Lysine Acetylation Regulates The Activity Of Vibrio Alginolyticus PykF Protein

Vibrio alginolyticus is a kind of opportunistic pathogen in the marine environment.Using of antibiotics in aquaculture has resulted in increased bacterial antibiotics resistance.Pyruvate kinase I(PykF),as one of the key metabolic enzymes,can regulate the metabolism of bacteria to adapt to the surrounding environment,and is a potential target of antibacterial drugs.The acetylated proteome of V.alginolyticus revealed that PykF protein had acetylation sites.Subsequently,PykF protein was successfully expressed and purified by prokaryotic expression system.Autoacetylation analysis was performed in vitro,and it showed that PykF could be autoacetylated by Ac P in vitro,while Ac Co A could not catalyze PykF autoacetylated in vitro.And within a certain range,with the increase of incubation time and incubation concentration,the acetylation level of PykF increased and its enzyme activity decreased correspondingly.The deacetylation experiments showed that Cob B could catalyze the deacetylation of PykF in vitro,and enzyme activity of PykF increased after Cob B treatment;The regulation of Cob B on PykF was analyzed in vivo,and the results showed that deletion of cob B gene increased the acetylation level of PykF,but there was no significant difference in pyruvate kinase activity compared with WT.Fast Mutagenesis System was used to mutate lysine residues into arginine and glutamine,and then the expression and purification were performed,and the enzyme activity was determined.The results showed that the different lysine sites had different effects on the acetylation level of PykF,and deacetylation of K52,K68,K317 or K382 sites significantly reduced the acetylation level of PykF.There were no significant changes in acetylation levels at other sites.Deacetylation of K13,K368,or K382 significantly enhanced pyruvate kinase activity,while deacetylation of K145 or K319 did not.On the other hand,deacetylation of K52 R or K317 R resulted in a loss of pyruvate kinase activity of 80%,while deacetylation of K19 R,K59R,K68 R or K340 resulted in almost no activity.The pyruvate kinase activity of ΔpykF decreased by about60% compared with WT.ΔpykF:K52R,ΔpykF:K68R and ΔpykF:K317R were significantly lower than ΔpykF:pykF.These indicated that deacetylation of K52,K68 and K317 is key to regulate pyruvate kinase activity of V.alginolyticus.Then we compared the differences of ΔpykF strain,complement strain and sitedirected mutagenesis complemented strains on the growth,extracellular protease and LD50 of V.alginolyticus.The results showed that the growth rate of WT:pykF overexpressed strain was similar to that of WT,while that of ΔpykF strain was significantly reduced.In addition,compared with ΔpykF:pykF,the growth rate of sitedirected complemented mutagenesis strain did not significant change.Compared with WT strain,the extracellular protease activity of WT:pykF was not significantly different,but ΔpykF was decreased significantly.Compared with ΔpykF:pykF,the extracellular protease activity of ΔpykF:K52R and ΔpykF:K68R decreased significantly,whileΔpykF:K317R did not change significantly.LD50 of ΔpykF was about 6 times that of WT.Compared with ΔpykF: pykF,LD50 of ΔpykF: K52 R and ΔpykF: K68 R was about3 and 4 times of that of ΔpykF: pykF,while ΔpykF:K317R did not change significantly.In summary,this study found that acetylation modification could regulate activity of PykF protein and thus affect virulence of V.alginolyticus.And provide a new idea for the development of new antimicrobial drug targets of vibrios.