Honors Program


Date of this Version

Spring 3-12-2018

Document Type



Thelen, B. (2018). A lysine to alanine mutant in Escherichia coli PutA remarkably reduces its PRODH activity. University of Nebraska-Lincoln. 2018.


Copyright Brandon Thelen. 2018.


The imino acid proline plays a critical function in the oxidative stress response in organisms. The catabolism of proline is catalyzed by two enzymes PRODH and P5CDH, which constitute a single protein known as PutA in gram-negative bacteria. PRODH functions by catalyzing the electron transfer from proline to flavin, creating a reduced flavin and generating P5C. Within PRODH is a highly conserved active-site lysine residue (Lys329) which is proposed to act as a proton acceptor for proline, thereby initiating the catalytic steps of PRODH reaction. Previous literature has demonstrated that mutation of this active-site lysine residue in Mycobacterium tuberculosis PRODH severely diminishes enzyme kinetic activity. In this study, the role of Lys329 is examined in the E. coli PutA PRODH domain construct containing residues 86-630 (EcPutA86-630) by site-directed mutagenesis of Lys329 (K329A). EcPutA86-630 wild-type (WT) and K329A mutant enzymes are purified and characterized by steady-state enzyme kinetic assays to determine parameters kcat and Km. The UV-visible spectra of EcPutA86-630 WT and K329A mutant were also recorded, indicating that both proteins are capable of non-covalent binding of the flavin cofactor. Additionally, a proline titration of both proteins was performed to investigate flavin reduction by proline. It was discovered that the EcPutA86-630 K329A mutant has a 2-fold lower kcat/Km value relative to WT enzyme. Furthermore, the flavin cofactor is only partially reduced by proline in the EcPutA86-630 K329A mutant whereas full reduction of the flavin is observed for EcPutA86-630 WT. Despite these reductions in activity, the impact of the Lys329Ala mutant is not as significant as that observed in other stand alone PRODH enzymes indicating that Lys329 is not essential for catalysis in the EcPutA PRODH reaction.