GB2302540A - 5'-Methylthioadenosine nucleosidase as cofactor for transmethylation - Google Patents

Description

- 1 COPACTOR FOR TRANSMETHYLATION 2302540 The present invention relates to

the use of a compound as a cofactor for carminomycin 4-0-methyltransferase (COMT enzyme).

The anthracyclines of the daunorubicin group, such as doxorubicin, carminomycin and aclacinomycin, are among the most widely employed agents in antitumoral therapy [F. Arcamone, Doxorubicin, Academic Press, New York, 1981, pp. 12-25; A.

Grein, Process Biochem, 16:34 (1981); Taneko, Chimicaoggi May:11 (1988)1. o The anthracycline doxorubicin is made by S. peucetius 27952 from acetate, propionate and glucose. E-Rhodomycinone, carminomycin and daunorubicin are established intermediates in this process [Grein, Advan., Appl. Microbiol. 32:203 (1987)l - Two steps in this pathway involve the 0-methylation of discrete intermediates: the conversion of aklanonic acid to methyl aklanonate and carminomycin to daunorubicin.

In particular, this last step is catalysed by an enzyme termed carminomycin 4 - 0-methyltransf erase (COMT). The COMT enzyme, the gene coding for it, and its use in various manners for the bioconversion process is described in Madduri et al, J. Bacteriol. 175, 3900-3904, 1993, Scotti C. et al, Biotechnol. Bioeng in press and claimed in our US Patent No. 5,364,781.

The complete conversion of carminomycin into daunorubicin is required to minimize as much as possible the presence of the undesired intermediate carminomycin in the preparation of highly pure daunorubicin.

The enzymatic reaction requires the presence of Sadenosyl-L-methionine, which acts as a methyl donor in the transmethylation according to the following scheme:

COMT Carminomycin + SAM - daunorubicin + SAHC wherein SAM is S-adenosyl-L-methionine, and SAHC is S-adenosyl-homocysteine.

Surprisingly, we have found that the enzymatic activity of the COMT enzyme is greatly enhanced by the addition of 51 methylthio-adenosine-nucleosidase.

Thus, the invention provides use of 5'-methylthio-adenosine nucleosidase as a cofactor for COMT.

51-Methylthio-adenosine nucleosidase is a known enzyme 51- (E.C. 3.2.2.-) which catalyzes the degradation of methylthioadenosine and cleaves S-adenosylhomocysteine, as described by A. J. Ferro et al. in Biochimica et Biophysica Acta, 438 (1976), p. 487-494. A partial purification of 51methylthio-adenosine nucleosidase can be carried out starting from Escherichia coli strains or from other microorganisms which produce the enzyme, through ion-exchange... column chromatography, followed by an ammonium sulfate precipitation and gel filtration chromatography. The partially purified preparation showed a specific activity of about 0.5-1 Amole min-'mg-' when the formation of adenine from S-adenosylhomocysteine was assayed spectrophotometrically at 265 nm.

The process according to the invention comprises treating carminomycin with COMT in the presence of 51-methylthioadenosine nucleosidase. Typically, the co-incubation of 51- 3 - methylthio-adenosine nucleosidase in the reaction mixture of carminomycin methylation in vitro, was found to increase the final yield of the reaction by increasing the reaction rate. In another common application, namely the elimination of small amounts of carminomycin which are often present as a minor contaminant in daunorubicin extracts, the presence of 51methylthio-adenosine nucleosidase allows a complete transformation to daunorubicin.

The process of the invention is typically carried out by providing a reaction mixture containing carminomycin substrate, S-adenosyl-Lmethionine co-substrate, COMT and 51 -methylthioadenosine nucleosidase. The reaction mixture generally also contains a buffer.

The 51 -methylthio-adenosine nucleosidase is generally used in a specific activity of from 0.07 to 7 jumolemin-1mg-1, for example about 0.7 Amolemin-'mg-1, as assayed with Sadenosylhomocysteine as substrate. The COMT is preferably purified recombinant COMT and may be used in a concentration of from 0.01 to 1.0 mg/ml, for example about 0.1 mg/ml. The process of the invention may be carried out using a wide range of substrate concentrations, but from 1 to 10,000 AM(e.g. from 10 to 1,000 AM) carminomycin is typically used. The S adenosyl-L-methionine cosubstrate is typically used in a concentration of from 0.3 mM to 30 mM, for example about 3 mM.

As mentioned above, the process of the invention may be carried out on a daunorubicin extract containing only a minor amount of carminomycin in order to convert contaminating carminomycin into the desired daunorubicin. The molar ratio of 4 - carminomycin to daunorubicin in the reaction mixture may be from 1:5 to 1:500, for example about 1:50.

