非解朊棲熱菌HG102耐熱β-糖苷酶的結構與功能研究

21卷1期生物工程學(xué)報Vol.21 No.12005年1月.Chinese Journal of BiotechnologyJanuary 2005非解朊棲熱菌HG102耐熱β_糖苷酶的結構與功能研究，The Structure-function Relationship of Thermostable β-glycosidase from the Thermophilic Eubacterium Thermusnonproteolyticus HG102楊雪鵬'2，楊壽鈞"，韓北忠”，金城'"YANG Xue-Peng'.2，YANG Shou-Jun' , HAN Bei-Zhong?2 and JIN Cheng'" .1.中國科學(xué)院微生物研究所微生物資源前期開(kāi)發(fā)國家重點(diǎn)實(shí)驗室,北京1000802.中國農業(yè)大學(xué)食品科學(xué)與營(yíng)養工程學(xué)院，北京，10001. Slate Key Laboratory of Microbial Reouces，Intite of Micobiology, Chine Academy of Sciences, Beijing 1000 China2. College of Food Science and Nuritionad Enginering. Bejing 100083 , China摘要非解朊棲熱菌HG102耐熱B-糖苷酶為(β/a)。桶狀結構,是具有水解功能和轉糖苷功能的單體酶。該酶可以作為一個(gè)很好的模型來(lái)研究糖苷酶的反應機制、底物特異性和耐熱的分子基礎。根據對該酶的晶體結構解析和同家族酶的結構比較，推測Glu164和Glu338分別是質(zhì)子供體和親核基團兩個(gè)活性位點(diǎn);在a~螺旋N端第一位的脯氨酸和蛋白質(zhì)外周的精氨酸是耐熱機制的關(guān)鍵住點(diǎn)和關(guān)鍵氨基酸殘基。為確定這些氨基酸殘基的功能，通過(guò)基因定點(diǎn)突變的方法分別把Clu164、Glu338、Pro316、Pro356 Pro344和Ang325 置換成GlnAla、Gly、Ala、Phe和Leu,同時(shí)還對Pro316和Pro356進(jìn)行了雙置換。突變酶經(jīng)過(guò)純化得到電泳純,用CD光譜進(jìn)行了野生酶和突變酶的結構比較。通過(guò)突變酶的酶功能和酶學(xué)性質(zhì)分析,結果表明Glu164 和Glu338 分別是質(zhì)子供體和親核基團,親核基團的突變酶TnglyE338A可以合威混合型糖苷鍵寡糖類(lèi)似物;在a-螺旋N端第一位的Pro316和Pro356 以及在蛋白質(zhì)外周形成離子鍵的Arg325 均是對耐熱性有貢獻的關(guān)鍵氨基酸殘基。關(guān)鍵詞}$_糖苷酶， 轉糖苷活性，熱穩定性，定點(diǎn)突變中圖分類(lèi)號Q814文獻標識碼A文章編號100-3061 (2005)01 0084-08Abstract p-Clycosidase (Tngly) from the thermophilic eubacterium Thermus nonproteolyticus HG102, which is a thermostablemonomerie protein and adopts the (B/a)g barrel fold, is an excellent model system to be invetigated for the thermnostable mecha-nism, activity and substrate specificity. Here, based on the analysis of structural basis for thermostability of Tngly ( Wang et al ,2003) and comparison of other proteins structure of homofamily，Glu164 and Glu338 may act as proton donor and nucleophile inthe hydrolysis reaction respectively; proline located at N1 of a-helix and arginine which can form ion link may contribute to thethermostability. We aim to futher identifty the critical sites and the amino acid residue(s) responsible for the activity, the ther-mal stability and the substrate specifcity . Mutations had been constructed by site-directed mutagenesis. They are Glu164Gln，Clu338Ala, Pro316Gly, Arg325Leu, Pro344Phe， Pro356Ala and Pro316Gly/Pro356Ala. All mutant proteins were purified toSDS-PAGE purity 。Changes in the conformnations were examined by means of CD. The Ghu338 Ala mutant showed no detectablehydrolysis activity, but can synthesize oligosaccharides, as expected for the residue acting as the nucleophile of the reaction. TheReeived: June 17, 2004; Acepted: July 26, 2004This work was suprted by CAS Inovation Progan(No 0103).+ Crreponding autor. Tel: 86-10-62587206; E-mail: jinc@ sun. im. ac. cn中國科學(xué)院知識創(chuàng )新工程項目基金資助(No.0103)。