短链壬基酚聚氧乙烯醚脱氢酶脱氢催化机理

【目的】为研究短链壬基酚聚氧乙烯醚脱氢酶(sNPEO-DH)的脱氢氧化机制(基因克隆于Ensifer sp.AS08),我们进行了以下实验。【方法】采用同源序列比对及同源建模的方法筛选出与其辅酶黄素腺嘌呤二核苷酸(FAD)异咯嗪基邻近的4个氨基酸残基。以定点突变方法分别构建了突变体,并进行了重组蛋白的表达纯化和酶活力测定。【结果】野生型和突变体的酶学动力学实验表明,突变体N90A和N509A对亲水性底物聚乙二醇(PEG1000)的相对活性分别降低为51%和89%,对疏水性底物sNPEO的活性分别降低为26%和40%,说明氨基酸残基N90和N509可能与底物的结合相关。突变体H465A的相对活性丧失了90%以上,突变体N507A完全丧失活性;瞬时"停-流"检测实验进一步证明N507A突变体阻断了底物向FAD传递质子的过程,突变体H465A阻断了对FAD还原形成的FADH2脱氢再生的过程。【结论】以上结果说明N507和H465为sNPEO脱氢酶活性中心中参与对底物氧化脱氢及FADH2脱氢再生进行下一次反应的催化位点。     

腾冲嗜热厌氧菌丙氨酸消旋酶底物通道氨基酸位点的功能

【目的】通过定点突变探究腾冲嗜热厌氧菌MB4中生物合成型丙氨酸消旋酶Tt Alr底物通道内氨基酸位点A172和S173的功能。【方法】利用定点突变PCR技术构建突变体,通过亲和层析法纯化酶蛋白,采用D-氨基酸氧化酶偶联法检测各突变蛋白的活性及其稳定性。【结果】通过定点突变PCR成功得到8个突变体,酶学特性分析发现,A172位点突变为丝氨酸(S)后酶蛋白的相对活性有所提升,但含有该位点突变的酶蛋白稳定性均大幅下降;S173位点突变为天门冬氨酸(D)后导致突变体蛋白的最适反应温度提升了15°C,半衰期大幅延长,但相对活性明显下降。【结论】丙氨酸消旋酶Tt Alr底物通道内A172和S173位点均是影响酶蛋白催化活性和稳定性的关键位点。     

内消旋-二氨基庚二酸脱氢酶关键氨基酸位点的改造提高对烷基取代2-酮酸的催化活力

【背景】高效实现D-氨基酸的生物合成一直是人们关注的热点。内消旋-二氨基庚二酸脱氢酶(meso-diaminopimelate dehydrogenase,DAPDH)能够直接催化2-酮酸和氨合成D-氨基酸。【目的】提高DAPDH对烷基取代2-酮酸的催化活力,并解释其催化机制。【方法】以来源于嗜热共生杆菌(Symbiobacteriumthermophilum)的内消旋-二氨基庚二酸脱氢酶(StDAPDH)为模板,在前期结构分析结合被选择位点突变结果的基础上,确定对H227位进行定点饱和突变,并以D-丙氨酸、D-2-氨基丁酸、D-正缬氨酸、D-谷氨酸为底物进行筛选。【结果】获得突变体H227Q和H227N。突变体H227Q对丙酮酸、2-氧代丁酸、2-氧代戊酸、2-酮戊二酸的比活力比野生型分别提高了10.9、11.5、8.6和7.6倍。动力学参数表明,突变体H227Q同时提高了酶对底物的亲和力及催化常数,使其对丙酮酸的催化效率(k_(cat)/K_m)相较于野生型提高了9.4倍。利用分子模拟技术分析突变体H227Q与产物氨基酸之间的相互作用表明,227位的谷氨酰胺通过与氨基酸的羧酸形成氢键,使得氨基酸产物Cα上的氢和辅酶烟酰胺环C4原子之间的距离缩短。【结论】利用定向进化技术提高DAPDH对烷基取代2-酮酸的催化活力,有助于开发新型的高效生物催化剂,这些工作也为下一步继续进行更具挑战性的D-氨基酸研究提供了基础。     

