Studies On Site-directed Mutation And Enzymatic Characterization Of YgjD Gene Of Vibrio Harveyi

Glycoprotease?Gcp?and its homologue proteins are a class of conserved proteins found in cells of bacteria,archaea,and eukaryotes,and play an important role in cell growth and vability.YgjD is a glycoprotease in bacterial cells.It is one of the most important proteins necessary for bacteria to adapt to the environment and survive.Recently,YgjD was found to form a complex with two other proteins,YjeE and YeaZ,which play an important role in the synthesis of N⁶-threonylcarbamoyladenosine of tRNA.In this paper,the expression,purification,and enzymatic properties of the YgjD protein of Vibrio harveyi were performed.The sites of the enzyme activity of the YgjD protein were studied using site-directed mutations.The effects of the YgjD recombinant protein on the bacterial growth and the recovery of the VBNC state were studied,and the main findings are as follows:The recombinant plasmid pET-28a-ygjD containing the ygjD gene of Vibrio harveyi was expressed in Escherichia coli,and purified by Ni affinity chromatography to obtain recombinant YgjD protein.The protease activity of the recombinant YgjD was determined with bovine serum albumin,ATEE,BTEE and BAPNA as substrates.It was found that YgjD had no significant hydrolysis effect on bovine serum albumin,but had significant hydrolytic activity on ATEE,BTEE and BAPNA,with the specific activities of 59000 U/mg,53700 U/mg and 8100 U/mg,respectively.The effects of metal ions and some chemical reagents on the protease activity of recombinant protein YgjD were studied.It was found that the protease activity of YgjD increased to155.55%after addition of 1 mM Zn²⁺,while Cu²⁺,Ca²⁺,Co²⁺,Mg²⁺,EDTA,EGTA and DTT partially inhibited the protease activities of YgjD.Mn²⁺and PMSF had the strongest inhibitory effect.The interaction between YgjD and YeaZ in vitro was analyzed by SDS-PAGE electrophoresis,and it was found that YeaZ could obviously hydrolyze YgjD,but YgjD had no obvious effect on YeaZ.YgjD and YeaZ had obvious interactions in vivo by using yeast two hybrid techniques.The amino acid residues His¹¹¹,Glu¹¹³ and His¹¹⁵ at the active site of the YgjD were mutanted with site-directed mutagenesis.The expression plasmids pET-28a-ygjD with different mutants were constructed,expressed in E.coli,and purifeied.The protease enzymes of different mutants of the YgjD were compared.It was found that the mutant proteins had reduced enzyme activities when compared with the wild-type YgjD.The protease activity of the mutant YgjD containing two mutation sites of H111A and H115A was completely lost,indicating that the two amino acids,His¹¹¹and His¹¹⁵,played an important role in maintaining protease activity.The recombinant YgjD has a significant growth-promoting effect on V.harveyi SF-1.Mutants containing H111A and E113A promoted the growth of SF-1,but the mutants of H115A and H111A+H115A had no significant growth-promoting effect.The recombinant YgjD could promote the growth of some bacteria,such as V.parahaemolyticus,V.alginolyticus and Staphylococcus aureus,but it has no obvious effect on Rhodococcus erythropolis and Bacillus subtilis.YgjD had no significant resuscitation effect on viable non culturable?VBNC?cells of V.harveyi.