楊雪鵬等:非解朊棲熱菌HG102耐熱β-糖苷酶的結構與功能研究8SGlu164 acts as the general acid/base catalyst in the hydrolysis reaction. Changes in stabilities of mutants compared with wild-typewere determined by means of heat inactivity experiment. These results indicate that the amino acid residue of proline that is lo-cated at N1 positions of a-helix, and Arg325 that form salt bridge between a-helices 5 and a-helices 6, are the critical sites toprotein thermostabilization.Key words B-glycosidase, transglycosylation, thermostability ，site- directed mutagenesis3_糖苷酶(E.C.3.2.1.21)生物來(lái)源廣泛,可以Glu338來(lái)鑒定β_糖苷酶Tngly水解反應時(shí)的質(zhì)子供水解多種β構型的糖苷鍵,具有廣闊的應用前景"。體 和親核基團;置換Pro316 、Arg325、Pro344和Pro356嗜熱細菌Thermus nonproteolyticus HG102 的-糖苷酶來(lái) 探討酶的耐熱分子基礎。Tngly基因已克隆、表達并進(jìn)行了酶學(xué)性質(zhì)的研1材料和方法究”。Tngly 屬于糖苷酶家族1,具有葡萄糖苷酶、半乳糖苷酶、巖藻糖苷酶和甘露糖苷酶活性,在高溫下1.1材料還具有轉糖苷活性，它的最適水解反應溫度和pH1.1.1菌株與質(zhì)粒: 大腸桿菌( E. coli)AS1.1739值分別為90C和5.6,在90C時(shí),酶的半衰期為2.5[K12r~ O(lacIPOZY) x 74]在中國科學(xué)院微生物研h。Tngly晶體結構解析表明,該酶為(B/a)。桶狀結究所購買(mǎi),重組質(zhì)粒pHY(在pUC18載體HindI克構,分別位于第四B-sheet和第七B-sheet上的Clu164隆位點(diǎn)含有 β_糖苷酶目的基因)為本實(shí)驗室構建。和Glu338,可能為水解反應時(shí)的質(zhì)子供體和親核基BMH71-18購自Promega 公司。團。1.1.2培養基及培養條件: LB培養基為大腸桿菌β-糖苷酶可用來(lái)合成寡糖'5.61 ,但反應要在有機完全培養基，固體培養基加入1.5%的瓊脂粉。重相中并需要高濃度的糖基供體。Mackenzie 等人將組菌培養時(shí)加入氨芐青霉素(100 pg/mL),培養溫度Agrobacterium sp. β-葡 萄糖苷酶的親核基團谷氨酸為37C ;誘導時(shí)液體培養基加入1%( WIV)乳糖,固殘基突變成甘氨酸，使得突變的酶只能合成糖苷鍵，體培養基涂布4山IPTG(200 mg/mL)40 μl X-gal(20不再具有水解功能，從而能使寡糖的產(chǎn)率達到mg/mL)。90%”。R_糖苷酶Tngly 可以在65C下水解乳糖或1.1.3酶及生化試劑: 定點(diǎn)突變試劑盒GeneEdi-纖維二糖生成三糖}21,說(shuō)明酶的活性中心適合轉糖tor"M in vitro Site- Directed Mutagenesis System Kit購自苷反應，可用來(lái)合成寡糖,置換耐熱β_糖苷酶的親核Promega公司; T4 Polynucleotide Kinase 購自Promega基團來(lái)合成寡糖更具有優(yōu)勢。已知結構的蛋白質(zhì)大公司;溶菌酶購自華美公司;CMP-3-Fluoro- neuraminic約10%為(β/a)。桶狀結構,或叫TIM結構)，因此以Acid購自Calbiochem-Novabiochem 公司;胰化蛋白胨Tngly作為模型來(lái)研究(β/a)。桶狀結構的穩定機制非(TRYPTONE)和酵母提取物(YEAST EXTRACT)購自常有意義。文獻報道，在a-helixN端第一位脯氨酸OXOID公司;IPTG、X-gal .ONPG和乳糖購自Sigma公的剛性結構和蛋白質(zhì)外周精氨酸形成的離子鍵可能司;蛋白質(zhì)分子量標準購自華美公司;其它試劑均為對蛋白質(zhì)熱穩定性有一定的貢獻”。分析純試劑。本實(shí)驗在Tngly晶體結構的基礎上,用基因定所用誘變寡核苷酸為上海生工公司合成，見(jiàn)點(diǎn)突變的方法置換Tngly的氨基酸殘基Glu164和表1。表1置換氨基酸位點(diǎn)及其在蛋白質(zhì)二級結構中的位置和相應的誘變寡核苷酸設計Table 1 Oligonucleotides and mutagenic position in proteinMutationPosition in proteinMutagentic olgonucleotidesGlul64ClnNo.4,B-sheet5'-ACCCTGAACCAGCCCTCCTGC-3'Glu338AlaNo.7, B-sheetS-TACTCGCCCCCCC-3'Pro316GlyNo.6, a-helix NI5'-GGGAGGTCTACGGCGACGCGCCTT-3'Anrg325Leulon link5-CTCTTCAAGCTCCCGCCGG-3'Po344PheNo.7, B-shet5-CCCCCCCTACTTCGACCTCTGGAC-3'Pro356AlaNo.7, a-helix NI5'-GTGGAGGACGCCGACGGGTG-3'Pro316Gly/No.6, a-helix N1/5'-GCGAGCTCTACGGCGACCCGCTT-3'5'-GTGGAGGACGCCGACCCGGTC-3'Bold and underdined mucleotides are the mutations sites.8Chinse Joumal of Biotehnology生 物工程學(xué)報2005, Vol.21 ,No.11.2 方法SWISS MODEL. html網(wǎng)站上完成8910)。