L1.LtrB内含子编码蛋白反转录结构域关键催化位点分析及功能验证

【目的】筛选影响Ll.LtrB内含子编码蛋白(Intron encoded protein,IEP)反转录功能的关键催化位点,并获得无反转录活性的IEP突变体。【方法】首先,利用NCBI数据库,通过序列比对及同源建模方法筛选影响IEP反转录功能的关键氨基酸催化位点;然后,对筛选获得的关键催化位点进行定点突变,同时以Targetron载体为模板,构建无反转录功能的突变型Targetron打靶系统;最后,以大肠杆菌lacZ基因为例,体内验证IEP突变体的功能及其对Ⅱ型内含子"归巢"效率的影响。【结果】筛选到C164和G214两个位点是影响内含子编码蛋白反转录功能的关键氨基酸残基,并获得C164K和G214W两个突变体。体内功能分析表明,此两个位点突变完全失活了Ⅱ型内含子的"归巢"功能。【结论】筛选并获得了失活反转录功能的Ll.LtrB内含子编码蛋白突变体,为深入研究Ⅱ型内含子的结构和"归巢"机理奠定了基础。     

人类线粒体肌酸激酶uMtCK的底物结合位点分析

研究人类线粒体肌酸激酶u Mt CK的结合位点,将其与底物肌酸和ATP结合有关的关键氨基酸进行突变,并对突变体进行酶动力学和圆二色谱数据分析,探讨这些关键氨基酸在底物识别和催化过程中的作用。结果显示,与野生酶相比,突变体Q313A和R336A的K_m~(Cr)分别提高了2.6和2.9倍,k_(cat)下降了19%和55%;同样地,与ATP结合相关的突变体R125A和R287A分别使得K_m~(ATP)升高了3.2和4.2,k_(cat)下降了72%和38%。以上结果表明突变体R125A、R287A、Q313A和R336A影响对底物的结合,同时也降低了酶促反应的速度。利用圆二色谱比较野生酶与不同突变体的二级结构并无明显变化,但进一步的结构模拟表明底物结合位点氨基酸在与底物之间的氢键对底物的识别和酶催化过程中发挥着重要作用。     

基于磷脂酶水解圈定向筛选高效聚对苯二甲酸乙二醇酯降解酶

【目的】目前自然环境中聚对苯二甲酸乙二醇酯(polyethylene terephthalate, PET)废弃物的积累严重威胁生态健康,因此PET的降解问题已成为全球性的热点问题。生物酶法降解PET技术以其绿色环保而备受关注,但天然PET降解酶的催化活性普遍偏低,亟待进一步定向改造。现阶段定向进化为快速提高PET降解酶催化性能提供了可能,其中筛选方法是成功获得高性能突变体的关键所在。本研究旨在提出一种新型高效灵敏的筛选方法并应用于褐色喜热裂孢菌(Thermobifida fusca)来源角质酶Tfu-0883的定向改造,以期快速获得PET降解活性提高的突变体。【方法】基于易错PCR构建突变体文库,涂布于卵黄磷脂平板,以水解圈的大小作为筛选指标获得PET降解活性提高的突变体;对突变体进行酶学定性并筛选出潜在的分子改造位点,最终获得高性能突变体。【结果】从卵黄磷脂平板中挑取水解圈直径最大的单菌落,即突变体H10(N2D/D94H/A149E),其PET降解能力是野生型的1.5倍,最适温度与pH分别为60℃和8.0。突变体H10中第2位和第149位氨基酸残基远离底物结合凹槽,其突变会导致酶蛋白稳定性下降;第94位氨基酸残基则位于底物结合凹槽附近,由负电荷氨基酸Asp突变为正电荷氨基酸His,有利于吸附在带负电荷的PET表面,是突变体H10降解能力提升的关键因素;随后将野生型的第94位氨基酸残基Asp分别突变为His及同为正电荷且空间位阻更小的Lys和Arg,突变体D94H、D94K和D94R对PET降解能力均有提升,其中,突变体D94K降解PET能力是野生型的3.6倍。【结论】本研究基于磷脂酶水解圈构建了一种新的PET降解酶定向筛选方法,以此获得了降解活性提高的突变体,并证实角质酶Tfu-0883第94位氨基酸残基位点具有提升其PET降解活性的潜在能力。     

