Identification of OxyE as an Ancillary Oxygenase during Tetracycline Biosynthesis

Abstract

The double hydroxylation of 6-pretetramid to 4-keto-anhydrotetracycline is a key tailoring reaction during the biosynthesis of the broad-spectrum antibiotic tetracyclines. It has been shown previously by heterologous reconstitution that OxyL is a dioxygenase and is the only enzyme required to catalyze the insertion of oxygen atoms at the C-12a and C-4 positions. We report here that OxyE, a flavin adenine dinucleotide (FAD)-dependent hydroxylase homologue, is an ancillary mono-oxygenase for OxyL during oxytetracycline biosynthesis in Streptomyces rimosus. By using both gene disruption and heterologous reconstitution approaches, we demonstrated that OxyE plays a nonessential, but important role in oxytetracycline biosynthesis by serving as a more efficient C-4 hydroxylase. In addition, we demonstrated that partially oxidized biosynthetic intermediates can undergo various glycosylation modifications in S. rimosus. Our results indicate that the synergistic actions of OxyE and OxyL in the double hydroxylation step prevent accumulation of shunt products during oxytetracycline biosynthesis in S. rimosus.

Oxytetracycline Biosynthesis

“…The potential quinone-forming activities of OxyG homologs led to initial speculation that it may be involved in the formation of 17; however, as described above, OxyL (with the help of OxyE) has been shown to be sufficient for catalysis of this step. This does not eliminate, however, the possibility of an auxiliary role of OxyG such as that shown for OxyE (64). An interesting aspect of the C5 hydroxylation is that because it is a step unique to OTC biosynthesis, one would expect the gene responsible to be absent from the CTC gene cluster, much as the CTC C7 halogenase (73) is absent from the OTC gene cluster.…”

“…The hydroxylation activities of OxyL were confirmed in vitro, as addition of purified OxyL to 13 produced a short-lived product identified as 17. A subsequent report by Wang et al (64) indicated that OxyE, another FAD-dependent oxygenase, is a C4 hydroxylase and plays an ancillary role in this key dihydroxylation step that converts 13 to 17. Although OxyL can perform the dihydroxylation of C12a and C4 alone, the presence of OxyE significantly increases the titer of OTC, likely by increasing the rate of this key tailoring step (Fig.…”

Biosynthesis of Oxytetracycline by Streptomyces rimosus: Past, Present and Future Directions in the Development of Tetracycline Antibiotics

“…However, during th e last decade, the most signifi cant insight into OTC biosynthesis has been gained by Tang´s research group at the University of California, Los Angeles (USA) (57)(58)(59)(60)(61)(62)(63).…”

“…The C6 methylation is probably followed by a double hydroxylation of ring A at C4/C12a by oxygenase pairs OxyL in OxyE, whereas OxyE is believed to be an ancillary monooxygenase for OxyL with a nonessential but important role in improving its catalytic effi ciency as a C4 hydroxylase (7 and 8 in Fig. 4b) (60). Hydroxylation at C4 is a prerequisite for the incorporation of an amino group at C4 by PLP-dependent aminotransferase OxyQ (9 in Fig.…”

Identification of the chelocardin biosynthetic gene cluster from Amycolatopsis sulphurea: a platform for producing novel tetracycline antibiotics

“…hydroxylase (Wang et al, 2009). In contrast to OTC, CHD does not contain hydroxyl groups at C-5 and C-6 and differs in the pattern of reduction of carboxyl groups at C-11 and C-12, thus resulting in the less polar structure of CHD (Fig.…”