Author ORCID Identifier

https://orcid.org/0000-0002-2466-4018

Year of Publication

2021

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Pharmacy

Department

Pharmaceutical Sciences

First Advisor

Dr. Jürgen Rohr

Abstract

Combinatorial biosynthesis is a powerful tool for generating new, more active drug analogues to combat disease. But in order for combinatorial biosynthesis to be employed to its full potential, a deep understanding of the enzymes that produce the parent molecule must be had. The goals of the work presented in this thesis are to characterize the reaction catalyzed by MtmW, the final enzyme in the mithramycin (MTM) biosynthetic pathway, and to discover the interaction between MtmW and MtmOIV.

MtmW is an aldol-ketoreductase responsible for reducing the most distal carbonyl on the MTM pentyl side chain. It forms an octamer that has structural homology to the Kvβ subunits of the Kv1 potassium ion channel. While performing in vitro reactions with MtmW, we discovered that an inactive C-2 epimer, iso-mithramycin (iso-MTM), was produced. After verifying that iso-MTM was the correct product both in vitro and in vivo, we determined that iso-MTM isomerized spontaneously at a slow rate in the presence of Mg2+ and rapidly when passing through the S. argillaceus cell membrane. Finally, we showed that MTM DK, the product of MtmOIV, is the substrate for MtmW, and that MtmW could react with MTM DK without the presence of MtmOIV.

There are, however, weaknesses to the combinatorial biosynthesis method including low yields and low substrate affinity. A contributing factor to these weaknesses, we propose, is loss of the native protein-protein interactions (PPIs) that are present in the biosynthetic pathways subjected to combinatorial biosynthesis. To demonstrate this, we have shown that a PPI exists between MtmOIV and MtmW, and we have characterized the nature of this interaction using 19F NMR and covalent labeling strategies combined with MS/MS.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2021.043

Funding Information

This study was supported by the National Institutes of Health (NIH) under the grant Novel Aureolic Acid-Type Antitumor Agents (CA 091901) which was originally awarded in 2010.

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