Tetracycline antibiotics are one of the most well known classes of natural products and have been produced commercially for nearly 60 years. Previously, we have identified and sequenced the gene cluster responsible for SF2575, an unusual tetracycline with novel structural features including a C-9 D-olivose deoxysugar and with a C4’-angelate and a C-4 salicylate. These new structural features are reflected in its bioactivity which lacks the typical antibacterial activity, but has potent anticancer activity. Structure activity relationship studies have shown the angelate and salicylate groups to be vital to this activity and thus these positions are of high value for diversification to produce novel compounds for further SAR studies. This current study focuses on the dual goals of understanding the fundamental biosynthetic mechanisms involved in SF2575 biosynthesis, and using this knowledge to produce analogs of this potent compound. Towards the first aim, heterologous expression and genetic knockout experiments have illuminated several steps in the previously unknown post PKS tailoring pathway that differentiates SF2575 from the known oxytetracycline biosynthetic pathway. Toward the goal of diversifying the SF2575 scaffold, we have identified two enzymes responsible for the salicylyl transfer reaction, SsfL1 a salicylyl-CoA ligase and SsfX3, a salicylyl transferase. Both have shown to have relaxed substrate specificity in vitro which has been exploited to generate an array of analogs of late stage biosynthetic intermediates. To further investigate the nature of this important acyltransferase, we have solved the crystal structure of SsfX3 revealing a didomain structure consisting of an SGNH hydrolase domain and a putative substrate recognition domain; this sets the stage for exploring the mechanistic basis of the broad substrate specificity. This pathway therefore offers new opportunities to diversify tetracycline scaffolds and shed further light on tetracycline biosynthesis.
This preliminary program is subject to change.
![[ Visit Client Website ]](images/banner.gif)
Elucidating the the Tailoring Pathway of An Anticancer Tetracycline
Tetracycline antibiotics are one of the most well known classes of natural products and have been produced commercially for nearly 60 years. Previously, we have identified and sequenced the gene cluster responsible for SF2575, an unusual tetracycline with novel structural features including a C-9 D-olivose deoxysugar and with a C4’-angelate and a C-4 salicylate. These new structural features are reflected in its bioactivity which lacks the typical antibacterial activity, but has potent anticancer activity. Structure activity relationship studies have shown the angelate and salicylate groups to be vital to this activity and thus these positions are of high value for diversification to produce novel compounds for further SAR studies. This current study focuses on the dual goals of understanding the fundamental biosynthetic mechanisms involved in SF2575 biosynthesis, and using this knowledge to produce analogs of this potent compound. Towards the first aim, heterologous expression and genetic knockout experiments have illuminated several steps in the previously unknown post PKS tailoring pathway that differentiates SF2575 from the known oxytetracycline biosynthetic pathway. Toward the goal of diversifying the SF2575 scaffold, we have identified two enzymes responsible for the salicylyl transfer reaction, SsfL1 a salicylyl-CoA ligase and SsfX3, a salicylyl transferase. Both have shown to have relaxed substrate specificity in vitro which has been exploited to generate an array of analogs of late stage biosynthetic intermediates. To further investigate the nature of this important acyltransferase, we have solved the crystal structure of SsfX3 revealing a didomain structure consisting of an SGNH hydrolase domain and a putative substrate recognition domain; this sets the stage for exploring the mechanistic basis of the broad substrate specificity. This pathway therefore offers new opportunities to diversify tetracycline scaffolds and shed further light on tetracycline biosynthesis.