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1.
三磷酸腺苷(adenosine triphosphate,ATP)是一种重要的辅助因子,参与许多需能的生物催化反应。多聚磷酸盐激酶(polyphosphate kinases,PPK)由于其底物聚磷酸盐廉价易得,可以为消耗ATP的反应提供能量。本研究选择哈氏噬纤维菌(Cytophaga hutchinsonii)来源的ChPPK,进行了底物谱和耐受性分析,通过分子对接和定点突变,理性改造多聚磷酸盐激酶的双底物通道腔来提高PPK酶的催化活性。与野生型相比,筛选得到突变体ChPPKK81H-K103V的相对酶活提高了326.7%,同时,双突变扩大了ChPPK的底物利用范围与耐受性,提高了该酶的耐热性与耐碱性。基于该ATP再生系统,本研究偶联谷胱甘肽双功能酶GshAB和ChPPKK81H-K103V,破细胞后采用无细胞催化生产谷胱甘肽,加入5 mmol/L ATP后,该体系6 h可以生产(25.4±1.9) mmol/L的谷胱甘肽,比突变前的催化体系提高了41.9%。优化无细胞催化体系的缓冲液、裂解液菌体量、补料时间后,无细胞体系可产生(45.2±1.8) mmol/L谷胱甘肽,底物l-半胱氨酸的转化率达到90.4%。提高ChPPK生产ATP的能力,可有效增强底物的转化率,降低催化成本,实现了无细胞催化生产谷胱甘肽的高产量、高转化率与高经济价值的统一。本研究提供了一种绿色高效的ATP再生系统,可为消耗ATP的生物催化反应平台提供可持续动力。  相似文献   

2.
黄欣  李益民  杜聪  袁文杰 《生物工程学报》2022,38(12):4669-4680
聚磷酸激酶(polyphosphate kinase,PPK)在体外催化合成ATP的反应中有着重要作用。为寻找能利用短链聚磷酸盐(polyphosphate,polyP)为底物高效合成ATP的聚磷酸激酶,本文以来源于泗阳鞘氨醇杆菌(Sphingobacterium siyangensis)的聚磷酸激酶(PPK2)为研究目标,利用pET-29a构建重组质粒,在大肠杆菌(Escherichia coli)BL21(DE3)中表达,并将其作为ATP再生系统的关键酶与l-氨基酸连接酶(YwfE)联用生产丙谷二肽(Ala-Gln)。ppk2长度为810bp,编码270个氨基酸;SDS-PAGE结果表明PPK2为可溶性表达,分子量为29.7kDa。对PPK2的最适反应条件进行了优化,结果发现其在22–42℃、pH7–10的范围内均可以保持较好活性,且在37℃、pH为7、镁离子(Mg2+)浓度为30mmol/L、底物ADP与六偏磷酸钠浓度分别为5mmol/L和10mmol/L时酶活最大,在0.5h时ATP产率可以达到理论值的60%以上。作为模式反应体系,当PPK2与YwfE联用生产Ala-Gln时,达到与直接添加ATP相同的效果。此聚磷酸激酶作为ATP再生系统具有较好的适用性,适用的温度和pH范围广,且能以廉价易得的短链polyP为底物高效合成ATP,为依赖ATP的催化反应体系的能量再生提供了新酶的来源。  相似文献   

3.
李元  刘珊  祝俊 《生物工程学报》2016,32(12):1745-1749
构建了共表达ATP再生和L-茶氨酸合成酶的重组大肠杆菌菌株,并将其应用于L-茶氨酸的合成中。合成多聚磷酸盐激酶(PPK)和γ-谷氨酰甲胺合成酶(GMAS)基因序列,分别利用p ETDuet-1和p ET-21a(+)载体,构建共表达重组质粒p ETDuet-ppk+gmas和p ET21a-ppk+gmas。将上述两种重组质粒转入大肠杆菌BL21(DE3)中,获得重组菌株TPG和APG。IPTG诱导表达后,SDS-PAGE结果表明,PPK和GMAS在两种重组菌中均可溶性表达。当用于催化L-茶氨酸合成时,来自APG的GMAS-PPK要优于TPG。利用APG所产的酶进行L-茶氨酸合成,在37℃、p H 7.0条件下,使用催化量ATP可实现L-茶氨酸的摩尔产率为86.0%。该结果一方面扩展了酶法ATP再生系统的应用,另一方面为生物催化合成L-茶氨酸提供了一种有效方法。  相似文献   

4.
多聚磷酸相关蛋白结构及生物学功能   总被引:1,自引:0,他引:1  
多聚磷酸(polyphosphate,polyP)是由几个到数百个磷酸基通过高能磷酸酐键连接而成的链状多聚体,存在于所有细胞生物中.多聚磷酸相关蛋白包括多聚磷酸相关酶和多聚磷酸结合蛋白.多聚磷酸相关酶如多聚磷酸激酶(polyphosphate kinase,PPK)催化polyPn生成polyPn+1的可逆反应;外切聚磷酸酶(exopolyphosphatase,PPX)、内切聚磷酸酶(endopolyphosphatase,PPN)能将polyP水解成磷酸残基;多聚磷酸依赖的激酶将polyP的磷转移到生物小分子上,如葡萄糖和烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide,NAD),使其分别磷酸化为6 磷酸葡萄糖和烟酰胺腺嘌呤二核苷酸磷酸(nicotinamide adenine dinucleotide phosphate,NADP).多聚磷酸结合蛋白可与多聚磷酸结合,发挥各种生物学功能.本文将简要介绍多聚磷酸相关蛋白的结构与主要生物学功能,以阐述多聚磷酸参与的细胞内生化过程.  相似文献   

