Partial purification and properties of carminomycin 4-O-methyltransferase from Streptomyces sp. strain C5

Abstract

A methyltransferase that acts on carminomycin and 13-dihydrocarminornycin, and that is postulated to be the terminal enzyme in the daunomycin biosynthesis pathway, was purified to near-homogeneity from the daunomycinand baumycin-producing Streptomyces sp. strain C5. The enzyme was obtained in approximately 5 % yield with a purification of 114-fold in specific activity over the sample precipitated with 30-50 % ammonium sulphate. Polyacrylamide gel electrophoresis under denaturing conditions indicated a subunit M, of about 41 000. The enzyme was shown by gel filtration chromatography to have an M, of approximately 166000, suggesting that it is a homotetramer. Kinetic analysis indicated an affinity for S-adenosyl-L-methionine typical of antibiotic methyltransferases ; the enzyme had a slightly higher affinity for carminomycin than for 13-dihydrocarminomycin. The reaction product from methylation of carminomycin was confirmed by chromatography and mass spectral analysis to be daunomycin. The purified enzyme did not catalyse methylation of the aglycones carminomycinone or 13-dihydrocarminomycinone. S-Adenosyl-L-homocysteine inhibited the methyltransferase, whereas homocysteine, adenosine, adenine, &-rhodomycinone, daunomycin, and daunomycinone showed little or no inhibitory activity.

In vivo and in vitro bioconversion of epsilon-rhodomycinone glycoside to doxorubicin: functions of DauP, DauK, and DoxA

“…The only previously proven reactions within this pathway were the 4-O-methylation of carminomycin and 13-dihydrocarminomycin to daunorubicin and 13-dihydrodaunorubicin, respectively (8,9,15,30,41), and the DoxA-catalyzed oxidation of daunorubicin to doxorubicin (14). The catalysts for all of the other reactions were either speculated, based on indirect evidence, or unknown.…”

“…The genes encoding carminomycin 4-O-methyltransferase have been isolated and sequenced from both Streptomyces sp. strain C5 (15) and S. peucetius ATCC 29050 (31), and the enzyme has been purified from both organisms (9,31,41). Cultures of S. lividans TK24(pANT502), containing only dauK (15) in the pANT849 expression vector (12,14), converted rhodomycin D to 4-O-methylrhodomycin D (52) (Table 2; Fig.…”

“…Control reactions were run with extracts prepared from S. lividans TK24(pANT849). The products were analyzed by TLC and autoradiography and by liquid scintillation as described previously (8,9,15 substrate, 25 g. The NADP ϩ , glucose-6-phosphate, and glucose-6-phosphate dehydrogenase constituted an NADPH-regenerating system. The assay mixture was incubated in a 1.0-ml total reaction volume for 1 to 24 h at 28ЊC in 16-mm wells of a cell culture plate (Corning Costar, Cambridge, Mass.)…”

Changes in GE2270 antibiotic production in Planobispora rosea through modulation of methylation metabolism

“…Sinefungin was found to inhibit methyltransferases involved in the biosynthesis of secondary metabolites, causing the accumulation of demethylated antibiotics, as seen for avermectins (Schulman et al, 1985(Schulman et al, , 1987, citreamicin (Pearce et al, 1991), tylosin , pradimicins (Kakinuma et al, 1993;Saitoh et al, 1995) and staurosporine (Yang et al, 1999). S-Adenosyl-L-homocysteine, which is the common nucleoside reaction product, is often also employed to inhibit S-adenosyl-L-methionine-dependent methyltransferases in antibiotic biosynthesis (Bauer et al, 1988;Connors & Strohl, 1993;Kreuzman et al, 1988;Shafiee et al, 1994). Other methylation inhibitors are aminopterin (4-aminofolate), amethopterin (10-methylaminofolate), D-methionine and DL-ethionine.…”

“…Methionine, via its activated form, S-adenosyl-L-methionine, is considered as the general source of methyl groups in secondary metabolism (Roth et al, 1996). O-, Nand C-methyltransferases that use S-adenosyl-L-methionine as methyl donor are ubiquitous in the biosynthesis of secondary metabolites such as staurosporine (Yang et al, 1999), citreamicin (Pearce et al, 1991), avermectins (Schulman et al, 1986), erythromycin (Haydock et al, 1991), avilamycin (Weitnauer et al, 2002), daunomycin (Connors & Strohl, 1993), FK-506 (Shafiee et al, 1994), tylosin Seno & Baltz, 1981) and A10255 , the latter being a thiopeptide antibiotic belonging to the same class as GE2270. Much less is known about different methyltransferases involved in the biosynthesis of other antibiotics such as fortimicin, bialaphos and fosfomycin that seem to utilize methylcobalamin as a direct methyl donor Kuzuyama et al, 1992Kuzuyama et al, , 1995 and not as a prosthetic group.…”