Studies Of Glycosyltransferase And Phosphatase Involved In Mycothiol Biosynthesis And Heterologous Expression Of Butelasel

Glycosyltransferase is a large family of enzymes involved in the biosynthesis of oligosaccharides,polysaccharides,glycolipids,glycoproteins and other glycosides.Glycosyltransferases utilize activated sugars such as uridine diphosphate glucose?UDP-Glc?,guanosine diphosphate mannose?GDP-Man?,cytidine monophosphate sialic acid?CMP-Sia?as glycosyl donors and various receptors,which can be saccharides,lipids,proteins,antibiotics and nucleic acids,to produce ?-or ?-linked glycosylation products.Glycosyltransferases follow the "one enzyme for one type of glycosidic bond" principle to exhibit high stereoselectivity and regioselectivity for efficient synthesis of specific glycosidic bonds.Therefore,glycosyltransferases have been widely used in the synthesis of oligosaccharides.Mycothiol is a low-molecular weight thiol found only in actinomyces.Similar to glutathione,MSH plays a pivotal role in protecting bacteria against oxidative stresses and xenobiotics detoxication.Mycobacteria contain the highest MSH concentration among actinobacteria,and its MSH metabolism is extensively studied.MSH-deficient mutants exhibited increased sensitivities to oxidative stresses,alkylating agents,and a broad range of antibiotics,suggesting the great significance of MSH in mycobacterial survival and pathogenicity.In addition,it has been demonstrated that increasing the intracellular MSH content in Corynebacterium glutamicum improved its resistance to alkylating agents,glyphosate,ethanol,antibiotics,heavy metals,and so on.Therefore,the MSH biosynthetic pathway represents a promising target for the development of new antibacterial therapeutics or new agents that can improve the efficacies of current antibiotics.MSH biosynthesis consists of five enzymatic steps mediated respectively by an inositol N-acetylglucosaminyltransferase?GlcNAc-T??MshA?,a phosphatase?MshA2?,a GlcNAc deacetylase?MshB?,an ATP-dependent ligase?MshC?,and an acetyltransferase?MshD?.Except for MshA2,the other four enzymes from different bacterial species have been identified.The first dedicated step for MSH biosynthesis is MshA-mediated transfer of a GlcNAc residue from uridine diphosphate GlcNAc?UDP-GlcNAc?to 1L-myo-insitol-l-phosphate?1L-Ins-1-P?,affording GlcNAc-Ins-3-P.Until now,only one MshA derived from C.glutamicum is expressed,whereas many biochemical properties of MshA have not yet been identified.Thus,the study of MshA and other related enzymes in MSH biosynthesis is of great significance.In this study,we prepared the mshA gene derived from C.diphtheria.The cdmshA gene was ligated into pET-22b vector and the resulting recombinant plasmid was then transformed into E.coli BL21?DE3?,which was induced to express the target protein that was purified with Nickle chelation chromatography to homogeneity.The molecular weight of the protein was about 50 kDa,in agreement with CdMshA.The enzymatic activity of CdMshA was characterized using UDP-GlcNAc and 1L-Ins-1-P as substrates with HPLC analysis.The optimal temperature for CdMshA was 40 ?,while it was stable between 20 and 50 ?.The optimal pH for CdMshA was 8.0,and it maintained over 80%enzymatic activity in the pH range of 7?10.Mental cations were not needed for CdMshA activity,while some cations like Mg2+,Ca2+ and K+ could slightly enhance its activity.CdMshA had a rigid substrate selectivity.Various glycosyl donors,such as UDP-Gal,UDP-Glc,UDP-GalNAc and UDP-GlcA,and glycosyl acceptors including myo-inositol,glucose-6-phosphate,mannose-6-phosphate,galactose-6-phosphate,glucose-1-phosphate and 1 D-myo-inositol-1-phosphate?1D-Ins-1-P?,were not accepted by CdMshA as substrates.The Km and Kcat values of CdMshA for UDP-GlcNAc were 0.185 ±0.047 mM and 5.184 ±0.442 Min-1.The Km and Kcat values of CdMshA for 1L-Ins-1-P were 0.485 ± 0.049 mM and 6.907 ± 0.347 Min-1.The Km value of CdMshA for UDP-GlcNAc was comparable to that of reported MshA,while the Km value of CdMshA for 1L-Ins-1-P was about two times higher than that of