Characterization of Aspartate Kinase from Corynebacterium Pekinense and the Critical Site of Arg169

Article Characterization of Aspartate Kinase from Corynebacterium pekinense and the Critical Site of Arg169

Weihong Min 1,2,*, Huiying Li 1,2, Hongmei Li 1,2, Chunlei Liu 1,2 and Jingsheng Liu 1,2

Received: 19 August 2015; Accepted: 11 November 2015; Published: 27 November 2015 Academic Editor: Qiang “Shawn” Chen

1 College of Food Science and Engineering, Jilin Agricultural University, Changchun 130118, China; [email protected] (H.L.); [email protected] (H.L.); [email protected] (C.L.); [email protected] (J.L.) 2 National Engineering Laboratory of Wheat and Corn Deep Processing, Changchun 130118, China * Correspondence: [email protected]; Tel.: +86-431-8453-3329; Fax: +86-431-8451-7235

Abstract: Aspartate kinase (AK) is the key enzyme in the biosynthesis of aspartate-derived amino acids. Recombinant AK was efficiently purified and systematically characterized through analysis under optimal conditions combined with steady-state kinetics study. Homogeneous AK was predicted as a decamer with a molecular weight of ~48 kDa and a half-life of 4.5 h. The enzymatic activity was enhanced by ethanol and Ni2+. Moreover, steady-state kinetic study confirmed that AK is an allosteric enzyme, and its activity was inhibited by allosteric inhibitors, such as Lys, Met, and Thr. Theoretical results indicated the binding mode of AK and showed that Arg169 is an important residue in substrate binding, catalytic domain, and inhibitor binding. The values of the kinetic parameter Vmax of R169 mutants, namely, R169Y, R169P, R169D, and R169H AK, with L-aspartate as the substrate, were 4.71-, 2.25-, 2.57-, and 2.13-fold higher, respectively, than that of the wild-type AK. Furthermore, experimental and theoretical data showed that Arg169 formed a hydrogen bond with Glu92, which functions as the entrance gate. This study provides a basis to develop new enzymes and elucidate the corresponding amino acid production.

Keywords: Corynebacterium pekinense; aspartate kinase; characterization; molecular docking

1. Introduction Aspartate kinase (AK) is the first identified important enzyme in the biosynthesis of several amino acids, such as Lys, Thr, and Met, which are essential to mammals. AK is a multimer and an allosteric enzyme that catalyzes the phosphorylation of aspartate to form aspartyl-P [1–3]. Allosteric regulation (feedback inhibition) of proteins controls the synthesis pathway of this enzyme [4]. AK is classified based on subunit organization as a homo-oligomer or heterotetramer. AK can also be classified into three types, namely AK-III, AK-II, and AK, depending on the source organism [5,6]. AK-III from Escherichia coli, AK-I from Arabidopsis thaliana, and AK from Methanocaldococcus jannaschii are homo-oligomers; furthermore, AK from Thermus thermophilus, AK-II from Bacillus subtilis, and AK from Corynebacterium glutamicum are heterotetramers [7–9]. AK from Corynebacterium pekinense (CpAK) shares 98.5% sequence identity with AK from C. glutamicum (AK-II), with an α2β2-type structure containing two α and β subunits [4,10,11] (Figure1). Each αβ dimer contains two lysine binding sites [12], in which one site is exclusively found in the dimer with A and B chains [13–15] located at the interface between α and β subunits. The presence of this exclusive site indicates that the lysine-binding site in the regulatory region of CgAK performs a vital function in AK allosteric inhibition [16,17].

Int. J. Mol. Sci. 2015, 16, 28270–28284; doi:10.3390/ijms161226098 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2015, 16, page–page Int. J. Mol. Sci. 2015, 16, 28270–28284 Int. InJ. Mol. this Sci. study, 2015, 16 we, page–page describe CpAK, a class II AK with allosteric effectors, namely Lys and Thr, and determine the regulatory functions of this enzyme. By combining experimental and theoretical data, InIn this this study, study, we we describe describe CpAK, CpAK, aa classclass IIII AK wi withth allosteric allosteric effectors, effectors, namely namely Lys Lys and and Thr, Thr, and and we reported an important residue, namely Arg169. This residue is involved in enzyme catalysis and determinedetermine the the regulatory regulatory functions functions ofof thisthis enzyme.enzyme. By combining combining experimental experimental and and theoretical theoretical data, data, forms a hydrogen bond with Glu92, which functions as the entrance gate of AK. This study provides wewe reported reported an an important important residue, residue, namely namely Arg169.Arg169. This residue residue is is involved involved in in enzyme enzyme catalysis catalysis and and a basis for the design of an allosteric control system for aspartate bioproduction. formsforms a hydrogena hydrogen bond bond with with Glu92, Glu92, whichwhich functionsfunctions as the the entrance entrance gate gate of of AK. AK. This This study study provides provides a basisa basis for for the the design design of of an an allosteric allosteric control control systemsystem for aspartate bioproduction. bioproduction.

Figure 1. Multiple sequence alignment of aspartate kinase (AK) with other members. CpAK from C. Figure 1. Multiple sequence alignment of aspartate kinase (AK) with other members. CpAK from C. Figurepekinense 1.; CgAKMultiple from sequence C. glutamicum alignment, 99%; ofCcAK aspartate from C. kinase callunae (AK), 94%; with CaAK other from members. C. accolens CpAK, 87%; pekinense; CgAK from C. glutamicum, 99%; CcAK from C. callunae, 94%; CaAK from C. accolens, 87%; fromGsAKC. from pekinense Gordonia; CgAK sputi, from77%; DcAKC. glutamicum from Dietzia, 99%; cinnamea CcAK, 76%; from andC. callunaeMgAK ,from 94%; Mycobacterium CaAK from GsAK from Gordonia sputi, 77%; DcAK from Dietzia cinnamea, 76%; and MgAK from Mycobacterium C.gilvum accolens, 74%., 87%; Percentages GsAK from representGordonia the degree sputi, 77%; of amino DcAK acid from sequenceDietzia identity. cinnamea In, 76%;ascending and MgAK order, fromgilvumMycobacterium, 74%. Percentages gilvum ,represent 74%. Percentages the degree representof amino acid the degreesequence of aminoidentity. acid In ascending sequence order, identity. sequencesequence similarity similarity is is highlighted highlighted inin yellow,yellow, sky blue,blue, pink, pink, and and blue. blue. In ascending order, sequence similarity is highlighted in yellow, sky blue, pink, and blue.