1.2.1基因定點(diǎn)突變: 以重組質(zhì)粒pHY單鏈DNA1.2.6轉糖苷反應:突變酶TnglyE338A和各種底物為模板,在誘變寡核苷酸介導下,用定點(diǎn)突變試劑盒在65C,pH6.8下反應2h,薄層層析檢測。相應的試劑進(jìn)行突變和篩選。篩選出單菌株提取質(zhì)1.2.7薄層 層析(TLC):展開(kāi)劑為正丁醇:乙酸:水粒送交TaKaRa Biotechnology (Dalian)測序鑒定突變=1:2:1;顯色劑為苯胺:二苯胺:磷酸=5:5:1(顯色結果。范圍為10 ug)。1.2.2突變基因 的表達和蛋白質(zhì)純化:用突變的在數據的測定當中都進(jìn)行3次或3次以上試重組質(zhì)粒轉化到大腸桿菌(E. coli)AS1 .1739,從過(guò)驗 ,誤差范圍在5%以?xún)?。夜培養的Amp-LB平皿上挑取單菌落,接種到5 m[2結果液體LB(Amp 100 pg/mL)培養12 h,1%接種量接種到100 mL Amp-LB液體培養基中培養12 h;再以1%2.1基因定點(diǎn)突變構建突變酶基因的接種量接種到4 L Amp-LB液體培養基(5 L發(fā)酵以單鏈重組質(zhì)粒為模板，由誘變寡核苷酸和選罐)中培養,加入1%的乳糖誘導, 37C ,300 r/min的擇寡核苷酸介導突變和篩選突變基因"。突變質(zhì)攪拌速度,通無(wú)菌空氣,培養28 h。粒與野生質(zhì)粒大小相同。測序結果顯示突變質(zhì)粒收集發(fā)酵罐中的4 L發(fā)酵液(4C, 6000g, 15pHYE164Q編碼164位上的Clu堿基密碼子GAG突min)離心,菌體用磷酸緩沖液(50mmol/L,pH6.6)懸變?yōu)镃In的密碼子CAG;突變質(zhì)粒pHYE338A編碼浮;冰浴超聲破碎,離心去除細胞碎片;在80C水浴338位上的Glu堿基密碼子GAA突變?yōu)锳la的密碼恒溫加熱15 min,離心(4C , 10000g, 15 min)取上清。子GCA;突變質(zhì)粒pHYP316G編碼316位上的Pro堿向上清液中緩慢加入固體硫酸銨,收集30% ~ 60%基密碼子CCC突變?yōu)镚ly的密碼子GGC;突變質(zhì)粒飽和度的沉淀,溶于磷酸緩沖液中(50mmol/L,.pHYP356A編碼356位上的Pro堿基密碼子CCC突pH6.6),用同種緩沖液透析過(guò)夜。除鹽的粗酶凍干變?yōu)锳la的密碼子GCC;突變質(zhì)粒pHYP344F編碼濃縮后,在A(yíng)KTAFPLC蛋白質(zhì)純化系統上用DEAE344位上的Pro堿基密碼子CCC突變?yōu)镻he的密碼離子交換柱進(jìn)行純化，緩沖液A為磷酸緩沖液(50子CTT;雙突變質(zhì)粒pHYP316G/P356A編碼316位和mmol/L,pH6.6),緩沖液B為1 mol/L NaCl溶于磷酸356位上的Pro堿基密碼子CCC分別突變?yōu)镚ly的緩沖液(50 mmol/L, pH6.6),洗脫條件為在5個(gè)柱體密碼子CGC和Ala的密碼子GCC; 突變質(zhì)粒積內B溶液比例上升到30%，測酶活和SDS-PAGEpHYR325L編碼325位上的Arg堿基密碼子CGC突.檢測目的蛋白純度,收集合并酶活峰,用凍干機凍干變?yōu)長(cháng)eu的密碼子CTC。濃縮,用蒸餾水溶解凍干的酶蛋白，在磷酸緩沖液2.2突變基因的表達和突變酶的純化(50 mmol/L, pH6.6)中透析,再用Superdex G-75分子含有突變基因的質(zhì)粒分別轉化到大腸桿菌篩柱層析純化,洗脫液為磷酸緩沖液(50mmol/L,AS1.1739中,經(jīng)乳糖誘導,發(fā)酵罐大量培養,目的基pH6.6)。以上步驟均在常溫下進(jìn)行。因得到大量表達,將表達產(chǎn)物分別經(jīng)加熱分離、硫酸1.2.3酶活測定: 0.1 mL 4 mmol/L ONPC,0.1 mL銨分級沉淀、DEAE和SuperdexG75分子篩層析純pH5.8磷酸緩沖液,0.7 mL H20,混勻后于85C水浴化,得到電泳純，純度均達90%以上(圖1);除保溫5 min,加入0.1 mL酶液,反應10 min,加入4TnglyE164Q和TnglyE338A外,其他突變酶在在85CmL Na,CO,溶液終止反應,420 nm測定吸光值。此時(shí)的比活均與野生酶相當(表2)。條件下每分鐘產(chǎn)生1 pmol對硝基苯酚的酶量為一2.3突變酶與野生 酶結構的比較個(gè)酶活單位。野生和突變純酶由CD光譜儀直接掃描得到觀(guān)1.2.4蛋白含量測定:以牛 血清白蛋白為標準，察值(mdeg)，把觀(guān)察值換算成平均殘基橢圓度(meanLowry 法測定。residuellplicit)。結果顯示突變酶與野生酶結構1.2.5 CD 光譜分析和同源建模: - .定濃度的各種相同，如圖2(因為CD光譜圖曲線(xiàn)擁擠,這里只顯示酶蛋白分別加入測量杯中，把測量杯放人光譜儀兩個(gè)突變酶和野生酶的CD光譜圖)。說(shuō)明單個(gè)氨(JASC0 J-715)中,用200 ~ 250nm的光進(jìn)行掃描。用基酸殘基的置換沒(méi)有改變蛋白質(zhì)的折疊。同源建模蛋白質(zhì)分子量和氨基酸殘基數目計算殘基橢圓度。結果也顯示置換位點(diǎn)的結構域與野生酶相同(見(jiàn)圖同源建模在ht://ww. expasy . org/wissmod/3),這與CD光譜結果一致。楊雪鵬等:非解朊棲熱菌HG102酎熱β糟苷酶的結構與功能研究.7表2 I 突變純酶的比活Table 2 The specific activity of purifted TnglysWild-TnglyPurified proteinstype_E338AE1640P316GP356AP344F P316G/P356AR325LSpecifie activity/( u/mng)17.600.9820.021.017.924.010005000......-- - TnglyP316G/p356A粵-500020一喜-00000.