Search results

(1 - 2 of 2)
Characterization of the role of His257 of vibrio cholerae ApbE in the flavin transfer reaction
Title
Characterization of the role of His257 of vibrio cholerae ApbE in the flavin transfer reaction
Creator
Yuan, Ming
Date
2019
Description
ApbE is a novel enzyme that transfers flavin cofactors into subunits NqrB and NqrC of the sodium-dependent NADH dehydrogenase (Na+-NQR). As...
Show moreApbE is a novel enzyme that transfers flavin cofactors into subunits NqrB and NqrC of the sodium-dependent NADH dehydrogenase (Na+-NQR). As the first enzyme of the bacterial respiratory chain, the function of Na+-NQR affects the survival and development of pathogenicity in many disease-causing bacteria, including Vibrio cholerae. Our preliminary studies indicate that His257 plays a key role in the catalytic activity of ApbE, and that it is an essential component in the transfer of FMN to NqrC. In order to further study how His257 is specifically involved in the catalytic reaction of ApbE, we produced and characterized four mutants: H257G, H257E, H257K, and H257T; in the presence of the activator, K+. Our data showed that mutants H257E and H257K present minimal flavin transfer activity. Interestingly, the mutants H257G and H257T showed activity several times higher compared to the other mutants, however, their activities were still smaller when compared to wild-type. The data suggests that His257 has a very important role for ApbE activity, but that it is not essential. Furthermore, steady-state kinetics showed that the mutants have similar substrate KM values with the wild-type. In addition, double reciprocal plots from bi-substrate titrations showed that ApbE follows a sequential kinetic mechanism where a ternary complex is formed during the reaction.
Show less
THE K+ ACTIVATION MECHANISM OF V. CHOLERAE APBE
Title
THE K+ ACTIVATION MECHANISM OF V. CHOLERAE APBE
Creator
Yang, Jun
Date
2019
Description
ABSTRACTNa+-translocating NADH: quinone oxidoreductase (NQR) is a protein complex that exists in the respiratory chain of Vibrio cholerae....
Show moreABSTRACTNa+-translocating NADH: quinone oxidoreductase (NQR) is a protein complex that exists in the respiratory chain of Vibrio cholerae. This complex can transport sodium ions to the outside of the plasma membrane. NQR has important influences on the survival and pathogenesis of V. cholerae. Two of the subunits of NQR, NqrC and NqrB, has a covalently bound FMN coenzyme. This FMN is necessary for the activity of NQR complex. A protein, alternative pyrimidine biosynthesis protein (ApbE) can transfer the FMN molecule to NqrB and NqrC covalently. And ApbE is also important to some other flavoproteins like the NOS and RNF. The ApbE protein use the FAD as the substrate to transfer the FMN group to the NqrC and NqrB apo-enzyme. Mg2+ is necessary for the activity of ApbE protein. Sodium and potassium ion are not necessary, but potassium ion can increase the activity of the ApbE by about ten times. In order to understand the mechanism of potassium activation of ApbE, several potassium binding sites were identified by molecular docking in this study. Point mutations of the amino acid residues constituting these sites were performed. The FMN transfer activity and affinity to potassium ions of these mutants were measured. The results suggest that when G125 was mutated, the binding of potassium ions was affected. Therefore, the structure composed of P126 and G125 may play a significant role in the activation of ApbE potassium ions.
Show less