2. 2.Results Results and and Discussion Discussion 2. Results and Discussion 2.1.2.1. Structural Structural Differences Differences between between thethe ActiveActive and InactiveInactive Forms Forms of of Aspartate Aspartate Kinase Kinase 2.1. Structural Differences between the Active and Inactive Forms of Aspartate Kinase WeWe constructed constructed AK AK modelsmodels byby usingusing two crystalcrystal structuresstructures of of AKs AKs (active (active form: form: PDB PDB code code We constructed AK models by using two crystal structures of AKs (active form: PDB code 3AB2; 3AB2;3AB2; inactive inactive form: form: PDB PDB code code 3AAW)3AAW) through SwSwississ modelermodeler online online (sequence (sequence identity identity = 98.5%)= 98.5%) inactive[11].[11]. Figure Figure form: 2a,b 2a,b PDB show show code that that 3AAW) thethe twotwo through structuresstructures Swiss werewere modeler highlyhighly online similar similar (sequence to to each each other, identityother, but but =Figure 98.5%)Figure 2c,d [2c,d11 ]. Figureindicateindicate2a,b that that show their their that protein protein the two contact contact structures potentialpotential were differed highly;; thatthat similar is, is, the the to active eachactive other,form form ofbut of AK AK Figure presented presented2c,d indicatea more a more thatcompactcompact their 3D protein 3D structure structure contact than than potential thethe inactiveinactive differed; form. that FiguFigu is,re there 3a3a active showsshows form the the ofsuperimposed superimposed AK presented structures structures a more compactof ofthe the 3Dactiveactive structure (purple) (purple) than and and the inactive inactive inactive (green)(green) form. forms.forms. Figure Resi3a showsduesdues 131131 the toto superimposed 171 171 differed differed between structuresbetween the the oftwo thetwo forms. activeforms. (purple)SequenceSequence and alignment alignment inactive results (green)results showed forms.showed Residues thatthat Asp92 131 funcfunc to 171tionstions differed as as a a catalytic catalytic between residue residue the two [10]. [10]. forms. As As shown Sequenceshown in in alignmentFigureFigure 3b, 3b, resultsGlu92 Glu92 showedformed formed thataa saltsalt Asp92 bridgebridge functions with Lys1 as6969 a catalyticinin thethe inactiveinactive residue form, form, [10]. but Asbut no shown no salt salt bridge in bridge Figure was 3wasb, Glu92foundfound formedin in the the active a active saltbridge form form (Figure with(Figure Lys169 3c).3c). ThisThis in the structur inactivealal form,differencedifference but nomay may salt result result bridge in in wasthe the fluctuation found fluctuation in the in activeinthe the catalytic efficiency of AK. formcatalytic (Figure efficiency3c). This of structuralAK. difference may result in the ﬂuctuation in the catalytic efﬁciency of AK.

Figure 2. Cont. FigureFigure 2.2. Cont.Cont. 2 2 28271 Int. J. Mol. Sci. 2015, 16, 28270–28284 Int. J. Mol. Sci. 2015, 16, page–page

FigureFigure 2.2. Three-dimensional (3D)(3D) structurestructure andand proteinprotein contactcontact potentialpotential ofof AKAK (active(active form:form: PDBPDB IDID 3AB2;3AB2; inactive form: PDB PDB ID ID 3AAW). 3AAW). (a (a) )3D 3D structure structure of of the the inactive inactive form form of of AK; AK; (b ()b 3D) 3D structure structure of ofthe the active active form form of ofAK; AK; color color purple purple represents represents for for β-sheet,β-sheet, and and color color green green represents represents for for α-heliex;α-heliex; (c) (proteinc) protein contact contact potential potential of ofthe the inactive inactive form form of of AK; AK; and and ( (dd)) protein protein contact potentialpotential ofof thethe activeactive formform ofof AK.AK. Red represents negative negative char charge,ge, and and blue blue represents represents positive positive charge. charge.

Figure 3. Structure of active/inactive forms of AK. (a) Superimposed structures of the active (purple) FigureFigure 3.3. StructureStructure ofof active/inactiveactive/inactive forms of AK. (a) Superimposed structuresstructures ofof thethe activeactive ((purplepurple)) and inactive forms (green); color blue represents for open state and color red represents for close andand inactiveinactive formsforms ( green(green);); color color blue blue represents represents for for open open state state and and color color red representsred represents for close for state;close state; (b) E92 formed a salt bridge with R169 in the inactive form; (c) E92 did not form a salt bridge (state;b) E92 (b formed) E92 formed a salt bridgea salt bridge with R169 with inR169 the in inactive the inactive form; form; (c) E92 (c did) E92 not did form not aform salt a bridge salt bridge with with R169 in the active form. R169with inR169 the in active the active form. form.