-15000圖1 SDS-PAGE 分析野生酶Tngly和突變酶的純化-20000 :Fig.1 SDS- PAGE pattern of purifed Tnglys2122232402501:Tnpgly E164Q; 2:Tngly P344F; 3:Tngly P316G;入num4:Tngly P356A; S:Tngly R325L; 6:Tngly P316G/P356A;圖2突變酶和野生酶 CD光譜圖.7; wild-type; Mstanderd marker proleins.Fig. 2 1 Cireular dichroie speclra of wild-type Tnglyand two mutant TnglyR325L, TnglyP316C/P356A in the2.4質(zhì)子供體 與親核基團的確定far-ultraviolet region突變酶TnglyE164Q的比活是野生酶比活的The secta were measured on a JASCO J-715 setrolarineter at 25C.5.5%，且幾乎不受反應液pH值的影響(見(jiàn)圖4);突The protein concentration of wild-type Tngly was 0. 166mng/mL, the Tng-變酶TnglyE338A檢測不到水解活性,在CMP-3-F-lyR325L was 0.053mn/ mL, the TngyP316G/P356A was 0.079mg/mL alNeu5Ac和甘露糖為底物的情況下表現出轉糖苷功pH6.6, 50 mmol/ mol phosporie acid bulfr.能(見(jiàn)圖5),用相應的糖苷酶Tngly和a-2-(3, 6)-和底物形成共價(jià)復合物,是雙置換反應的關(guān)鍵步驟。NANase II都沒(méi)能把反應產(chǎn)物水解。根據以上結果突變酶TnglyE338A失去水解功能而只有轉糖苷功確定Glu164為水解反應時(shí)的質(zhì)子供體,介導水的攻能,可作為糖苷合成酶用于寡糖的合成。擊，Glu338為水解反應時(shí)的親核基團,其側鏈羧基345。 3434356443575435T 34626 !2S324359203231932215D圖3突變點(diǎn)的結構和野生酶的比較立體圖Fig. 3 Stereo drawings showing the mutant structure in the vicinity of the mutant sitesWild- type and mutants stutures are superimposed. A:Ar325L; B:P344F; C:P316G; D:P356A88Chinese Jourmal of Biotechnology生物工程學(xué)報 2005, Vol.21,No, I .0.8 .Wild-type Tngly0.5-←wT一- TnglyP344F...... TnglyR325L一TnglyE164Qa6-+- Tgl1g6P3360.4-TnglyP316GTnglyP356Ag0.2+0.1-0.20.0-0.0070809100圖4突變酶TnglyE164Q和野生酶的活性受反應液pH值Temperature/C的影響圖6突變酶的最適反應溫度Fig. 4 Comparison of the pH dependence on the ONPG substrateFig. 6 Elects of temperature on ste-diretedwild-type Tngly and TnglyE164Q at the same condition of reactionmutagenesis Tngly hydrolysis activitiesThe hyddrolysis activity was determined as descnibedin Materials and Mcthods at diferent tempernturesfrom s0C to 95C.0.7-.. **. TnglyP344F0.6-TnglyR325L●。0.51Tngly316G/P356A-Highigtnder3+ulravioletradiation0.2個(gè)圖5突變酶TnglyE338A的轉糖昔反應TLC圖譜Fig. 5 Transglycosylation activity of mutant0.0+TnglyE338A at 65C ,pH6.8pF1:mannose; 2:sialic acid; 3:CMP-3-F-Neu5Ac; 4: mannose and CMP-3-F-Neu5Ac reacted with mutant TnglyE338A; 5: mannose and CMP-3-F.圖7突變酶的最適反應pHNeu5Ac with no enzyme; 6: production of reaction of lane4 reacted withFig. 7 Elects of pH on sile-directed mutagenesiswild-type Tngly; 7: production of reaction of lane4 reacted with a-2-(3,Tngly hydrolysis activities6)-NANase I (raction of condition rely on the enxyme of opimum con-The hydrolysis activity was determined as dernbeddition) .in Materials and Methods at diferet pH from 4.5 to 9.0.2.5熱穩定 性相關(guān)的突變酶酶學(xué)性質(zhì)分析2.5.2最適pH值:如圖7所示,6個(gè)突變酶的最適2.5.1最適溫度:在50~95C的溫度范圍內測定.pH值都是大約pH5.8,與野生酶相似。說(shuō)明置換的野生酶和突變酶的最適反應溫度,發(fā)現突變酶.氨基酸不影響酶活性中心的環(huán)境,也表明置換的點(diǎn)TnglyP344F的最適反應溫度與野生酶都為90C,其在蛋白的外周不影響酶蛋白質(zhì)的活性。