the reported enzyme,indicating CdMshA had slightly lower affinity towards 1L-Ins-1-P than other similar enzymes.Finally,the enzymatric reaction product was isolated and purified by ion-exchange HPLC to verify the formation and yield?95%?of GlcNAc-?l,1-D-myo-inositol-3-phosphate?GlcNAc-Ins-3-P?,and its structure was characterized with HR ESI-MS and NMR data.For example,the coupling constant of the anomeric proton?JH1-2 = 3.6 Hz?of GlcNAc confirmed its a-configuration.Based on the CgMshA crystal structure and the results of multi-sequence allignment,D23,M27,N28,K81,Y113,T137 and R157 were identified to be relevant to the binding with 1L-Ins-1-P.We mutated these amino acids to alanine and prepared the mutant proteins.Their enzymatic activities were evaluated with UDP-GlcNAc and 1L-Ins-1-P under the optimal conditions.Results showed that N28A,K81A,and R157A mutations abolished almost completely the enzymatic activity,and the T137A mutant retained only ca.30%of activity compared to wild type of CdMshA.The D23A,M27A and Y113 A mutants exhibited a mild reduction in catalytic activity.It was proposed that K81/R157 and N28 should form hydrogen bonds with the phosphate and 4-OH groups of 1L-Ins-1-P,respectively,which would help keep 1L-Ins-1-P in proper orientation at the active site of CdMshA and hold its 3-OH group close to the pyrophosphate group of UDP-GlcNAc.This should be critical for the enzymatic reaction.T137 could also form a hydrogen bond with the phosphate group of 1L-Ins-1-P and,in the meantime,work together with M27 and Y113 to create a small pocket to accommodate the axial 2-OH group of 1L-Ins-1-P and limit its freedom to facilitate the reaction.All these results indicated that N28,K81,R157 and T137 played a critical role in the enzymatic activity of CdMshA.The phosphatase MshA2 involved in MSH biosynthesis has not been identified.According to the previous report,Rv3137,an inositol monophophatase gene derived from M.tuberculosis,can be a promising candidate.Therefore,we conducted the BLAST with Rv3137 in the C.glutamicum and found four homologous sequences with Genbank accession numbers of WP₀11013898.1,WP₀03861968.1,WP₀11265625.1 and WP 003862394.1,which were designated as ImpA,ImpB,ImpC and ImpD,respectively.The impA,impB,impC and impD gene contains 783,828,876 and 759 bp,respectively,and theoretical molecular weights for the encoded proteins were 28.9,30.2,31.9 and 27.3 kDa.These four genes were ligated to vectors to construct the expression vectors and the resulting vectors were transformed into E.coli BL21?DE3?.These recombinant proteins were expressed and purified to homogenity via affinity chromatography,of which ImpA,ImpB and ImpC contained a His-tag whereas ImpD contained a GST-Tag because ImpD was folded into inclusion bodies when expressed with His-tag.The phosphatase activities of the four purified proteins were conducted with GlcNAc-Ins-3-P as the substrate in Tris-HCl buffer containing 50 mM Tris and 2 mM Mg2+ at pH 8.0.Results showed that ImpC had good activity to hydrolyze the phosphate of GlcNAc-Ins-3-P whereas ImpA had very low activity and ImpB and GST-ImpD had no activity at all.However,studies on Imp A confirmed that it had fructose-1,6-bisphosphatase and 1D-Ins-1-P monophosphatase activities but was not active to GlcNAc-Ins-3-P and 1L-Ins-1-P.Above results indicate that ImpC is an MshA2 candidate,mediating the dephosphorylation of GlcNAc-Ins-3-P to produce GlcNAc-Ins and inorganic phosphate.Thus,its biochemistry was studied in detail.First,we optimized the reaction conditions of ImpC.ImpC had no enzymatic activity under acidic conditions and was stable in the pH range 7?9 with optimal pH at 8.0.Its enzymatic activity reduced dramtically when pH was over 10.0.The optimal temperature for ImpC was 40 ?.While it was stable at 25?50 ?,its activity reduced significantly at over 55 ?.Mg2+ was crucial for the activity of ImpC,and it was not active without Mg2+.The optimal Mg2+ concentration for ImpC was 5 mM,and the high concentration would inhibit ImpC activity.For example,only 70%enzymatic activity remained