2.2. Binding Position of AK 2.2.2.2. Binding PositionPosition ofof AKAK The structure of the substrate aspartic acid was obtained using Gaussian View 4.0 [12] and TheThe structurestructure ofof the the substrate substrate aspartic aspartic acid acid was was obtained obtained using using Gaussian Gaussian View View 4.0 [ 124.0] and[12] thenand then optimized with Gaussian 09 software through the B3LYP methods at 6-311G* setting [13]. optimizedthen optimized with Gaussianwith Gaussian 09 software 09 software through thro theugh B3LYP the methodsB3LYP methods at 6-311G* at setting6-311G* [13 setting]. Aspartic [13]. Aspartic acid was then docked to AK by using Auto Dock Vina. acidAspartic was thenacid was docked then to docked AK by to using AK Autoby using Dock Auto Vina. Dock Vina. Figure 4a shows the substrate docked in the AK structure, with aspartic acid in the groove. FigureFigure4 a4a shows shows the the substrate substrate docked docked in in the the AK AK structure, structure, with with aspartic aspartic acid acid in in the the groove. groove. Aspartic acid possessed a negative charge and was located in the groove with a positive charge, AsparticAspartic acidacid possessedpossessed aa negativenegative chargecharge andand waswas locatedlocated inin thethe groovegroove withwith aa positivepositive charge,charge, thereby facilitating charge transfer. therebythereby facilitatingfacilitating chargecharge transfer.transfer. Four hydrogen bonds were present between Asp and AK (Figure 4b). S172 formed two FourFour hydrogenhydrogen bonds bonds were were present present between between Asp andAsp AK and (Figure AK 4(Figureb). S172 4b). formed S172 two formed hydrogen two hydrogen bonds with aspartic acid (1.97 and 1.70 Å), whereas D173 formed a hydrogen bond with bondshydrogen with bonds aspartic with acid aspartic (1.97 and acid 1.70 (1.97 Å), and whereas 1.70 Å), D173 whereas formed D173 a hydrogen formed a bond hydrogen with the bond O atomwith the O atom with aspartic acid. Another hydrogen bond (2.26 Å) was formed between aspartic acid withthe O aspartic atom with acid. aspartic Another acid. hydrogen Another bondhydrogen (2.26 bond Å) was (2.26 formed Å) was between formed aspartic between acid aspartic and K25.acid and K25. Moreover, E92 and S59 formed a hydrogen bond with aspartic acid. Figure 4b shows that Moreover,and K25. Moreover, E92 and S59 E92 formed and S59 a hydrogen formed a bondhydrogen with bond aspartic with acid. aspartic Figure acid.4b shows Figure that 4b V231,shows L88, that V231, L88, and G171 formed electronic contact with aspartic acid, whereas D173, S59, S72, K25, E92, andV231, G171 L88, formed and G171 electronic formed contactelectronic with contact aspartic with acid, aspartic whereas acid, D173, whereas S59, D173, S72, K25, S59, E92,S72, K25, T65, E92, and T65, and R169 formed van der Waals interactions with aspartic acid. Therefore, the binding pocket R169T65, and formed R169 van formed der Waals van der interactions Waals interactions with aspartic with acid. aspartic Therefore, acid. Therefore, the binding the pocket binding between pocket between aspartic acid and AK may contain V231, L88, G171, D173, S59, S72, K25, E92, T65, and R169, asparticbetween acid aspartic and AKacid may and contain AK may V231, contain L88, V231, G171, L88, D173, G171, S59, D173, S72, K25, S59, E92,S72, T65,K25, and E92, R169, T65, withand R169, D92 with D92 as the catalytic site-binding residue, which is in agreement with the experimental data aswith the D92 catalytic as the site-binding catalytic site-binding residue, which residue, is in agreementwhich is in with agreement the experimental with the dataexperimental [10]. data [10]. [10]. The 11 selected residues of the active binding site were used for computational alanine scanning The 11 selected residues of the active binding site were used for computational alanine to exploreThe 11 the selected function residues of each residueof the onactive substrate binding binding. site were Figure used4c shows for computational the mutated residues. alanine scanning to explore the function of each residue on substrate binding. Figure 4c shows the mutated residues. The R169A mutation decreased the binding affinity of the substrate by 3.72 kcal·mol−1. The 28272 3 Int. J. Mol. Sci. 2015, 16, 28270–28284

TheInt. R169A J. Mol. Sci. mutation 2015, 16, page–page decreased the binding afﬁnity of the substrate by 3.72 kcal¨ mol´1. The other main binding components included residues S172 and G171, which present minimal effects when other main binding components included residues S172 and G171, which present minimal effects mutatedwhen tomutated alanine. to Therefore,alanine. Therefore, R169, S172, R169, and S172, G171 an mayd G171 be themay key be the residues key residues in the substrate in the substrate binding site.binding We selected site. We R169 selected for further R169 for study. further study.

FigureFigure 4. Substrate 4. Substrate binding binding site site of of AK. AK. ( a(a)) Aspartic Aspartic acidacid locatedlocated in the active groove. groove. Red Red represents represents negativenegative charge, charge, and and blue blue represents represents positive positive charge; charge; ((bb)) ImportantImportant residues ar aroundound aspartic aspartic acid acid binding;binding; color color purple purple represents represents for electrostaticfor electrostati contactsc contacts and and color color green green represents represents forvan for dervanwaals der ´1 contacts;waals (contacts;c) Computational (c) Computational alanine mutantsalanine mutants (kcal¨ mol (kcal·mol) calculated−1) calculated for aspartic for aspartic acid-AK. acid-AK.

2.3.2.3. Expression Expression and and Purification Purification of of Wild Wild Type Type and and Mutant Mutant AK AK High-activityHigh-activity mutants, mutants, namely,namely, R169Y,R169Y, R169P,R169P, R169D,R169D, and R169H, were were obtained obtained by by high-throughputhigh-throughput screening. screening. After After native native polyacrylamide polyacrylamide gel gel electrophoresis electrophoresis (PAGE), (PAGE), a 130 a kDa130 kDa band wasband observed was observed (Figure5a, (Figure Figure 5a, S1); Figure this finding S1); this indicated finding that indicated AK is athat heterotetramer. AK is a heterotetramer. Purified AK wasPurified subjected AK towas sodium subjected dodecyl to sodium sulfate dodecyl (SDS)-PAGE, sulfate (SDS)-PAGE, and two bands and weretwo bands observed were throughout observed thethroughout purification the progress purification (Figure progress5b, Figure (Figure S2). 5b, Figure The 47 S2). and The 18 47 kDa and bands18 kDacorresponded bands corresponded to the to the AK α- and β-subunits, respectively, which is consistent with the experimental results AK α- and β-subunits, respectively, which is consistent with the experimental results reported by reported by Kalinowsk et al. [18]. Kalinowsk et al. [18].

