Expression and purification of Class D flavin dependent monooxygenase N‐oxygenases

Abstract

Members of the Class D flavin monooxygenase family are found in numerous natural product biosynthetic pathways, including those of valanimycin and daunorubicin, two medicinally useful natural products. Our lab is interested in understanding catalysis by this enzyme family. We are investigating several representative Class D monooxygenases, with the specific goal of understanding structure‐function relationships in their active sites. Our ultimate goal is to enable rational design of the enzymes in this family. The research presented here summarizes our efforts towards expression, purification and initial biochemical characterization of nitrososynthase from Streptomyces peucetius (DnmZ), and two isobutylamine N‐hydroxylase homologs (from Streptomyces setae, vlmH6, and Streptomyces viridochromogenes, vlmH7). These three Class D monooxygenases catalyze a biosynthetic step common to multiple natural products – flavin‐dependent hydroxylation of a primary amine. Streptomyces setae vlmH was successfully overexpressed in E. coli cells. The highest levels of protein were obtained when cultures were induced with 0.5 mM IPTG and incubated at 18°C. DnmZ is likewise expressed in E. coli with an optimal IPTG concentration and induction temperature of 0.1mM and 25°C, respectively. Both proteins will be purified by nickel affinity chromatography in preparation for preliminary characterization.Support or Funding InformationThis research was made possible by funding through the National Institutes of Health (NIH) grant #1SC3GM122652.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.