Study On The Bioactivity And Homology Modeling Of Ferredoxin-NSDPH Reductase And Isochorismate Synthase In Aeromonas Hydrophila XS91-4-1

Aeromonas hydrophila is a well-known pathogen for anthropozoonosis and could cause great economic losses of aquaculture. Ferredoxin-NADP+Reductases (FNR) are ubiquitous flavoenzymes that play an important role as an electron carrier in basic metabolic pathways. FNR catalyzes reversible electron tansfer between ferredoxin (Fd) or flavodoxin (F1d) and NADP (H). Isochorismate synthases (ICS) exist in as bacteria, plants, fungi, apicomplexan parasites and many other organisms but not in humans. It’s responsible for converting chorismate to isochorismate and is necessary for the biosynthesis of both the repiratory chain component menaquinone (MK) and the siderophore 2,3-dihydroxybenzoate (DHB).In this study, we have investigated the FNR and ICS (EntC and MenF) of Aeromonas hydrophila. FNR and ICS genes were cloned from A. hydrophila XS91-4-1, and recombinant plastimid pET42a-fnr, pET28a-ent C and pET28a-menF were constructed and overexpressed in Escherichia coli BL21. FNR-GST, EntC and MenF interest proteins were purified by nickel column affinity chromatography. Commasie brilliant bule staining was used to study the protein content. According to Michaelis-Menten equation and double reciprocal plot, the enzyme activity of FNR was assayed using NADPH and EDTA-Fe3+ as the substrate, and EntC and MenF were assayed using chorismic acid as substrate. Then the bioinformatics analysis of FNR, EntC and MenF was performed and three-dimensional structures of FNR, EntC and MenF were predicted by MEGA 5.0 and Swiss-Model. At the same time, the secondary structures of EntC and MenF were analyzed by Espript 3.0 and the active sites of the two proteins were forecasted. At last, this stduy first constructed the F328Y mutant of the EntC. Results are as follows:1. Recombinant plastimid pET42a-fnr, pET28a-entC and pET28a-menF were constructed and could be highly sloble expressed in Escherichia coli BL21.2 FNR, EntC and MenF could be purified well by nickel column affinity chromatography. The specific activity of all interest proteins was higher after purification.3. Based on its sequence and phylogenetic relationship, the FNR of A. hydrophila XS91-4-1 was closely related to Bacterial-class FNR. Our study suggested that the FNR of A. hydrophila XS91-4-1 belongs to Bacterial-class FNR, and is similar to FNRs in many fundamental characteristis. According to the phylogenetic tree, three-dimensional and secondary structure analysis of EntC and MenF, there were differences between the two kinds of IS. Our results also showed that the EntC or MenF of different species had high similarity.Our stduy had preliminary discussed the structure and function of FNR, EntC and MenF in A. hydrophila XS91-4-1, and lay the foundation for furrher research about drug terget of A. hydrophila. We hope to provide a new idea for finding or developing new drugs for prevention A. hydrophila harm.