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Figure 5. Native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate Figure 5. Native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the recombinant AK and its mutants. (a) Native polyacrylamide gel electrophoresis (SDS-PAGE) of the recombinant AK and its mutants. (a) Native PAGE of the recombinant AK and the mutants. M: molecular weight marker; lane 1: purified PAGE of the recombinant AK and the mutants. M: molecular weight marker; lane 1: purified recombinant R169Y; lane 2: purified recombinant R169P; lane 3: purified recombinant R169D; lane 4: recombinant R169Y; lane 2: purified recombinant R169P; lane 3: purified recombinant R169D; purified recombinant R169H; and (b) SDS-PAGE of the recombinant AK and the mutants. M: lane 4: purified recombinant R169H; and (b) SDS-PAGE of the recombinant AK and the mutants. high-molecular weight protein marker; lane 1: purified recombinant R169Y; lane 2: purified M: high-molecular weight protein marker; lane 1: purified recombinant R169Y; lane 2: purified recombinant R169P; lane 3: purified recombinant R169D; lane 4: purified recombinant R169H; lane recombinant R169P; lane 3: purified recombinant R169D; lane 4: purified recombinant R169H; lane 5: 5: supernatant of induced sample; and lane 6: Western blot of the purified AK. supernatant of induced sample; and lane 6: Western blot of the purified AK. 2.4. Kinetic Assay of the Wild Type (WT) and AK Mutants 2.4. Kinetic Assay of the Wild Type (WT) and AK Mutants As shown in Table 1, kinetic parameters, namely, Vmax, Km, and nH (Hill coefficient), with the substrateAs shown L-aspartate, in Table were1, kinetic 96.07 U/mg·s parameters,−1, 4.56 namely,mol/L, andV max2.82,, Krespecm, andtively. nH Based (Hill on coefficient), the dynamic with thedata substrate fitting Lresults,-aspartate, the fitting were 96.07curve U/mgdid not¨ s ćo1,nform 4.56 mol/L,to the Michaelis–Menten and 2.82, respectively. equation Based but onwas the dynamicconsistent data with fitting the results, Hill equation. the fitting This curve finding did not indicated conform that to theAK Michaelis–Mentenis an allosteric enzyme equation with but waspositive consistent cooperativity. with the HillAt the equation. site of the This R169 finding mutant, indicated the Vmax thatvalues AK were is an 4.71, allosteric 2.25, 2.24, enzyme and 2.12 with positivetimes higher cooperativity. than those At of the the site wild of type the R169(WT) mutant,for R169Y, the R169P,Vmax R169D,values and were R169H, 4.71, 2.25,respectively. 2.24, and 2.12However, times higher positive than cooperativity those of the decreased wild type (WT)in all mutants. for R169Y, The R169P, nH values R169D, of and R169Y, R169H, R169P, respectively. R169D, However,and R169H positive were cooperativity1.13, 1.95, 0.91, decreasedand 1.09, respectively, in all mutants. which The are nH consistent values ofwith R169Y, Michaelis–Menten R169P, R169D, andenzymes. R169H wereThe K 1.13,m of each 1.95, mutant 0.91, and increased 1.09, respectively, relative to WT which, which are indicated consistent the with weakened Michaelis–Menten affinity to L-aspartate. Of the four mutants, the R169Y mutant with the highest activity was selected for enzymes. The Km of each mutant increased relative to WT, which indicated the weakened affinity further study. to L-aspartate. Of the four mutants, the R169Y mutant with the highest activity was selected for further study. Table 1. Kinetic parameters of wild type (WT) and mutants.

ParametersTable 1. KineticWT parametersR169Y of wild R169P type (WT) and R169D mutants. R169H Vmax (U/mg) 96.07 452.78 216.55 208.77 204.25 KmParameters (mol·L−1) 4.56 WT 4.98 R169Y R169P4.95 R169D6.29 R169H8.79

VmaxnH(U/mg) 2.82 96.07 1.13 452.78 216.551.95 208.770.91 204.251.09 ´1 Km (mol¨ L ) 4.56 4.98 4.95 6.29 8.79 2.5. Optimum Temperature,nH pH, and Thermo 2.82stability 1.13 of Wild 1.95Type (WT) 0.91and R169Y1.09

L-Aspartate was used as the substrate to study the optimum catalytic conditions of WT and 2.5.R169Y. Optimum As shown Temperature, in Figure pH, 6a, and the Thermostability optimum reaction of Wild temperatures Type (WT) and of R169YWT and R169Y were 26 and 35 °C, respectively. The optimum pH values of WT and R169Y were 7.0 and 9.0, respectively. L-Aspartate was used as the substrate to study the optimum catalytic conditions of WT and R169Y.Compared As shown with in neutral Figure 6WT,a, the R169Y optimum became reaction a typical temperatures alkaline ofenzyme WT and when R169Y the were optimum 26 and temperature increased by 9 °C. 35 ˝C, respectively. The optimum pH values of WT and R169Y were 7.0 and 9.0, respectively. The results of thermal stability experiments performed at 26 °C and pH 8.0 are shown in Figure Compared with neutral WT, R169Y became a typical alkaline enzyme when the optimum temperature 6c. The half-life of WT was 4.5 h, and the half-life of the mutant R16 9Y increased to 5.5 h. This increased by 9 ˝C. result indicated that the 3D structure of R169Y is more stable than that of WT. The results of thermal stability experiments performed at 26 ˝C and pH 8.0 are shown in Figure6c. The half-life of WT was 4.5 h, and the half-life of the mutant R16 9Y increased to 5.5 h. This result indicated that the 3D structure of R169Y5 is more stable than that of WT.

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Figure 6. Optimum temperature ( a),); pH (b);), and thermostability (c) curves ofof WTWT andand R169Y.R169Y.

