Date of Award


Document Type

Open Access Dissertation


Chemistry and Biochemistry

First Advisor

F. Wayne Outten


Fe-S clusters are one major type of the sulfur-containing cofactors, which conduct essential functions in organisms. The Suf pathway is one of the three main pathways for the biosynthesis of Fe-S clusters. In E. coli, the Suf pathway is utilized under iron limitation and oxidative stress. This ability is important for pathogens to survive. Also, the Suf pathway is found to be exclusive in bacteria, so it is a good target for novel antibiotic design. SufS is a cysteine desulfurase in the Suf pathway to extract sulfur from L-cysteine. It needs the enhancement of SufE. To better understand the catalytic mechanism of the reaction between SufS and L-cysteine in the presence of SufE, we applied 31P NMR, stopped flow spectroscopy, and site directed mutagenesis. The results show that binding of SufE causes a conformational change of the pyridoxal 5’- phosphate (PLP) cofactor in SufS. The reaction of L-cysteine and PLP is a biphasic process including the fast phase (formation of external aldimine) and slow phase (shift to ketimine). The binding of SufE facilitates the formation of the ketimine. We mutated SufS H123A, which removed the enhancement of SufE to the activity of SufS and the formation of Cys ketimine. Finally, binding of SufE increased the formation of the persulfide in SufS. Characterization of the interaction of SufS and SufE may provide insight for the design of protein-protein interaction inhibitor. We made the Y345A/D346A mutation in SufS. We applied PLP quantification, analytical gel filtration, UV-visible absorption spectroscopic analysis and circular dichroism to confirm this mutant still keeps the structural integrity. The basal activity of SufS Y345A/D346A is similar as that of wild-type SufS. However, SufE cannot enhance the activity of this mutant. The result of the isothermal titration calorimetry (ITC) shows that there is reduced interaction between this mutant and SufE. Based on the research above, a structural modeling of the SufS-SufE interaction was made through proteinprotein docking, which clarifies more details in this interaction. SufS has an essential cofactor PLP that can be a target for the PLP-based inhibitor like DCS and LCS. To investigate if DCS/LCS can inhibit the activity of SufS, we checked the activity of SufS in the presence of either D-cycloserine (DCS) or L-cycloserine (LCS). The results show that there is a dose-dependent inhibition of SufS activity by DCS. The 50% inhibitory concentration (IC50) was calculated to be 1.98 mM. A dose-dependent inhibition of SufS by LCS was also observed and the IC50 is 306.1 μM Compared with DCS, LCS shows much better inhibitory effects. The small-molecular docking shows that the nitrogen of DCS to start the nucleophilic attack towards the Schiff base of the PLP and Lys226 is far away from its target, which is not a proper orientation for the transimination reaction. The docking of LCS shows that the nitrogen of LCS for the nucleophilic attack is close to the Schiff base of Lys226 and PLP, which is a proper orientation for the following transimination reaction. The UV-visible absorption spectra of SufS and DCS shows the degradation of internal aldimine and a new intermediate at 380 nm is formed. The spectrum of LCS shows the 380 nm peak is reduced but the 320 nm peak keeps growing, which indicates the intermediate at 320 nm is a stable adduct and it is rarely get rescued by excessive L-cysteine. A reaction mechanism is proposed to depict the reaction between SufS and DCS/LCS.

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