Immobilization and properties of carminomycin 4‐O‐methyltranferase, the enzyme which catalyzes the final step in the biosynthesis of daunorubicin in Streptomyces peucetius

Abstract

The carminomycin 4-O-methyltransferase enzyme from Streptomyces peucetius was covalently immobilized on 3M Emphaze ABI-activated beads. Optimal conditions of time, temperature, pH, ionic strength, enzyme, substrate (carminomycin), and cosubstrate (S-adenosyl-L-methionine) concentrations were defined for the immobilization reaction. Protein immobilization yield ranged from 52% to 60%. Including carminomycin during immobilization had a positive effect on the activity of the immobilized enzyme but a strongly negative effect on the coupling efficiency. The immobilized enzyme retained at least 57% of its maximum activity after storage at 4 degrees C for more than 4 months. The properties of the free and immobilized enzyme were compared to determine whether immobilization could alter enzyme activity. Both soluble and bound enzyme exhibited the same pH profile with an optimum near 8.0. Immobilization caused an approximately 50% decrease in the apparent K(m) (K'(m)) for carminomycin while the K'(m) for S-adenosyl-L-methionine was approximately doubled. A 57% decrease in the V(max) value occurred upon immobilization. These changes are discussed in terms of active site modifications as a consequence of the enzyme immobilization. This system has a potential use in bioreactors for improving the conversion of carminomycin to daunorubicin. (c) 1995 John Wiley & Sons, Inc.

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

“…1; Table 3). Thus, the substrate specificity for DauK is much wider than was previously thought (8,9,31,41). With the expanded substrate range, we propose to rename this enzyme anthracycline 4-O-methyltransferase.…”

“…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.…”

Morphologically structured model for antitumoral retamycin production during batch and fed‐batch cultivations of Streptomyces olindensis

“…Retamycin complex is a red powder with low solubility in water and high solubility in organic solvents (methanol, chloroform), and is produced as a secondary metabolite by submerged cultivations of Streptomyces olindensis . The isolation of new anthracyclins has been pursued in attempts to overcome its undesirable cardiotoxicity (Scotti and Hutchinson, 1995). Only a few studies have been published about this antibiotic, but retamycin showed promising results in the intercalation into DNA, which makes this antibiotic suitable to be used as a chemotherapy agent (Latorre, 2001).…”

Design of novel nano-carriers for multi-enzyme co-localization

“…In multi-covalent binding, the attachment points on the enzymes are relatively randomized, which implies lack of control on the position of enzyme structures. In some cases, covalent binding could induce unfavorable conformational changes in the protein structure, which could reduce enzymatic activity significantly (Jordao et al, 1996;Murtinho et al, 1998;Scotti and Hutchinson, 1995).…”