GH42家族保守氨基酸位点累积突变对Geobacillus stearothermophilus来源β-半乳糖苷酶BgaB催化活性的影响

【背景】β-半乳糖苷酶转糖苷活性弱,产物低聚半乳糖(galactooligosaccharides, GOS)易被水解,致其催化得率普遍较低。【目的】以GH42家族Geobacillus stearothermophilus来源β-半乳糖苷酶BgaB为对象,探讨家族保守氨基酸位点突变对β-半乳糖苷酶BgaB催化活性的影响。【方法】在单点突变体功能研究基础上,采用定点突变与化学修饰相结合的方法,对保守氨基酸位点E303与F341进行累积突变。【结果】与野生型酶相比,所构建双点突变体Ox-E303C/F341S水解活性降低为30%;GOS最大得率由0.75%提高到19.50%。【结论】家族保守氨基酸位点累积突变能够使单点突变体功能得到共同进化,降低β-半乳糖苷酶水解活性和底物抑制作用,能够提高其转糖苷催化活性。     

嗜酸热脂环酸杆菌中甘露聚糖酶活性位点的确立

【目的】通过定点突变确定嗜酸热脂环酸杆菌中甘露聚糖酶的活性催化位点。【方法】根据序列比对和GH53家族的结构信息选择可能的催化活性位点,利用重叠PCR法构建定点突变体,采用薄层层析(TLC)法和3,5-二硝基水杨酸(DNS)法检测各酶蛋白活性。【结果】通过重叠PCR法成功构建了7个位点的突变体,其中第150和159位的氨基酸突变对活性改变甚少或几乎没有,而第151和231位谷氨酸的羧基基团的改变以及双位点突变体E2Q则导致其对各种底物催化活性的丧失,说明位于β4和β7折叠的C末端的E151和E231的羧基基团作为功能基团参与了催化反应。【结论】E151和E231分别是新型甘露聚糖酶AaManA的酸碱催化位点和亲核催化位点。     

β-葡萄糖苷酶的糖耐受和促活性质研究

【目的】以葡萄糖耐受并促活的β-葡萄糖苷酶Bgl2A为出发材料,寻找与β-葡萄糖苷酶的葡萄糖耐受和促活性质相关的重要氨基酸残基位点并对其进行突变;对突变酶性质进行检测,结合分子对接,探究突变对酶的糖耐受和促活性质的影响及机制;进而对葡萄糖不耐受的Bgl3A (Bgl2A:A22S/V224S)进行分子改造,以获得应用潜能更好的突变酶。【方法】通过序列和结构比对、统计耦联分析和结构分析,选取Bgl2A底物通道口、蛋白质表面以及活性中心附近可能间接影响葡萄糖耐受和促活性质的残基作为突变位点,构建了多个突变酶,并对其酶学性质进行检测。【结果】以Bgl2A为出发酶,D322I、W325A、W126Y、F172N、C173I和N226V的糖耐受和促活性质显著提升。分子对接提示,这些突变可能是通过变构效应影响活性中心与葡萄糖结合的自由能,从而改变酶葡萄糖耐受和促活性质。据此,在Bgl3A分子上对应构建多个突变体,筛选获得了较出发酶在糖耐受和促活性质提升的同时保持较高酶活和稳定性的突变酶N226V和F172N。【结论】除了酶与葡萄糖直接结合的位点,不与葡萄糖直接相互作用的位点也可通过远程作用间接影响...     