它突變酶最適反應溫度都有所下降(如圖6),2.5.3熱穩定性: 突變酶和野生酶在85C溫浴不TnglyP316C和TnglyP3S6A 最適反應溫度大約為同的時(shí)間，然后測剩余酶活,當剩余酶活是原來(lái)的879 ,Tngly-P316G/P356A和TnglyR325L最適反應溫50%時(shí),各種酶經(jīng)歷的時(shí)間(1n2)分別是野生酶Tngly度大約為85C。結果表明,位于第六a-helix的N端63 min,突變酶TngyP344F 57 min , TnglyP316G 43 min,第一位的P316、位于第七a-helix的N端第一位的TnglyP356A 45 min,TnglyR325L 33 min和TnglyP316G/P356和位于第六a-helix的R325置換都降低了酶蛋P356A 15 min(見(jiàn)圖8)。把各種酶在不同的溫度溫白的剛性結構，但在Loop區的P344對酶的結構剛浴15min測定酶的剩余酶活,得出相似的結果,Tm性影響較小。值是酶剩余50%活力時(shí)所對應的溫度如圖9,結果楊雪鵬等:非解朊棲熱菌HG102耐熱β_糖苷酶的結構與功能研究89110 1力學(xué)常數,底物濃度由2mmol/L到40mmol/L。結果- Wild-type100 '. Pro316Gly/Pro356Ala顯示動(dòng)力學(xué)常數基本相同(見(jiàn)表3),表明突變酶置)0一. Pro356Ala30Pro316Gty換的氨基酸不影響酶的親和性。Ang325Leu. Pro344Phe60 t表3野生酶及其突變酶 熱穩定性和反應動(dòng)力學(xué)常數50 |Table 3 Thermodynamic and kinetic Parameters forwild-type Tngly and its mutants01Protetin'K.(kem/K)/m19(1(mmol/L) /[U(mol-s)]Wild-ype 63+0.5 94.0+0.5.377.90.010.0 20.0 30.0 40.0 50.0 60.0 700/minPro316G]y 43+0.5 91.0+0.5 -3.0+0.5 1.277.1圖8野生酶和突變酶的熱穩定性分析Pro356Ala 45+0.5 91.5+0.5 -2.5+0.5 1.277.2Fig. 8 Kinetics of inctvivation of wild-type TnglyPro344Phe 57+0.5 93.0+0.5 -1.0+0.5 1.378.1and its mutants at 85C Values whose error ranges wereArg325leu 33+0.5 89.0+0.5 -5.0+0.578.6within 5% were averagedPro316Cly/15+0.5 86.5土0.5 -7.5+0.5.478.211Pr356Ala100a: Determined by kinetics of irevernible heat inetivity at 85C.b: Mecling temperature from Fig.9.)te: btained by catalyie reaction of enzyme at optimun temperature.專(zhuān)603討論曾5+Wlild-ype糖苷酶家族1 β_糖苷酶采用保留型雙置換催化營(yíng)40 +◆Pro316Gly.機制,在酶的活性部位存在兩個(gè)重要的羧酸部分,一正3甘Pro316Gly20 →Pro356Ala個(gè)質(zhì)子化,稱(chēng)為質(zhì)子供體,另一個(gè)發(fā)生離子化,稱(chēng)為. Ang325Leu親核基團,所切糖苷鍵的氧原子被質(zhì)子化的羧基攻r t Pro34PheoL擊，糖苷鍵斷裂,形成的碳正離子與離子化的另-一個(gè)75.0 80.085.010.0Incubation temperature/C羧基以離子鍵或共價(jià)鍵的形式形成中間產(chǎn)物，此中圖9野生酶和突變酶的熱穩定性分析間產(chǎn)物不穩定，當親核試劑進(jìn)行親核攻擊時(shí),此中間Fig. 9 Efect of temperature on the stability of產(chǎn)物不存在,取而代之的是一個(gè)新的糖苷鍵 ,當親核試劑是水時(shí),發(fā)生水解反應,當親核試劑是醇或某個(gè)Each purtied enyme was teated t difret teperntures for 15 miutes.糖的羥基時(shí)，則發(fā)生轉糖苷反應8.121。質(zhì)子供體在The remaining activities were expressed 越percentages of the original ac-雙置換當中起到酸/堿催化的雙功能,在第-.步提供tivities. Values whose error ranges were within 5% wene avenged.質(zhì)子,在第二步介導水的攻擊明,親核基團是形成顯示野生酶和突變酶TnglyP344F 、Tngly-P316G、酶與底物中間物的關(guān)鍵氨基酸殘基。TngIyP356A .TnglyR325L、Tng!yP316G/P356A的T.值Agrabacterium faecalis β-葡萄糖苷酶的質(zhì)子供體分別是94. .93.91.91、89和86C。以上結果表明位和Sufolobus solfataricus β_糖苷酶兩個(gè)活性位點(diǎn)的谷于第六a-helix 的N端第一位的P316和位于第七氨酸殘基都已確定[15.16)。目前,在糖苷酶家族1中a-helix的N端第-.位的P356剛性結構及位于第六除了葡萄糖硫苷酶外，質(zhì)子供體和親核基團兩個(gè)活a-helix的R325所形成的離子鍵對酶蛋白的熱穩定性位點(diǎn)都是谷氨酸,分別在第四B-sheet 和第七B-性有一定的貢獻，而在Loop區的P344對蛋白質(zhì)的sheet.上。這兩個(gè)活性位點(diǎn)氨基酸殘基在家族1中很.穩定性影響較小。保守,靠近蛋白質(zhì)N端是T(F/LM)NE(P/L/I) ,靠近2.5.4突變酶米氏常 數(K )和轉化數(..)的測C端是(IV)TENG。