2.6. Effect of Metal Ions and Organic SolventsSolvents onon WTWT andand R169YR169Y The effect ofof metalmetal ionsions onon thethe activityactivity ofof WTWT andand thethe R169YR169Y mutantmutant isis shownshown inin TableTable2 .2. TheThe concentrations ofof metal metal ions ions investigated investigated were 0.2,were 1, 5,0.2, and 1, 10 5, mM. and For 10 WT, mM. different For concentrationsWT, different 2+ ofconcentrations Ni2+, especially of Ni 0.2 mM,, especially induced 0.2 the mM, activation. induced At the this activation. concentration, At this enzymatic concentration, activity enzymatic increased + nearlyactivity 1.6-fold. increased K +nearlyimproved 1.6-fold. the activityK improved only atthe low activity concentrations only at low (0.2 concentrations mM). Other metal (0.2 mM). ions, 2+ 2+ 2+ includingOther metal Ca ions,2+, Zn including2+, and MnCa2+, ,Zn all, exerted and Mn inhibitory, all exerted effects. inhibitory For the effects. R169Y For mutant, the R169Y enzymatic mutant, + 2+ activityenzymatic decreased activity indecreased most of metalin most ions of ofmetal various ions concentrations,of various concentrations, except for K except+, Zn2+ for, Cu K2+, ,Zn and, 2+ 2+ + + NiCu2+, and which Ni promoted, which promoted the activation the activation of enzymatic of enzymatic activity at activity 5 mM. Naat +5 andmM. K Na+ slightly and K increased slightly activityincreased at 1activity mM. In at summary, 1 mM. In the summary, inhibition the of inhibi the R169Ytion of mutant the R169Y decreased mutant relative decreased to WT. relative to WT. The effects of organic solvents on the activity of WT and R169Y are shown in Table3. The concentrationsThe effects of of organic organic solvents solvents investigated on the activity were 1%,of WT 5%, and 10%, R169Y and 20%. are Forshown WT, in concentrations Table 3. The ofconcentrations nearly all organic of organic solvents solvents severely investigated inhibited were the enzyme,1%, 5%, 10%, with and inhibition 20%. For by WT, glycerol concentrations being the mostof nearly signiﬁcant. all organic Only solvents ethanol severely signiﬁcantly inhibited activated the enzyme, the enzyme with at inhibition concentrations by glycerol of 1%, being 5%, andthe 10%.most However,significant. all Only organic ethanol solvents significantly inhibited activa the enzymaticted the enzyme activity at of concentrations R169Y. of 1%, 5%, and 10%. However, all organic solvents inhibited the enzymatic activity of R169Y.

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Table 2. Effects of metal ions on WT and R169Y.

WT R169Y Enzyme Relative Activity (%) Relative Activity (%) Metal ions (mM) 0.2 1 5 10 0.2 1 5 10 Control 100 100 100 100 100 100 100 100 Na+ ND 75.44 ˘ 0.47 11.40 ˘ 1.44 5.26 ˘ 0.33 46.11 ˘ 0.88 101.48 ˘ 2.23 89.20 ˘ 0.43 39.75 ˘ 0.74 K+ 137.72 ˘ 1.54 62.28 ˘ 1.03 55.26 ˘ 1.31 45.61 ˘ 1.04 54.51 ˘ 1.09 118.10 ˘ 1.94 105.61 ˘ 0.98 84.03 ˘ 1.43 Ca2+ 57.02 ˘ 1.09 38.60 ˘ 0.89 14.91 ˘ 0.33 10.53 ˘ 0.43 33.40 ˘ 0.95 41.60 ˘ 0.74 92.33 ˘ 1.45 66.24 ˘ 1.47 Mg2+ 54.39 ˘ 0.87 55.26 ˘ 1.53 57.90 ˘ 1.77 43.42 ˘ 0.74 35.34 ˘ 1.54 40.74 ˘ 2.03 76.12 ˘ 1.78 62.84 ˘ 1.45 Zn2+ 90.35 ˘ 1.89 82.46 ˘ 1.34 78.07 ˘ 1.64 71.93 ˘ 0.45 49.39 ˘ 0.43 75.54 ˘ 1.54 164.02 ˘ 1.95 95.87 ˘ 0.74 Mn2+ 78.07 ˘ 1.54 70.18 ˘ 2.04 40.35 ˘ 0.54 10.53 ˘ 0.32 64.27 ˘ 1.54 36.71 ˘ 0.87 18.14 ˘ 0.32 ND Cu2+ 57.02 ˘ 0.43 42.11 ˘ 1.54 40.64 ˘ 0.68 32.46 ˘ 0.39 24.74 ˘ 1.43 93.12 ˘ 1.87 106.84 ˘ 0.45 73.51 ˘ 1.54 Fe3+ 78.07 ˘ 0.58 44.74 ˘ 1.54 33.33 ˘ 1.81 16.67 ˘ 0.85 86.10 ˘ 1.73 68.01 ˘ 0.39 55.73 ˘ 1.82 ND Ni2+ 159.94 ˘ 1.54 143.57 ˘ 1.05 129.30 ˘ 1.74 122.22 ˘ 2.57 21.34 ˘ 0.38 75.66 ˘ 1.47 164.90 ˘ 0.78 62.08 ˘ 1.94 ND, enzyme activity could not be determined.

Table 3. Effects of organic solvents on WT and R169Y.

WT R169Y Enzyme Relative Activity (%) Relative Activity (%) Concentration (%) Concentration (%) Organic solvents 1 5 10 20 1 5 10 20 Control 100 100 100 100 100 100 100 100 methanol 45.56 ˘ 1.74 64.44 ˘ 0.64 113.33 ˘ 0.54 46.67 ˘ 0.92 43.04 ˘ 0.29 5.0 ˘ 0.32 ND ND ethanol 143.33 ˘ 0.43 182.22 ˘ 1.54 154.44 ˘ 2.01 22.22 ˘ 0.41 30.91 ˘ 1.94 26.67 ˘ 1.02 18.84 ˘ 0.32 7.71 ˘ 0.55 isopropyl alcohol 30.0 ˘ 1.56 46.67 ˘ 2.04 74.45 ˘ 1.09 67.78 ˘ 1.87 39.41 ˘ 0.54 14.07 ˘ 0.25 ND ND n-butanol 23.33 ˘ 0.31 20.0 ˘ 0.51 ND ND 30.95 ˘ 0.93 ND ND ND acetonitrile 41.11 ˘ 0.43 11.11 ˘ 0.23 13.33 ˘ 0.96 ND 42.39 ˘ 2.06 36.33 ˘ 1.03 29.48 ˘ 0.62 16.96 ˘ 1.09 dimethyl sulfoxide 62.22 ˘ 1.73 31.11 ˘ 1.47 13.33 ˘ 0.95 ND 26.24 ˘ 1.53 18.46 ˘ 0.34 16.15 ˘ 0.49 11.44 ˘ 1.55 glycerol 25.56 ˘ 1.07 ND ND ND 66.61 ˘ 1.33 53.83 ˘ 0.52 37.01 ˘ 1.54 3.94 ˘ 0.21 ND, enzyme activity could not be determined.