原玻璃蝇节杆菌D-氨基酸氧化酶及突变体的酶学特性

【目的】研究原玻璃蝇节杆菌(DSM 20168)中D-氨基酸氧化酶的酶学特性。【方法】通过PCR从原玻璃蝇节杆菌(DSM 15035,20168)中克隆获得D-氨基酸氧化酶基因apdaao-1和apdaao-2,构建原核表达载体,以表达质粒pET-ApDAAO-2为模板,采用QuickChange Site-Directed Mutagenesis技术构建定点突变体,经过原核表达及纯化获得重组型和突变体酶蛋白,分析其酶学特性。【结果】通过原核表达及纯化成功获得了2个重组蛋白和4个突变体酶蛋白,SDS-PAGE检测显示其分子量均约为36 kDa;酶学特性分析表明,ApDAAO-2和突变体蛋白的最适反应温度为30℃;ApDAAO-2和T286A的最适反应pH范围为7.0-11.0,其它突变体为8.0-11.0;ApDAAO-2和突变体都具有较广泛的底物特异性,除T256K的最适底物为D-Phe外,其余均为D-Met;动力学参数测定结果显示,以二级表观常数kcat/Km表示,对于底物D-Met或D-Phe,ApDAAO-2和4个突变体的kcat/Km值均比ApDAAO-1和pKDAAO高数倍以上。【结论】ApDAAO-2及突变体具有比ApDAAO-1和pKDAAO更广泛的底物特异性和较高的催化效率,有一定的商业应用价值。     

Crystal structure of D-psicose 3-epimerase from Agrobacterium tumefaciens and its complex with true substrate D-fructose: a pivotal role of metal in catalysis, an active site for the non-phosphorylated substrate, and its conformational changes

D-psicose, a rare sugar produced by the enzymatic reaction of D-tagatose 3-epimerase (DTEase), has been used extensively for the bioproduction of various rare carbohydrates. Recently characterized D-psicose 3-epimerase (DPEase) from Agrobacterium tumefaciens was found to belong to the DTEase family and to catalyze the interconversion of D-fructose and D-psicose by epimerizing the C-3 position, with marked efficiency for D-psicose. The crystal structures of DPEase and its complex with the true substrate D-fructose were determined; DPEase is a tetramer and each monomer belongs to a TIM-barrel fold. The active site in each subunit is distinct from that of other TIM-barrel enzymes, which use phosphorylated ligands as the substrate. It contains a metal ion with octahedral coordination to two water molecules and four residues that are absolutely conserved across the DTEase family. Upon binding of D-fructose, the substrate displaces water molecules in the active site, with a conformation mimicking the intermediate cis-enediolate. Subsequently, Trp112 and Pro113 in the beta4-alpha4 loop undergo significant structural changes, sealing off the active site. Structural evidence and site-directed mutagenesis of the putative catalytic residues suggest that the metal ion plays a pivotal role in catalysis by anchoring the bound D-fructose, and Glu150 and Glu244 carry out an epimerization reaction at the C-3 position.     

Crystal structures of D-psicose 3-epimerase from Clostridium cellulolyticum H10 and its complex with ketohexose sugars

D-Psicose 3-epimerase (DPEase) is demonstrated to be useful in the bioproduction of D-psicose, a rare hexose sugar, from D-fructose, found plenty in nature. Clostridium cellulolyticum H10 has recently been identified as a DPEase that can epimerize D-fructose to yield D-psicose with a much higher conversion rate when compared with the conventionally used DTEase. In this study, the crystal structure of the C. cellulolyticum DPEase was determined. The enzyme assembles into a tetramer and each subunit shows a (β/α)₈ TIM barrel fold with a Mn²⁺ metal ion in the active site. Additional crystal structures of the enzyme in complex with substrates/ products (D-psicose, D-fructose, D-tagatose and D-sorbose) were also determined. From the complex structures of C. cellulolyticum DPEase with D-psicose and D-fructose, the enzyme has much more interactions with D-psicose than D-fructose by forming more hydrogen bonds between the substrate and the active site residues. Accordingly, based on these ketohexosebound complex structures, a C3-O3 proton-exchange mechanism for the conversion between D-psicose and D-fructose is proposed here. These results provide a clear idea for the deprotonation/protonation roles of E150 and E244 in catalysis.     