The process of the invention is typically carried out at a pH of from 7 to 9, for example 8. The buffer is chosen so as to provide this pH and may be tricine buffer.

The process may be carried out at a temperature from room temperature (200C) to 400C, but the temperature of 370C is preferred. The process is typically carried out for from 20 min to 3 hours, preferably from 30 min to 1 hour. Almost complete reaction is generally obtained after about 45 min.

The following examples illustrate the invention. Brief Description of the Drawings

Figure 1: in vitro methylation of carminomycin to daunorubicin catalyzed by recombinant COMT. Estimated kinetic parameters were as follows: Km = 11 AM and Vmax=3.5 nmol min-' mg-I in the control experiment (full circles) and Km = 22 and Vmax = 35 nmol min-' mg-I in the presence of 51methylthio-adenosine nucleosidase (full squares).

Figure 2: efficiency of the in vitro methylation of 2.5?c carminomycin in the presence of a large excess of daunorubicin (97.51k), in a control experiment (full circles) and in the presence of 51-methylthio-adenosine nucleosidase (full squares).

ExamDle 1.

The yield of in vitro carminomycin methylation catalyzed by purified recombinant COMT, was studied in the absence and in the presence of a partially purified preparation of 51methylthio-adenosine nucleosidase according to the following protocol. A reaction mixture containing 100 yM carminomycin substrate, 3 mM S-adenosyl-L-methionine cc-substrate, 0.1 mg/ml of purified recombinant COMT, 170 mM tricine buffer, pH 8 was incubated at 37C for up to 30 min.

A parallel incubation also contained 0.06 mg/ml of 51methylthio-adenosine nucleosidase (specific activity about 0.7gmole min-'mg-' when assayed with S-adenosylhomocysteine as substrate).

So Al of each reaction mixture was sampled after 1, 3, 6, 10, 20, and 30 min and the enzymatic reaction was stopped by treatment with 15 Al of 5011 trichloroacetic acid, followed by centrifugation to eliminate the precipitated proteic material.

The separation of the methylated product daunorubicin from the substrate carminomycin was assayed on 40 Al of each supernatant by reverse-phase high pressure liquid chromatography (RP-HPLC) on a PTC- C18 microbore Hypersyl (Trade Mark) column (2.1x220 mm) eluted at a flow rate of 0.4 ml/min with a linear gradient from 550-. phase A-4516 phase B to 1511 phase A-85k phase B. Mobile phase A composition was 1.20-6 sodium dodecylsulfate-15 mM phosphate buffer pH 2.5 while mobile phase B composition was 9596 acetonitrile-0.07i trifluoroacetic acid. The elution profile was monitored at 485 nm. The amount of daunorubicin formation was estimated from the peak areas in comparison to a standard solution.

As shown in figure 1, the presence of 51-methylthioadenosine nucleosidase increased the Vmax value from 3.5 nmol min-1mg-' to 35 nmol min-1mg-' with approximately a three fold increase of the amount of carminomycin produced during the 30 min reaction.

- 6 is Exam'Dle 2 The efficiency of carminomycin methylation in the presence of a large excess of daunorubicin was studied in the absence and in the presence of partially purified 51-methylthio-adenosine nucleosidase according to the following protocol.

A reaction mixture containing 100 AM carminomycin substrate, 3.8 mM of daunorubicin, 3 mM S-adenosyl-L-methionine, 0.3 mg/ml of purified recombinant COMT, 170 mM tricine buffer, pH 8 was incubated at 37C for up to 40 min.

A parallel incubation also contained 0.06 mg/ml of 51methylthio-adenosine nucleosidase (specific activity about 0.7 Amole min-Img-1).

Al of each reaction mixture were sampled after 10, 30, and 40 min and the enzymatic reaction was analyzed by RP-HPLC as described in the example 1.

shown in figure 2, in the control reaction only approximately half of the initial amount of carminomycin (2.5%) was transformed, while in presence of 51-methylthio-adenosine nucleosidase a complete methylation of carminomycin was observed.

As

Claims (3)

1. Use of 5'-methylthio-adenosine nucleosidase as a cof actor for carminomycin 4-0-methyltransferase (COMT) enzyme.

2. A process for converting carminomycin into daunorubicin, which process comprises treating carminomycin with COMT in the presence of 5'methylthio-adenosine nucleosidase.

3. A process according to claim 2, wherein undesired contaminant carminomycin present in a daunorubicin solution is converted into daunorubicin.