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2.7. Effect of Inhibitors on WT and R169Y CpAK is an allosteric enzyme inhibited by end-products such as Lys, Met, and Thr. Table4 shows the effect of inhibitors on enzymatic activity when applied at concentrations ranging from 0.2 to 10 mM. The end-products, namely Lys, Met, and Thr, inhibited the enzymatic activity of WT. Inhibitors arranged in ascending order based on inhibitory strength were Thr, Met, and Lys. The results showed that cooperativity inhibition occurred between Met and Lys. For the other groups, such as Thr + Lys, Thr + Met, and Thr + Lys + Met, inhibition decreased compared with the end-products alone. For the R169Y mutant, the inhibition of end-products evidently decreased; by contrast, Lys and Met improved the enzymatic activity by 1.86- and 1.74-fold, respectively. The inhibitor combination Met + Lys exhibited activated cooperativity at different concentrations. The effect of inhibitors on R169Y was weaker than that on WT.

2.8. Enzymology Properties of Arg169 Similar to CgAK, CpAK is an allosteric enzyme with Lys, Met, and Thr inhibition and specifically regulated by Lys or Thr, whereas AK in bacilli is regulated by Lys or Lys and Thr [19]. CpAK was successfully expressed in E. coli. This neutral enzyme has an optimum temperature of 26 ˝C and a half-life of 4.5 h. Both Ni2+ and ethanol activated AK, and this activation could be used to guide the industrial application of the enzyme. Arg169 influenced enzyme catalysis, which regulates the catalytic domain through a hydrogen bond and therefore affects enzymatic activity. Increased enzyme activity was observed in the R169Y, R169P, R169D, and R169H mutants (Table1) because resistance from the hydrogen bond between Glu92 and the mutants disappeared; hence, Arg169 participated in nucleophilic catalysis. Table4 shows that that AK activity gradually decreased with increasing inhibitor concentration; hence, Lys was found to be the strongest inhibitor. The inhibitor binding site of Lys was found in the substrate binding cavity [20]. Other inhibitors were bound to locations other than in the substrate cavity, which allows them to more easily combine with the substrate than Lys. These inhibitors minimally influenced enzymatic activity mainly because the substrate is not involved in the competition for binding. The arrangement of inhibitors in ascending order based on the strength of the inhibitory effect is as follows: Thr, Met, and Lys. A two-step concerted inhibition of AK by Thr and Lys is therefore proposed. First, Thr binding triggers the interaction of the β-subunit with the regulatory domain in the α-subunit. Second, Lys binding induces a slight rotation in the regulatory domains in the α-subunit [21]. Therefore, the inhibitory effect of this combination mainly depends on Thr. Similarly, our experimental results indicated the absence of evident inhibition differences among the combinations of Thr + Lys, Thr + Met, and Thr + Lys + Met. However, few reports on the inhibitor Met and Met + Lys demonstrated cooperativity [22]. The inhibitory site of Met is probably identical to that of Lys, resulting in a stronger inhibitory effect of Met + Lys than those of Lys or Met alone.

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Table 4. Effects of inhibitors on WT and R169Y.

WT R169Y Enzyme Relative Activity (%) Relative Activity (%) Concentration (mM) Concentration (mM) Inhibitors 0.2 1 5 10 0.2 1 5 10 Control 100 100 100 100 100 100 100 100 Thr 73.97 ˘ 0.46 55.86 ˘ 1.63 27.07 ˘ 0.54 18.19 ˘ 0.59 94.25 ˘ 1.45 79.20 ˘ 0.54 50.34 ˘ 1.04 33.82 ˘ 0.29 Lys 38.47 ˘ 0.92 20.36 ˘ 1.03 19.18 ˘ 0.41 ND 83.09 ˘ 1.93 186.05 ˘ 1.22 68.46 ˘ 1.29 50.63 ˘ 0.34 Met 53.36 ˘ 0.56 50.89 ˘ 0.43 49.58 ˘ 0.94 43.62 ˘ 1.65 99.88 ˘ 1.98 167.43 ˘ 1.93 116.36 ˘ 1.19 68.62 ˘ 1.01 Thr + Lys 88.30 ˘ 1.14 33.95 ˘ 1.45 28.63 ˘ 0.19 30.18 ˘ 0.82 84.23 ˘ 0.17 41.13 ˘ 0.26 37.76 ˘ 1.38 27.93 ˘ 0.42 Thr + Met 73.32 ˘ 1.04 46.52 ˘ 1.82 29.38 ˘ 1.05 15.05 ˘ 0.04 73.04 ˘ 0.32 48.50 ˘ 0.91 32.88 ˘ 0.96 27.46 ˘ 1.04 Met + Lys 30.86 ˘ 1.74 22.63 ˘ 0.43 ND ND 102.38 ˘ 2.03 138.47 ˘ 1.92 146.76 ˘ 1.03 183.12 ˘ 1.13 Thr + Lys + Met 93.96 ˘ 1.54 64.35 ˘ 1.83 49.04 ˘ 1.62 35.92 ˘ 0.81 192.5 ˘ 2.04 141.99 ˘ 0.63 30.68 ˘ 0.14 16.87 ˘ 0.26 ND, enzyme activity could not be determined.

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Int. J. Mol. Sci. 2015, 16, page–page 2.9. “Triple Function” of Arg169 Int. J. Mol. Sci. 2015, 16, page–page 2.9.The “Triple so-called Function” triple of Arg169 function of Arg169 depends on enzyme catalysis with a hydrogen bond (Glu92–Arg169).2.9. “TripleThe so-called Function” R169 triple formedof Arg169 function a salt of bridgeArg169 withdepend thes on catalytic enzyme residue catalysis Glu with 92 a inhydrogen the inactive bond form. (Glu92–Arg169). R169 formed a salt bridge with the catalytic residue Glu 92 in the inactive form. As As shownThe in so-called Figure7 ,triple Glu92 function formed of a Arg169 salt bridge depend withs on Lys169 enzyme in catalysis the inactive with a form hydrogen (3.0 Å), bond but no shown in Figure 7, Glu92 formed a salt bridge with Lys169 in the inactive form (3.0 Å), but no salt salt bridge(Glu92–Arg169). was found R169 in formed the active a salt form bridge (8.6 with Å). the However, catalytic residue in the mutantGlu 92 in R169Y the inactive (Figure form.7), theAs salt bridge was found in the active form (8.6 Å). However, in the mutant R169Y (Figure 7), the salt bridgeshown in the inactive Figure form 7, Glu92 was formed 11.86 Å. a Thissalt bridge structural with differenceLys169 in the may inactive lead toform differences (3.0 Å), but in theno salt catalytic bridge in the active form was 11.86 Å. This structural difference may lead to differences in the efﬁciencybridge of was AK. found in the active form (8.6 Å). However, in the mutant R169Y (Figure 7), the salt catalytic efficiency of AK. bridge in the active form was 11.86 Å. This structural difference may lead to differences in the catalytic efficiency of AK.