Characterization of an Agrobacterium tumefaciens D-psicose 3-epimerase that converts D-fructose to D-psicose

The noncharacterized gene previously proposed as the D-tagatose 3-epimerase gene from Agrobacterium tumefaciens was cloned and expressed in Escherichia coli. The expressed enzyme was purified by three-step chromatography with a final specific activity of 8.89 U/mg. The molecular mass of the purified protein was estimated to be 132 kDa of four identical subunits. Mn2+ significantly increased the epimerization rate from D-fructose to D-psicose. The enzyme exhibited maximal activity at 50 degrees C and pH 8.0 with Mn2+. The turnover number (k(cat)) and catalytic efficiency (k(cat)/Km) of the enzyme for D-psicose were markedly higher than those for d-tagatose, suggesting that the enzyme is not D-tagatose 3-epimerase but D-psicose 3-epimerase. The equilibrium ratio between D-psicose and D-fructose was 32:68 at 30 degrees C. D-Psicose was produced at 230 g/liter from 700-g/liter D-fructose at 50 degrees C after 100 min, corresponding to a conversion yield of 32.9%.     

以D-果糖为原料利用新型异构酶转化生产D-阿洛糖

稀少糖是自然界中含量稀少、化学合成困难的一类低热量单糖。D-阿洛糖是一种重要的稀少己醛糖,其具有减少活性自由基、抑制癌细胞增殖等独特的生理学功能。因此,以微生物发酵生产D-阿洛酮糖-3-差向异构酶(DPE)和L-鼠李糖异构酶(L-RhI)转化生产D-阿洛糖,成为近几年来国际研究的热点之一。文中分别克隆了来源于解纤维梭菌Clostridium cellulolyticum H10的DPE基因以及来源于枯草芽胞杆菌Bacillussubtilis 168的L-RhI基因,并分别使其在宿主菌B.subtilis及大肠杆菌Escherichia coli BL21(DE3)中得到了表达。进一步利用镍亲和层析和阴离子交换色谱等手段对这两种酶进行了纯化,并对这两种纯化后酶的转化能力进行了分析测定。结果表明,以D-果糖为原料利用两种异构酶依次转化获得D-阿洛酮糖及D-阿洛糖,其两步转化效率分别为27.34%和34.64%。     

Beta-glucosidase from Trichoderma reesei. Substrate-binding region and mode of action on [1-3H]cello-oligosaccharides

To determine the mode of action of the beta-glucosidase from Trichoderma reesei a method was developed for synthesizing [1-3H]cello-oligosaccharides with specific radioactivities of approximately 3000 Ci/mol. The beta-glucosidase removed glucosyl residues from the non-reducing end of the [1-3H]cello-oligosaccharides in a multiple attack mode with little tendency to attack the substrates repetitively. Values of Km were lower for longer cello-oligosaccharides, whereas values of V remained essentially constant. A subsite map, constructed using values of V/Km for the cello-oligosaccharides, showed that the substrate-binding region comprises primarily three subsites.     

D-阿洛酮糖的功能及其生物合成研究进展

随着肥胖、糖尿病等代谢性疾病的发病率在全球范围急剧上升,人们对食品营养和健康等问题日益关注。D-阿洛酮糖作为重要的天然稀有己酮糖,不仅保持良好的甜度,而且具有降血糖、降血脂、抗氧化等功效,逐渐成为食品、保健和医疗领域的研究热点。文中阐述了D-阿洛酮糖的主要生理功能,综述了D-阿洛酮糖的生物合成研究进展及其酮糖3-差向异构酶的晶体结构,为筛选D-阿洛酮糖的产生菌株及提高合成酶的热稳定性和转化率提供理论指导,以满足工业化生产的需求。     

Biosynthesis of streptomycin. dTDP-dihydrostreptose synthase from Streptomyces griseus and dTDP-4-keto-L-rhamnose 3,5-epimerase from S. griseus and Escherichia coli Y10.