在Tngly結構當中,E164所在定:野生酶及突變酶可以水解ONPCle、ONPGal、-TLNEP-區 域和E338所在ITENG-區域，分別位于第ONP-GFuc . PNPGle、PNPGal、PNPFuc和PNPMan等多四B-sheet和第七B-sheet 上,可推測E164和E338可種底物,這里只選擇ONPGIe作為底物來(lái)測定酶的動(dòng)能為R-_糖苷酶Tngly的兩個(gè)活性位點(diǎn)，在水解反應當9Chinese Journal of Biotechnology 生 物工程學(xué)報2005, Vol.21,No.1中前者作為質(zhì)子供體，后者作為親核基團。Glu164P344的B值分別為25.27.28.51和29.97。B值和.和Clu338分別置換為GIn和Ala的突變試驗結論與原子的穩定性有關(guān),B值越小原子群的穩定性越大。上述推測- -致。β-糖苷酶Tngly熱穩定性可能不只是含有脯氨糖苷酶水解反應時(shí)糖苷鍵的水解有立體選擇酸的結果,在蛋白質(zhì)- .級序列當中含有9.6%的精性，但合成糖苷鍵時(shí),合成的鍵型是多樣的,可能是氨酸,在3D結構當中發(fā)現精氨酸大部分位于蛋白β鍵或a鍵,也可能是1-3、1-4或1-6等[”。耐熱突質(zhì)外周,在a-helix之間形成離子鍵網(wǎng)絡(luò )。這樣的離變酶TnglyE338A在65C溫度下可催化底物CMP-3-子鍵在其他耐熱蛋白質(zhì)中都被證明為重要的穩定因F-Neu5AC和甘露糖的合成，合成產(chǎn)物不能用相應的30011231。較早Penutz等人比較了同一種酶不同糖苷酶Tngly和a-2-(3,6)-NANase I水解,表明產(chǎn)物來(lái)源的耐熱酶和常溫酶的結構發(fā)現,耐熱酶的分子的糖苷鍵鍵型可能發(fā)生了改變。這樣產(chǎn)生的混合鍵表面離子鍵比常溫酶的多0。在以后的耐熱酶研型連接的寡糖(mixed-linkage)有可能作為寡糖類(lèi)似究中也發(fā)現類(lèi)似的情況，比如,蘋(píng)果酸脫氫酶{4、甘物用于糖苷水解酶的抑制劑[8。油醛-3-磷酸~脫氫酶'35]和DNA聚合酶[6]等。我們蛋白質(zhì)的耐熱因素,包括離子鍵作用、氫鍵作把6-a-helix的Arg325 和5-a-helix的Asp235 之間形用疏水作用、金屬鍵、二硫鍵、包裝效應、Proline理成的離子鍵打斷,突變酶R325L的熱穩定性參數r。論、a-螺旋的穩定作用和氨基酸組成等都經(jīng)過(guò)深入值下降大約5C,表明蛋白質(zhì)外周的離子鍵對野生廣泛的分析(9,20.21 ,從大量的研究中發(fā)現,一個(gè)蛋白酶Tngly熱穩定性也有貢獻。質(zhì)的耐熱機制可能是多種因素的結果。各個(gè)突變酶與野生酶純化方法相同,從SDS-Proline 與其他的a-氨基酸不同，由于其N(xiāo)原子PAGE電泳結果分析各個(gè)突變酶與野生酶純度相位于吡略環(huán)上,使得前一個(gè)氨基酸與它形成肽鍵時(shí)當,然而突變酶TnglyP316G、TnglyP356A、TnglyP316G/(C*-N)不能自由旋轉,另外吡咯環(huán)還具有疏水作P356A和TnglyR325L的比活與野生酶相比有所上用。Proline 分子的這些特點(diǎn),在形成肽鏈時(shí)導致它升,可能的原因是在85C時(shí)突變酶結構與野生酶相比其它氨基酸的構型熵小2.20),從而降低蛋白質(zhì)比“剛性”下降“柔韌性”增加,有利于突變酶催化反的折疊熵。蛋白質(zhì)的折疊熵降低可以提高其穩定應。突變酶TnglyP316G、TnglyP356A、TnglyP316G/性5.26。較早,從統計的結果表明蛋白質(zhì)中脯氨酸P356A、TnglyP344F和TnglyR325L動(dòng)力學(xué)常數與野生含量增多,可以明顯提高蛋白質(zhì)的熱穩定性田,但酶基本相同,說(shuō)明被置換的氨基酸殘基與酶的活性脯氨酸在蛋白質(zhì)二級結構中的位置不同對穩定性的無(wú)關(guān),置換上來(lái)的氨基酸殘基也不影響酶活性中心貢獻不一樣,動(dòng)力學(xué)模擬試驗證明脯氨酸位于ar結構的正確折疊。helix的N端第一位最有利于蛋白質(zhì)的穩定性。統計結果也表明脯氨酸偏向位于a-helix的N端第REFERENCES(參考文獻)-.位和B-tum的第二位9],這可能有利于蛋白質(zhì)的[1] IehikawaY, Look cC, Wong CH. Enxyme calyed oigogechae穩定。在β_糖苷酶Tngly 436個(gè)氨基酸殘基當中,有ride synthesis. Anal Biochem 1992, 202: 215-23835脯氨酸殘基,其中有5個(gè)位于a-helix N端第一[2] He xYr, Zhang s, Yang s. Coming ad expesion od a te位,其它位于p-tum的第二位和Loop區。從以上理stable Bgycosidaee Gene from論可推測位于a-helixN端第一位的脯氨酸可能對β_harocteriation of the recombinant enzyme . Applied Biocemistry andBiotechnology, 2002,94(3); 243 - 255糖苷酶Tngly的穩定性有較大的貢獻。我們分別把[3] WangXQ, He XY，Yang SJ et al. Sinuctural basis for thermosta-位于第六個(gè)a-helix 和第七個(gè)a-helix N端第一位的bility of beta- glycosidase from the thermophilie enbacterium thermosP316和P356置換為Gly和Ala, 還對兩者進(jìn)行了雙nonproteolytieus HG102. Journal of Bateriology, 2003, 185(14):突變試驗,結果突變酶TnglyP316G、TnglyP356A和雙4248- 4255突變TnglyP316G/P356A酶的熱穩定性參數T.