FigureFigure 7. Distance 7. Distance in wildin wild type type and and R169Y R169Y calculatedcalculated by by PyMOL PyMOL (http://pym (http://pymol.sourceforge.net/).ol.sourceforge.net/).

AsFigure shown 7. Distancein Figure in 8a,b, wild typeR169 and was R169Y located calculat at theed bysubstrate PyMOL (http://pymchannel inol.sourceforge.net/). the active and inactive forms;As shown hence, in this Figure residue8a,b, can R169 prevent was the located substrate at from the substrate sliding. In channel particular, in R169 the activewas absolutely and inactive As shown in Figure 8a,b, R169 was located at the substrate channel in the active and inactive forms;conserved hence, among this residue all AK canfamily prevent members the (Figure substrate 1). from sliding. In particular, R169 was absolutely forms; hence, this residue can prevent the substrate from sliding. In particular, R169 was absolutely conserved among all AK family members (Figure1). conserved among all AK family members (Figure 1).

Figure 8. Substrate binding channel of AK. (a) Substrate binding in the inactive form of AK, color cyan represents for β-sheet, color yellow represents for α-helix, and color red represents for the Figure 8. Substrate binding channel of AK. (a) Substrate binding in the inactive form of AK, color Figurechannel; 8. Substrate (b) substrate binding binding channel in the of active AK. ( aform) Substrate of AK, color binding green in represents the inactive for formβ-sheet, of AK,color color red cyan cyan represents for β-sheet, color yellow represents for α-helix, and color red represents for the representsrepresents for forβ-sheet, α-helix, color and color yellow cyan represents represents forfor αthe-helix, channel. and color red represents for the channel; channel; (b) substrate binding in the active form of AK, color green represents for β-sheet, color red (b) substrate binding in the active form of AK, color green represents for β-sheet, color red represents represents for α-helix, and color cyan represents 10for the channel. for α-helix, and color cyan represents for the channel. 10

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CgAK is an allosteric enzyme that shares 98.5% homology with CpAK and exists in two states, namely, relaxed (R) and tense (T) states. In the T state, R151 (α) forms bidentate ionic bonds with E74 (α), and this binding is responsible for aspartate binding. The inhibitor binding structure showed that E3-β1 (β), E74 (α), S41 (α), R151-β5 (α), G152-β5 (α), and S154-β5 (α) were involved in Lys binding [1–4], and bidentate ionic bonds were formed between R151 and E74. According to homology modeling with CgAK (3aaw) as the template, the R151 (α) and E74 (α) of CgAK correspond to the R169 (α) and E92 (α) of CpAK. For the mutant R169Y, the inhibitory effect of Lys nearly vanished, and this observation may be related to the competition of aspartate and Lys binding. Overall, the binding site of the substrate is similar to that of Lys. Therefore, Arg169 may function as an evolutionary trace.

3. Experimental Section

3.1. Reagents, Strains, and Plasmids All restriction enzymes were bought from Promega (Madison, WI, USA). The His-Tag™ monoclonal antibody and the rabbit anti-mouse peroxidase-conjugated secondary antibody were purchased from Merck (San Diego, CA, USA). E. coli was obtained from Novagen (Madison, WI, USA). The recombinant plasmid pET-28a-AK was provided by our laboratory.

3.2. Construction of Mutant Strains The genomic DNA of C. pekinense was isolated with a genomic DNA extraction kit. The aspartokinase gene was then amplified by PCR, ligated to plasmid PMD 18-T, and then transformed to E. coli DH5α. The plasmids were extracted and sequenced. After digestion with the restriction enzymes, namely, BamHI and NdeI, the single band between 1000 and 2000 bp was recycled; this band corresponds to the target gene size of 1453 bp. The recombinant plasmid pET-28a-AK was constructed by subcloning the structure gene to pET-28a with His-Tag. Based on the DNA sequence of AK, two primers, namely, 51-CAGTGGTGTCAGAACCACCNNNACCCAACGTG-31 and 51-GTCACCACGTTGGGTNNNGGTGGTTCTGACAC-31, were designed and synthesized to amplify the gene encoding the mutant R169 site through PCR. The recombinant wild-type plasmid pET-28a-AK was used as template. The following thermal profile was used in PCR: initial denaturation at 94 ˝C for 4 min, followed by 18 cycles of denaturation at 94 ˝C for 1 min, annealing at 56 ˝C for 1 min, and extension at 72 ˝C for 10 min, and then a final extension of 72 ˝C for 20 min. The PCR products were digested with DpnI, transformed into E. coli, and then cultured in Luria–Bertani (LB) solid medium. The monoclones were inoculated into 96-well plates with 200 µL of the LB medium per well and then transformed into new 96-well plates at a ratio of 2% after overnight culture (37 ˝C, 180 rpm). The mixture was induced by adding 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 30 ˝C for 8 h. The cells were isolated through centrifugation at 3500 rpm for 45 min. The intracellular products were released through multigelation in 50 mM phosphate-buffered saline (PBS, pH 7.4) and then centrifuged at 3500 rpm for 45 min. The supernatant contained the crude enzyme. Enzymatic activity was detected using a microplate reader at 540 nm, and mutations with high activity were screened for sequencing.

3.3. Expression and Purification of the Recombinant AK and Mutants WT and mutant strains were cultured in the LB medium until an absorbance of 0.5 to 0.8 at 600 nm [23,24]. The strains were induced with 1 mM IPTG at 30 ˝C for 8 h. The cells were isolated through centrifugation at 8000 rpm for 10 min and then suspended in PBS (pH 7.4). The suspension was disrupted through sonication, followed by centrifugation at 8000 rpm for 10 min. The supernatant was filtered using a 0.45 µm membrane to remove residual proteins, loaded onto a Ni2+-NTA column, and then eluted with imidazole gradient. The purified enzyme was obtained with 500 mM imidazole. The purity of the WT and mutant samples was analyzed with SDS-PAGE.