dTDP-dihydrostreptose synthase from Streptomyces griseus was purfied about 50-fold by removal of protein with polyethyleneimine, (NH4)2SO4 fractionation and gel filtration on Ultrogel AcA44. The synthase preparation was free of dTDP-4-keto-L-rhamnose 3,5-epimerase (dTDP-4-keto-6-deoxy-D-glucose 3,5-epimerase, EC 5.1.3.13) activity. A new enzyme assay using Escherichia coli Y10 as source for the epimerase and dTDP-glucose 4,6-dehydratase (dTDP-glucose 4,6-hydro-lyase, EC 4.2.1.46) was developed. In the presence of excess epimerase the apparent Km for dTDP-4-keto-6-deoxy-D-glucose was determined to be 25 microM. The molecular weight of epimerase and synthase were determined by their elution volumes from a Sephadex G-100 column to be approx. 67,000 and 32,000, respectively. The pH optimum for the epimerase was between 7.5 and 8.5. The intermediate formation of dTDP-4-keto-L-rhamnose in the epimerase reaction could be shown by detection of 6-deoxy-[3H]talose after NaB3H4 reduction. Results which indicate the existence of dTDP-4-keto-6-rhamnose as a free intermediate in the epimerase reaction are reported.     

Effects of amino acid alterations on the transglycosylation reaction of endoglucanase I from Trichoderma viride HK-75

Endoglucanase I (EGI) from Trichoderma viride HK-75 catalyzes not only hydrolysis but also transglycosylation reactions of cellooligosaccharides. In order to characterize the important amino acid residues in transglycosylation of EGI, three Tyr, one Leu, and two Glu residues of EGI were replaced by Trp or Asp. The seven resulting EGI, except for L200W, had reduced activities toward carboxymethyl-cellulose compared to that of wild type EGI. The results from the mutations in the catalytic residues of E196 and E201 indicate that the space just around the catalytic residues is not directly related to the transglycosylation reactions of EGI. Analyses of the enzymes with mutations in the substrate-binding residues showed that Y146, Y170, and L200 of EGI are closely involved in the mode of transglycosylation and that several amino acid residues within the active site are involved in the transglycosylation reaction of EGI.     

Recent advances on applications and biotechnological production of d-psicose

D-Psicose is a hexoketose monosaccharide sweetener, which is a C-3 epimer of D-fructose and is rarely found in nature. It has 70 % relative sweetness but 0.3 % energy of sucrose, and is suggested as an ideal sucrose substitute for food products. It shows important physiological functions, such as blood glucose suppressive effect, reactive oxygen species scavenging activity, and neuroprotective effect. It also improves the gelling behavior and produces good flavor during food process. This article presents a review of recent studies on the properties, physiological functions, and food application of D-psicose. In addition, the biochemical properties of D-tagatose 3-epimerase family enzymes and the D-psicose-producing enzyme are compared, and the biotechnological production of D-psicose from D-fructose is reviewed.     

Structure of L-xylulose-5-Phosphate 3-epimerase (UlaE) from the anaerobic L-ascorbate utilization pathway of Escherichia coli: identification of a novel phosphate binding motif within a TIM barrel fold

Three catabolic enzymes, UlaD, UlaE, and UlaF, are involved in a pathway leading to fermentation of l-ascorbate under anaerobic conditions. UlaD catalyzes a beta-keto acid decarboxylation reaction to produce L-xylulose-5-phosphate, which undergoes successive epimerization reactions with UlaE (L-xylulose-5-phosphate 3-epimerase) and UlaF (L-ribulose-5-phosphate 4-epimerase), yielding D-xylulose-5-phosphate, an intermediate in the pentose phosphate pathway. We describe here crystallographic studies of UlaE from Escherichia coli O157:H7 that complete the structural characterization of this pathway. UlaE has a triosephosphate isomerase (TIM) barrel fold and forms dimers. The active site is located at the C-terminal ends of the parallel beta-strands. The enzyme binds Zn(2+), which is coordinated by Glu155, Asp185, His211, and Glu251. We identified a phosphate-binding site formed by residues from the beta1/alpha1 loop and alpha3' helix in the N-terminal region. This site differs from the well-characterized phosphate-binding motif found in several TIM barrel superfamilies that is located at strands beta7 and beta8. The intrinsic flexibility of the active site region is reflected by two different conformations of loops forming part of the substrate-binding site. Based on computational docking of the L-xylulose 5-phosphate substrate to UlaE and structural similarities of the active site of this enzyme to the active sites of other epimerases, a metal-dependent epimerization mechanism for UlaE is proposed, and Glu155 and Glu251 are implicated as catalytic residues. Mutation and activity measurements for structurally equivalent residues in related epimerases supported this mechanistic proposal.