值下[4 ] Hocker B, Jurgens C. Wilmanns M et al. Stability , ctalytie versa-降3C到7;而對位于loop區的脯氨酸突變,熱穩tility and erolution of the (Bx) 8-barel fold. Curen Opinion inBiotehnology , 2001, 12: 376-381定性和野生酶基本相同。這從試驗的角度證明了脯[5 ] Watt GM, Lowden PAS, Fisch SL. Enzyme ealyzed frmation of氨酸在不同的位置對蛋白質(zhì)的穩定性貢獻不同。這glycosidie linkages. Cur Opin Struet Biol, 1997，, 7: 652 - 660個(gè)結論也和野生酶的晶體熱因子B值(erystallo-[6] Eling L. lyobioiechnology: enzymes for the synhesis of mueleoti-graphie thermal factor B values) 相一致,P356、P316 和de suguns. Ado Biochem Eng Biorechnol, 1997, 58:89- 144楊雪鵬等:非解朊棲熱菌HGI02耐熱B糖苷酶的結構與功能研究91[7] Mackenie LF, Wang Q, Waren RAJ ot al. Clyeoeyontuses mutant[23] Hutchinson EG, Thortion JM. A revised set of potentials for beta-glycosidases for oligosaccharide eyntbeis. J Am Chem Soe, 1998,tum fomation in proteins. Protein Sei, 1994, 3; 2207 - 2216120: 5583- 5584[24] Sripnpundh D, Vielle C, Zeikus JG. Molecular deteminants of[8] Schwede T, KoppJ, Guex N et al . SWIsS MODEL: an mutomatedylose isomernse thermal stability and activity: analysia by site-di-protein homology modeing server. Nucleic Acids Research, 2003,rected mutagenesis. Prolein Eng ,000, 13: 259 - 26531: 3381 - 3385Mtthews BW, Nicholson H, Becktel WJ. Enhanced protein ther-[9] Guex N, Peitsch MC. SWISS MODEL and the Swise Padb Viewer:motability from site directed mutations that deese the entopy ofAn environment for comparotive protein modeling. Electrophoreis ,unfold. Proc Nal Acad Sci USA, 1987. 84: 666 - 66671997, 18: 2714-2723[26] Hardy F, Vriend G, Velman OR er al . Subiliaion of Bacillus[ 10] Peitech MC. Protein modeling by E mail. Bio/ Technology, 1995,stearothermophilus neutral protease by introduction of pralines.13: 658- 60FEBS let, 1993, 317: 89-92[11] Venkatachalm KV, Huang W. LaRocco M e al , Characterization[27] Watanabe K, Masuda T. Ohashi H et al。Muliple proline substitu-of TEM-1 beta-lacamase mutants from positionis 238 to 241 with in-creased ceatalytic fficiency for cftaxidime. J Biol Chem，1994,6-glucosidase. Irefrngble proo spprting the proline nule. Eur J269: 23444 - 234S0Biochem , 194, 226(2):277 - 283[12] Crout DHG, Vic G. Glycosidaes and gyosgl tansferases in glyco-[28] Yun RH, Andenon A, Hermans J. Proline in alpba- helix: stabilityside and olgaccharide Bynthesis. Cur Opin Chem Biol, 1998, 2:and confomation studied by dynamics simulation. Proeins, 1991 .98-11110: 219-228[13] Burmeister WP, Cottaz s, Driguer H e al. The eryal structure of[29] Heatwole LC, Soeliman J, Shobam M. Crystal stnucture of a ther-Sinapis alba myrosinaseprovide insights into the substrate recogition and active-site machi-nants of substrate seifieity and thermostability. Protein Structnery of an sgycosidase. Sructure, 1997, 5(5); 663-675Funet Cenet， 199， 37: 619- 627[14]’Wither SG. Mechanism of elycosyl tranferases and hydrolases. Car-[30] Perutz