Title of Oral/Poster Presentation

Characterizing the Adenylation Domain of a BbBSLS Construct

Presenter Information

Miekan StonhillFollow

Class

Article

Department

Chemistry and Biochemistry

Faculty Mentor

Joan Hevel

Presentation Type

Poster Presentation

Abstract

BbBSLS, a 350kDa nonribosomal peptide synthetase (NRPS) found in Beauveria bassiana, includes an unusually situated methyltransferase domain, which catalyzes the methylation of leucine in the growing peptidyl natural product. This domain is embedded in an adenylation domain, but the reason for this and the effects of the domains on each other are unknown. Understanding the function and communication of NRPS domains is a topic of interest because NRPSs produce a variety of useful products such as antibiotics. Using a radiolabeled methylation assay, we have shown that the methyltransferase domain alone has significantly altered substrate specificity when embedded in the entire NRPS compared to when it is isolated as a separate protein. We have also shown that the methyltransferase domain shows more typical substrate preference when at least part of the adenylation domain is included. However, the effect of the methyltransferase domain on the activity and substrate specificity of the adenylation domain is unknown. We hypothesize that the methyltransferase insertion affects the activity of the adenylation domain. In order to test our hypothesis, we have engineered several different protein constructs of the NRPS. The BSLS-A2dMT construct includes the entire adenylation domain without the embedded methyltransferase. Testing the adenylation activity of this construct compared to the full NRPS will provide important information about substrate specificity.

Start Date

4-9-2015 3:00 PM

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Apr 9th, 3:00 PM

Characterizing the Adenylation Domain of a BbBSLS Construct

BbBSLS, a 350kDa nonribosomal peptide synthetase (NRPS) found in Beauveria bassiana, includes an unusually situated methyltransferase domain, which catalyzes the methylation of leucine in the growing peptidyl natural product. This domain is embedded in an adenylation domain, but the reason for this and the effects of the domains on each other are unknown. Understanding the function and communication of NRPS domains is a topic of interest because NRPSs produce a variety of useful products such as antibiotics. Using a radiolabeled methylation assay, we have shown that the methyltransferase domain alone has significantly altered substrate specificity when embedded in the entire NRPS compared to when it is isolated as a separate protein. We have also shown that the methyltransferase domain shows more typical substrate preference when at least part of the adenylation domain is included. However, the effect of the methyltransferase domain on the activity and substrate specificity of the adenylation domain is unknown. We hypothesize that the methyltransferase insertion affects the activity of the adenylation domain. In order to test our hypothesis, we have engineered several different protein constructs of the NRPS. The BSLS-A2dMT construct includes the entire adenylation domain without the embedded methyltransferase. Testing the adenylation activity of this construct compared to the full NRPS will provide important information about substrate specificity.