel small molecule inhibitor K858. K858 was selected in a phenotype-based forward chemical genetics screen as an antimitotic agent, and subsequently characterized as an inhibitor of Eg5. K858 blocked centrosome separation, activated the spindle checkpoint, and induced mitotic arrest in cells accompanied by the formation of monopolar spindles. Long-term continuous treatment of cancer cells with K858 resulted in antiproliferative effects through the induction of mitotic cell death, and polyploidization followed by senescence. In contrast, treatment of nontransformed cells with K858 resulted in mitotic slippage without cell death, and cell cycle arrest in G 1 phase in a tetraploid state. In contrast to paclitaxel, K858 did not induce the formation of micronuclei in either cancer or nontransformed cells, suggesting that K858 has minimal effects on abnormalities in the number and structure of chromosomes. K858 exhibited potent antitumor activity in xenograft models of cancer, and induced the accumulation of mitotic cells with monopolar spindles in tumor tissues. Importantly, K858, unlike antimicrotubule agents, had no effect on microtubule polymerization in cell-free and cellbased assays, and was not neurotoxic in a motor coordination test in mice. Taken together, the Eg5 inhibitor K858 represents an important compound for further investigation as a novel anticancer therapeutic. [Cancer Res 2009;69(9):3901-9]
S-adenosyl-L-methionine:norcoclaurine 6-0-methyltransferase (norcoclaurine 6-0-methyltransferase), which catalyzes the transfer of the S-methyl group of S-adenosyl-L-methionine to the 6-hydroxyl group of 1,2,3,4-tetrahydro-l-[(4-hydroxyphenyl)methyl]-6,7-isoquinolinediol (norcoclaurine), was purified from cultured Coptisjuponica cells and its enzymic properties were characterized. Purified norcoclaurine 6-0-methyltransferase had apparent PI 4.7, a native molecular mass of 95 kDa (determined by gel filtration) and subunit molecular mass of 40 kDa (SDSFAGE). The enzyme did not require a divalent cation for activity, and the addition of Fez+, Cuz+, Co2+, Zn2+, Mn '+, or Ni2' at 5 mh4 severely inhibited enzyme activity. Neither p-chloromercuribenzoate, Nmethylmaleimide nor iodoacetamide inhibited enzyme activity at 1 mM. 5,6-Dihydro-9,10-dimethoxybenzo[g]-l,3-benzodioxolo[5,6-u]quinolizinium (berberine, the end-product of the biosynthetic pathway in which norcoclaurine 6-0-methyltransferase catalyzes an intermediate step) also inhibited the activity by 50% at 10 mM. Norcoclaurine 6-0-methyltransferase methylated both (S)-norcoclaurine and (R)-norcoclaurine and (R,S)-norlaudanosoline. Further characterization of substrate-saturation kinetics and product inhibition of the purified enzyme indicated that norcoclaurine 6-0-methyltransferase follows a bi-bi ping-pong mechanism with K, values of 2.23 mM and 3.95 mM for (R,S)-norlaudanosoline and S-adenosyl-L-methionine, respectively, while Ki values for S-adenosylhomocysteine versus S-adenosyl-L-methionine 0.18 mM, respectively. [5,6-u]quinolizinium (berberine), a benzylisoquinoline alkaloid obtained from Berberis (Berberidaceae) and Coptis (Ranunculaceae) rhizomes, is used as an antibacterial agent and to treat stomach ache. Berberine is synthesized from two molecules of tyrosine, via the reaction of three O-methyltransferases (Rueffer et al., 1983;Muemmler et al., 1985;Frenzel and Zenk, 1990b; Sat0 et al., 1993), an N-methyltransferase (Frenzel and Zenk, 1990a), a hydroxylase (Loeffler and Zenk, 1990), a berberine bridge enzyme (Steffens et al., 1984), a methylenedioxy ring-forming enzyme (Rueffer Enomes. S-Adenosyl-L- 5,6-Dihydro-9,l0-dimethoxybenzo[g]-1,3-benzodioxolo-
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