in the presence of 100 mM of Mg2+.Some other divalent cations such as Zn2+,Mn2+ and Co2+ could be used to replace Mg2+ but the activity recued over 80%.Ni2+ could rescued 50%fo the activity,while Ca2+,Cu2+ and Fe2+ had no positive influence of the activity.Li+ was a potent inhibitor of ImpC,and the IC50 was about 2 mM,and only 10%enzymatic activity remained with 20 mM of Li+,but high concentrations of Na+and K+ had no significant influence.ImpC showed broad substrate selectivity.It could dephosphorylate various phosphate compounds,such as GlcNAc-Ins-3-P,1L-Ins-1-P,1L-Ins-1-P,1D-Ins-1-P,fructose-1,6-diphosphate,glucose-1-phosphate,glycerol-phosphate,and p-nitrophenyl-phosphate?pNPP?,but GlcNAc-Ins-3-P,1L-Ins-1-P,and glucose-1-phosphate were the best substrates.ImpC showed selectivity in the conformation of inositol monophosphate,as its activity to hydrolyze 1D-Ins-1-P was much lower than that of 1L-Ins-1-P or GlcNAc-Ins-3-P,i.e.,its activity to 1D-Ins-1-P was only 35%of that to GlcNAc-Ins-3-P and 25%of that to 1L-Ins-1-P.The Km and Kcat values of ImpC for GlcNAc-Ins-3-P were be 1.29±0.52 mM and 37.11±4.66 s-1,respectively.The calcd MS m/z for C14H25NO11Na+[GlcNAc-Ins+Na]+was 406.13,and it was found to be 406.82.Via multi-sequence alignment,three amino residues were found conservative and thus were mutated to D62N,L96A,and D117N by site-directed mutation.The mutant proteins were purified to homogenity and their enzymatic activities were measured with GlcNAc-Ins-3-P.Results showed that L96A and D117N mutations almost completely eliminated the enzymatic activity,while D62N mutantion still retained ca.70%enzymatic activity compared to the wild type of ImpC,suggesting that L96 and D117 played a key role in the catalysis by ImpC.Peptide ligases are enzymes that can catalyze peptide-peptide ligation reactions with site specificity and substrate selectivity,which are highly desirable tools for protein engineering.Butelase 1?Btl1?was a ligase recently derived from butterfly pea Clitoria ternatea.Btll recognizes and react with a specific short peptide signal Asn/Asp-His-Val at the protein C-terminus,of which only Asn/Asp will remain after the reaction.Btll exhibits highly catalytic efficiency?1,340,000/M/s?for ligation,mild reaction conditions,and broad substrate selectivity,For example,it can accept most amino acids except proline linked to the signal peptide at the N-terminus.Thus,Btll have been used for protein N-terminal labelling,protein cyclization,synthesis of circular bacterioncins and protein thioesters,and macrocyclization of D-amino acid-containing peptides.However,access to Btll is still limitated,as it is purified from fresh pea pods with a yield of 5 mg/kg.Thus,recombinant expression of Btll is of great significance.Here,we constructed a btl1 E.coli expression system and Pichia expressions ystem.In E.coli,btll gene was ligated to different vectors,including pET-28b-btl1,pET-32 EK/LIC-btl1,pET-41 EK/LIC-btl1 and pMAL-c5E-btl1,to construct expression plasmids.The pET-series plasmids were transformed in E.coli BL21?DE3?and E.coli rosetta2 pLysS,whereas pMAL-c5E-btl1 was transformed in E.coli BL21?DE3?.pET-32 EK/LIC-btll and pET-41 EK/LUC-btl1 were induced to express target proteins in E.coli rosetta2 pLysS in inclusion bodies,while there was no protein expression in E.coli containing pET-28b-btl1.Btll was well expressed in x E.coli BL21?DE3?containing pMAL-c5E-btl1,and purified to homogenity in soluble form.In the Pichia expression system,the btll gene was ligated to pPICZ-B vector for intracellular expression or ligated to pPICZ-aC for secreted expression.The resulting plasmids were transformed into Pichia postoris KM71H,and the recombinant proteins were induced by methanol to express proteins in a soluble form.We used synthetic peptide KALVINHV and GIGGIR to test the enzymatic activity of the recombinant proteins.Unfortunately,they did not result in the desired product KALVINGIGGIR.These results indicating that the soluble recombinant Btll might have some difference in protein conformation compared with the natural Btl1.