Purification and Characterization of a Monooxygenase Involved in the Biosynthetic Pathway of the Antitumor Drug Mithramycin

Abstract

A monooxygenase encoded by the mtmOIV gene from the mithramycin gene cluster of Streptomyces argillaceus was purified 21-fold by a three-step purification procedure. This monooxygenase catalyzes the oxidative cleavage of the fourth ring of premithramycin B. The enzyme was dependent on NADPH and flavin adenine dinucleotide for activity with optimal pH at 9.5, and the K m values for NADPH and premithramycin B were 269.22 and 23.35 M, respectively. The reaction catalyzed by MtmOIV yields two possible isomers of the same basic shortened aliphatic chain molecule. One of the reaction products showed important biological activity, thus highlighting the importance of the cleavage of the fourth ring of the aglycon for biological activity.Mithramycin is a member of the aureolic acid group of antitumor compounds (8,12). It shows antibacterial activity against gram-positive bacteria and also a remarkable cytotoxicity against a variety of tumor cell culture lines. Mithramycin has been clinically used for the treatment of certain tumors, such as disseminated embryonic cell carcinoma, as well as for Paget's bone disease (12) and is also used for control of hypercalcemia in patients with malignant disease (1). The biosynthetic gene cluster for mithramycin has been fully characterized (2-6, 9), and it has been found that although mithramycin is a tricyclic aromatic polyketide, its biosynthesis proceeds through tetracyclic intermediates. Many of these intermediates have been isolated from mutants blocked at different steps in the biosynthesis (2-6). Insertional inactivation of one of the genes of the mithramycin cluster, mtmOIV, generated a non-mithramycinproducing mutant that accumulated a fully glycosylated tetracyclic intermediate, premithramycin B (6). This gene codes for an oxygenase responsible for the oxidative cleavage of the fourth ring of premithramycin B, and after this oxidation step, the final mithramycin molecule is produced by a ketoreduction of the 4Ј position at the generated aliphatic chain (6). Since only mithramycin, and not the biosynthetic intermediates, possesses antibiotic and antitumor properties, the opening of the fourth ring appears as a very important step in the biosynthesis of this antitumor compound.Purification and characterization of the MtmOIV oxygenase. The MtmOIV oxygenase was purified from Streptomyces albus LP04, a strain that overexpresses MtmOIV. Enzyme activity was monitored at 30°C by using premithramycin B as a substrate to determine NADPH oxidation at 340 nm. The enzyme was purified about 21-fold with a three-step purification procedure (including ammonium sulfate precipitation and two chromatographic steps) using affinity chromatography on the immobilized reactive dye Blue 4 and anionic exchange on Mono Q HR 5/5 (Table 1; Fig. 1). The enzyme was purified as a single band, as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), migrating at 56.1 kDa (Fig. 1). The enzyme was eluted from the Blue 4 column at 40 mM NaCl and from the Mono Q HR 5/5 column at 230...

Baeyer−Villiger Monooxygenases: More Than Just Green Chemistry

“…In 1959, more than 10 years after the first indications of the existence of an enzymatic version of the BaeyerÀVilliger oxidation during the bioconversion of steroids, Bradshaw et al reported the microbial degradation of (þ)-camphor (26) with Pseudomonas putida isolated from sewage sludge. 104 Although no biochemical studies were presented in this initial paper, the isolation of two lactone metabolites indicated the existence of BVMO activity in P. putida.…”

“…25 In the biosynthetic pathway of an anticancer antibiotic called mithramycin by the soil bacterium Streptomyces argillaceus ATCC 12956, a BVMO named MtmOIV was found to be responsible for the conversion of a biologically inactive precursor, premithramycin B (3) to the corresponding lactone 4 that is further converted to mithramycin DK presumably by lactone opening and decarboxylation, and finally into the active drug mithramycin (5). 20,26 MtmOIV is believed to catalyze more than the fourth ring modification of premithramycin but also the various followup reaction steps. 20 MtmOIV is regarded as the first example of a naturally occurring BVMO with its cognate substrate ( Figure 4).…”

Mithramycin Analogues Generated by Combinatorial Biosynthesis Show Improved Bioactivity

“…Its aglycon including the 3-side chain is biosynthesized from 10 acyl-CoA units by a type II polyketide synthase, and all five deoxyhexose moieties are successively attached to the tetracyclic intermediate premithramycinone, resulting in the fully glycosylated intermediate premithramycin B via premithramycin A 3 , 1-7 before an oxidative cleavage reaction, catalyzed by Baeyer-Villigerase MtmOIV, 3,5,7 followed by the reduction of a side chain keto group by MtmW, finishes the biosynthesis (Scheme 1). The glycosylation steps remain partly obscure, although the sequence could be established unambiguously: initially, the trisaccharide chain is formed through successive glycosylation steps, before the disaccharide chain is attached, also in a stepwise sequential order.…”

MtmMII-mediated C-Methylation during Biosynthesis of the Antitumor Drug Mithramycin Is Essential for Biological Activity and DNA-Drug Interaction

“…All of these biosynthetic intermediates have been isolated from mutants blocked at different steps in biosynthesis and share a common feature, which is the pres-ence of a tetracyclic aglycon. The final tricyclic structure of MTM is obtained after the oxidative cleavage of the fourth ring of premithramycin B, in a reaction catalyzed by a monooxygenase and followed by the ketoreduction of the 4Ј-position of the generated aliphatic chain (9,12). This renders the fully biologically active compound.…”

“…It has been clinically used for the treatment of testicular carcinoma, Paget's bone disease, and other bone growth disorders and is also used for control of hypercalcemia in patients with malignant disease (4). The biosynthetic pathway for MTM biosynthesis has been extensively studied by biochemical and genetic means (2,(5)(6)(7)(8)(9)(10)(11)(12). Although MTM is a tricyclic aromatic polyketide with a side chain, its biosynthesis proceeds through tetracyclic tetracycline-like intermediates (6).…”