Reactivate A Sialyltransferase By The Mutation Y141D And The Unique Role Of A Hydrogen Bond Chain Around The Activity Site

Sialic acids are a family of negatively charged α-keto acids with a nine-carbon backbone and typically found at the outermost end of glycan chains of all cell types.Sias are involved in a wide range of biological and pathological processes, such as cell-cell communication, signal transduction, bacterial and viral infection, as well as cancer metastasis.Sialyltransferases are key enzymes involved in the biosynthesis of biologically and pathologically important sialic acid-containing molecules in nature.Among them, bacteria-derived SiaTs have been widely used to synthesize a variety of Sia-containing structures owing to their flexible substrate specificity and productive expression in Escherichia coli.In this study, a putative GT80 sialyltransferase(Pm0106) harbouring a natural mutation of D141 Y in the conserved DDG motif, which has been identified in GT52 and GT80 families, was demonstrated to be inactive. The activity of Pm0106 was restored by a reverse mutation of Y141 D. In addition, a hydrogen bond chain(Asp141.Asn109.Asp33 in PmST1) connecting the second Asp of DDG motif, the ends of Nβ 4 and Nβ1, and Nα1 helix was found to be conserved in structures of four GT80 sialyltransferases. To explore the mutation of Y141 D and the hydrogen bond chain for Pm0106,mutatants of Y141 D,M144D141D,D141 N,D141N-N109 D,D141N-N109D-D33 N,N109A141D,D33A141 D and D33L141 D were made.Firstly, we used the pET-15 b plasmid as a template to amplify the Pm0106 gene.NdeI and XhoI as the restriction enzyme cutting site, through double enzyme digestion recombinant plasmid. A large amounts of exogenous recombinant protein was produced by induced expression of 0.1mmol L IPTG. We analyzed the mutant proteins were produced by SDS-PAGE and detect the proteins concentration. Establish a HPLC method for the detection of eachmutant enzyme activity, through the establishment of different pH reaction system, HPLC analysis of each product peak, to get the most suitable pH for the reaction. TLC was used to detect the enzyme activity of Y141 D, N109A141 D and D33A141 D at 45 ℃ and 50 ℃after 30 min, comparing the stability of each mutant.The analysis of the reactions were result in that the proportion of reaction product and the remaining substrate is appropriate for Y141 D,M144D141D, N109A141D1/80 times,however for D141 N, D33A141 D, D33L141 D 1 times participate in the reaction,While D141N-N109 D, D141N-N109D-D33 N almost no reaction. Through the HPLC testing, in the buffer solution of pH was up to 8 when the reaction reached the maximum. Through the TLC we can see both a-Lac and β-Lac as receptor, wild type Pm0160 is not active, while the mutant strains Y141 D, M144D141 D, N109A141 D, D33A141 D,D33L141D and PmST1 have good reaction activity, mutant D141 N, D141N-N109 D, D141N-N109D-D33 N have no reaction activity. Y141 D, N109A141 Dand D33A141Dwere induced at 45℃and 50 ℃for 30 min before addtion to reactions. Though three mutants were still active towards β-Lac after induced at 45 ℃ for 30 min, only Y141 D mutant remained obvious(around 50%)activity after three enzymes were induced at 50 ℃ for 30 min.Our mutagenesis experiments indicated that the hydrogen bond formed by Asp141 and the neighbouring Asn109 positioned at the end of Nβ4 seems to play an essential role in maintaining protein structural stability other than keeping the general base Asp141 in a productive orientation for transferring sialic acid, while the interaction between Asp33 and Ala39 located on Nα1 helix probably plays a role in regulating the enzyme activity.