MF, Raidt H. Sereochemical basis of heat sabilit in bacte-bolydrate Polymers ,2001, 44:325 -337ril frredorins and in hemoglobin A2. Nature, 1976, 255: 256 -[15] WangQ, Trimbur D, Crahamn R et al. Ienifcation of the acid/259base calalyst in Agrobaterim facalis betr glucosidase by kinetie[31] Pappenberger C, Schurig H, Jenicke R. Disruption of an ionieanalysis of mutants. Biochemisry, 1995, 34: 14554- 14562network leades to aceated thermal denaturaion of D-gyenlde-[16] MoracciM, Capalbo L, Ciramella M at al. Ienificatinin of twohyde 3-phosphate dehydrogenase from the hyperthemophilie bacte-glulamic acid residues essential for catalysis in the B-glycosidaserium Thermotoga maritime. J Mol Biol, 1997, 274: 676 - 683from the thermoacidophilie archaeon Sufolobus solfataricus. Protein[32] Merz A, Knochel T, Jansonius JN et al . The hperthemosable in-Enginering，1996, 9(12): 1191 - 1195doleglyceral phosphate synhase from thermnotoga maritime is destabi-[17] Wymer N, Toone EJ.Engym-catalyed eynthesie of earbolhydratee .lized by mutational disruption of two solvent-exposed sal bridges. JCurent Opinion in Chenmical Biology, 2000, 4: 110- 119Mol Biod, 199，288: 753- 763[18] FortS, Varot A, Schulein M et al . Mixed-linkage lloliosaege[33]Takano K, Tsuchimori K, Yamagota Y at al . Contribution of salcharides; A new class of glycoside hydrolase inhibitors. Chenmbio-bridges near the surface of a protein to the conformational stability .chem, 2001, 2: 319- 325Biochemistry, 2000, 39: 12375- 12381[ 19] Sailagyi A, Zavodsky P. Structurul diferences between mesophilic,[34] Kelly CA, Nihiyama M，Ohnishi Y et al . Deteminante of proteinmoderately thermophilie and extremely thermophilic protein sub-stbilitly in the 1.9-A crysal structure of malate dehydrogenuse fromunit: resuls of a comprehensive survey. Structure Fold Des, 2000,the thermophilie bacterium Thermus Jlauu. Biochemistry, 1993,8: 493-50432:3913- 3922[ 20] Ville C. Zeikus JG. Thermoxymues: denifying moleulaer detemi-[35]Kond rfer 1, Seipe B, Huber R et al . The erytal structure of ho-nants of protcin structural and functionl stability, TIBTECH,lgycaldehydere-posphale dehydrogenase from the hypether-1996, 14: 183- 190mophilie bacterium Thermotoga maritima at 2.5A resolution. J Mol[21]Vieille C. Zeikus JG. Hypenthermophilie enxymes: Sources, useasBiol,1995, 246: 511- 521and molecular mechanisms for terostability .Microbiology and[36]KorolevS, Nayal M, Barmes WM a al. Crstal structure of theMoleular Biologoy Reriews, 2001, 65:1-43large fragment of Thermus aquaticus DNA polymerase lat2.5A res-[22] MacArhur Mw, Thomton JM. Ifluence of proline residues on pro-olution: structural besis for termosbilily. Proe Nal Acad Scitein cnformation. J Mol Biol, 1991, 218(2): 397 -412USA, 1995, 92: 9264 - 9268