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3.4. Analysis of Native PAGE, SDS-PAGE, and Western Blot Native PAGE was performed using a polyacrylamide gel consisting of 6% (w/v) resolving gel and 5% (w/v) stacking gel. Staining with silver and Coomassie blue were performed to obtain better staining results. SDS-PAGE was performed according to the Laemmli method. In brief, a polyacrylamide gel consisting of 10% (w/v) resolving gel and 5% (w/v) stacking gel was used. The gel was then stained with Coomassie Brilliant Blue for visualizing the proteins in the gel [25]. For Western blot analysis, the proteins in the 10% (w/v) resolving gel were transferred onto a polyvinylidene ﬂuoride membrane through a wet trans-blot system (Bio-Rad, Munich, Germany). The membrane was blocked with 2% nonfat dry milk for 2 h and then incubated for 2 h with HRP Mouse Anti-6X His in TBS at a dilution of 1:2500. The blot was developed with 3,31-diaminobenzidine tetrahydrochloride.

3.5. Steady-State Kinetics Assay of WT and Mutant AKs The kinetic parameters of WT and mutant AKs were determined through the generation of aspartyl hydroxamate. Each assay tube contained 10 mM L-aspartate, 10.4 mM ATP, 10 mM β-mercaptoethanol, 100 mM Tris-HCl buffer solution (pH 8.0), 1.6 mM MgSO4, 800 mM NH4OH, 800 mM KCl, and 0.1 mg/mL purified enzyme sample. The assay tubes were incubated at 299 K in a thermocycler for 30 min [26]. The reaction was terminated using isopycnic FeCl3. Absorbance was determined at 540 nm. The maximum velocities were measured with L-aspartate concentrations ranging from 1 to 10 mM. The content of AK in the purified enzyme for enzymatic determination was measured using the Bradford method [27]. Km value and maximum velocity were determined by fitting the Hill Equation (1) to data points with nonlinear least-squares method. All measurements were performed in triplicate. V rSs V “ max (1) K1 ` rSsn

3.6. Characterization of AK from WT and Mutant R169Y The optimum temperature was measured by incubating the reaction mixtures from 15 to 50 ˝C at 5 ˝C intervals. The optimum pH was determined using 100 mM Tris-HCl under a wide-range pH buffer of 6 to 10 with a pH 0.5 scale interval [28]. All measurements were performed in triplicate. Standard deviation was used for determination of enzymatic activity. The effect of metal ions on AK activity was determined by adding reagents containing Na+,K+, Ca2+, Mg2+, Zn2+, Mn2+, Cu2+, Fe3+, and Ni2+ [29]. The ions were analyzed at final concentrations of 0.2, 1, 5, and 10 mM. Enzymatic activity without added metal ions was defined as 100%. All measurements were performed in triplicate. The effect of organic solvents on AK activity was determined by adding methanol, ethanol, isopropyl alcohol, n-butanol, acetonitrile, dimethyl sulfoxide, and glycerol to the enzyme [30]. These organic solvents were investigated at final volume ratios of 1%, 5%, 10%, and 20%. Enzymatic activity without the addition of organic solvents was defined as 100%. All measurements were performed in triplicate. The effect of inhibitors on AK activity was determined by adding Thr, Lys, Met, Thr + Lys, Thr + Met, Lys + Met, and Thr + Lys + Met to the enzyme. These inhibitors were analyzed at final concentrations of 0.2, 1, 5, and 10 mM. The enzymatic activity without inhibitors was defined as 100%. All measurements were performed in triplicate.

3.7. Homology Modeling and Structural Analysis The sequence of AK was obtained from UniProtKB/Swiss-Prot. AK from C. glutamicum (PDB ID 3aaw sequence identity, 99%) was used as the template protein. The BLAST was used for searching, and Swiss Model was used to build the 3D structure [31–33]. The distance between the residue of

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169 and E92 was calculated with the program PyMOL (http://pymol.sourceforge.net/) for further structural analysis of WT and mutant proteins.

3.8. Molecular Docking The substrate and ATP were docked to the homology modeled AK [10] by using the Lamarckian Genetic Algorithm provided by AutoDock 4.2 software [28,34]. A cubic box was built around the protein with 36 Å ˆ 36 Å ˆ 36 Å points.

3.9. Molecular Dynamics (MD) Simulation and Molecular Mechanics-Poisson-Boltzmann Surface Area (MM-PBSA) Calculations Eleven 10 ns structures of the complex were used as starting points for calculations of binding free energy. All simulations were performed using the Amber 11 package for 10 ns, with the amber 99 sb as the ﬁeld-force parameter [25]. Binding free energies were calculated using the MM-PBSA method [35]. In addition, the two substrates used in the present study are highly similar. According to previous studies [36,37], the entropy differences should be minimal such that the correlation between the experimental value and the calculated binding free energy may not be substantially improved. Therefore, the solute entropy term was neglected in the present study. For each MD-simulated complex, we calculated the ∆Gbind values for 1000 snapshots of the MD simulation trajectory. The ﬁnal ∆Gbind value was the average of the ∆Gbind values for the snapshot trajectory.

4. Conclusions Recombinant AK from C. pekinense is a member of the AK superfamily. Experimental data showed that the optimum temperature and pH of AK were 26 ˝C and pH 7, respectively. The half-life was 4.5 h under the optimum conditions, and ethanol and Ni2+ strongly increased the enzymatic activity of CpAK. The steady-state kinetics study conﬁrmed that AK is an allosteric enzyme, and enzymatic activity was inhibited by allosteric inhibitors, such as Lys, Met, and Thr. The results of molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) showed that the residue Arg169 participated in substrate binding, catalytic domain, and inhibitor binding. These ﬁndings can be used to develop new enzymes and provide a basis for amino acid production.

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/16/ 12/26098/s1. Acknowledgments: Funding for this work was provided by the national “863” plan project (No. 2013AA102206), the authors also would like to thank Jilin Provincial Science & Technology Department for supporting key project (No. 20130101139JC) and key project (No. 20150519012JH). Author Contributions: Weihong Min conceived and designed the experiments. Huiying Li performed the experiments. Huiying Li and Chunlei Liu analyzed the data. Weihong Min, Hongmei Li, and Jingsheng Liu provided reagents/materials/analysis tools. Weihong Min and Huiying Li wrote the paper. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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