Expression and functional characterization of active nicotinic acid dehydrogenase from Pseudomonas fluorescens Pf5

Abstract

Many compounds created as a byproduct of industrial processes or synthetic products are having a significant, negative affect on the environment. N‐Heteroaromatic compounds (N‐HACs), make up many of these environmental pollutants. A proposed solution to environmental damage by N‐HACs is bioremediation: using engineered microorganisms to clean areas affected by pollution. Nicotinic acid (NA) is an N‐HAC which is present in a wide range of synthetic products and is an environmental pollutant. Many microorganisms have evolved the ability to catabolize N‐HACs, including several capable of using NA as their sole source of carbon. One organism that degrades NA is Bacillus niacini. B. niacini is of interest because it degrades NA by a pathway unlike other organisms. Characterizing B. niacini’s NA degradation pathway is potentially important for accomplishing the long‐range goal of developing effective bioremediation strategies. The first step in B. niacini's putative NA catabolism pathway is through the nicotinic acid dehydrogenase NicAB, which is encoded by several ORFs in the gene cluster hypothesized to be responsible for NA degradation. Previous attempts to characterize the activity of NicAB expressed in E. coli have been unsuccessful and have demonstrated that the B subunits of NicAB are deficient in molybdenum, an essential metal for its MCD cofactor. The activity of NicAB will be characterized upon expression in Pseudomonas fluorescens Pf5, a closely related organism to the NA degrading bacteria Pseudomonas putida. P. fluorescens Pf5 may have MCD maturation proteins more compatible with NicAB. We show that P. fluorescens Pf5 has no endogenous NA dehydrogenase activity via HPLC. Future work will test whether knock‐in of the genes encoding NicAB into P. fluorescens Pf5 will show whether NA dehydrogenase hydroxylates NA and 6HNA. The activity and substrate binding of functional NA dehydrogenase will be characterized by kinetic analysis.Support or Funding InformationHenry J. Copeland Fund for Independent StudyThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.