5.
【目的】构建己糖激酶与葡萄糖-6-磷酸脱氢酶的大肠杆菌共表达体系,以葡萄糖为底物实现辅酶NADPH的高效再生。【方法】通过分子生物学方法,克隆己糖激酶HKgs、HKpp基因,并于Escherichia coli BL21(DE3)中表达,再将己糖激酶HKgs、HKpp分别与葡萄糖-6-磷酸脱氢酶Gpd PP共表达,实现NADPH的原位再生。比较两个共表达工程菌的辅酶再生效果,并针对催化活力较高的工程菌BL21(HKgs+Gpd PP)进行表达条件优化。【结果】NADPH再生活力达到856 U/L。该辅酶再生体系与醇脱氢酶Adh R联合催化,使不对称还原4-氯乙酰乙酸乙酯的催化活力提高至原始值的2.5倍。【结论】通过己糖激酶与葡萄糖-6-磷酸脱氢酶在大肠杆菌中的共表达,构建了一个新的NADPH高效再生体系,并用于醇脱氢酶催化的不对称还原反应。  相似文献   

6.
E.coli热诱导赖氨酰-tRNA合成酶(LysU,EC 6.1.1.6)是高效的Ap4A/Ap3A合成酶,已知反应模式为双重动态过程:2ATP→Ap4A+2Pi→Ap3A+3Pi。为进一步研究LysU"中间物可逆"催化模型,表达纯化了LysU蛋白并验证结构稳定性,构建了二腺苷多磷酸产物检测系统并分离了各阶段催化产物,观察了AMPPCP和AMPCPP阻断Ap3A/ADP合成的反应。圆二色光谱和荧光光谱扫描证明纯化后的LysU蛋白结构完整。LysU首先催化ATP合成83%的Ap4A,接着可逆生成67%的Ap3A。实验中发现,Ap3A并非LysU二腺苷多磷酸催化反应的终产物,Ap3A可继续逆生成80%的ADP。以AMPPCP或AMPCPP代替ATP为起始底物,发现无Ap3A转化ADP反应。上述结果证明LysU具有三重催化活性:2ATP→Ap4A+2Pi→Ap3A+3Pi→2ADP+2Pi,符合"磷酸捕获机制"催化模型:活化的赖氨酰-腺苷中间物捕获核苷酸或磷酸小分子,形成对应的二腺苷多磷酸化合物。这些研究结果可为阐明不同形式功能性腺苷酸衍生物间的相互转化提供更多的信息,有助于进一步认识功能性腺苷酸分子在生命活动中的作用。  相似文献   

7.
腺苷甲硫氨酸合成酶的基因及结构研究进展   总被引:3,自引:0,他引:3  
腺苷甲硫氨酸合成酶催化ATP和L-甲硫氨酸合成腺苷甲硫氨酸,在不同生物体和不同组织中腺苷甲硫氨酸合成酶的存在形式和编码酶的基因都有差别,本文综述了不同生物的腺苷甲硫氨酸合成酶的基因、酶结构、酶反应动力学及应用前景。  相似文献   

8.
副溶血弧菌(Vibrio parahaemolyticus)能够通过Ⅲ型分泌系统(typeⅢsecretion systems,T3SSs)分泌效应蛋白Vop S,催化三磷酸腺苷(ATP)分子中的单磷酸腺苷(AMP)通过磷酸二酯键共价连接至宿主细胞Rho鸟苷三磷酸激酶(Rho GTPases)成员蛋白Rho A、Rac1和Cdc42的特定的苏氨酸残基上,导致宿主细胞肌动蛋白骨架崩解,细胞变圆。该发现推动了一种蛋白质翻译后修饰方式——单磷酸腺苷酸化(AMPylation)修饰的迅速发展,其中催化AMPylation修饰的蛋白质称为单磷酸腺苷酸化酶(AMPylator)。目前的研究表明,与蛋白质的磷酸化修饰类似,蛋白质AMPylation在真核以及原核生物中都是一种重要的调控蛋白质功能的翻译后共价修饰调节机制。与AMPylation相对应的是去单磷酸腺苷酸化(de-AMPylation),即去单磷酸腺苷酸化酶(de-AMPylase)催化修饰后的蛋白质脱去AMP基团的过程,使底物蛋白重新恢复其原有的生物学功能。现就蛋白质AMPylation修饰的催化过程、AMPylation修饰的研究进展以及AMPylator/de-AMPylase的种类、物种来源、结构、功能和底物等方面的内容进行综合阐述。此外,就目前已有的AMPylation检测方法进行了总结,其中详细阐述了一种化学标记法的原理和过程。  相似文献   

9.
摘要:【目的】腺苷酸激酶(adenylate kinase, ADK)和多聚磷酸盐激酶(polyphosphate kinase, PPK)偶联催化的ATP扩增反应结合生物发光检测法能够对微量微生物进行检测。但是PPK当中结合的内源性的ADP会产生背景干扰,影响测定。本文旨在融合表达ADK和PPK,并建立一种方便有效的内源性ADP的去除方法,降低背景,使之与传统生物发光法结合,实现高灵敏生物发光法检测微量ATP及微生物。【方法】PCR扩增得到PPK、ADK基因,插入表达载体pET28a (+)中构建重组表达质粒pET28a (+)-PPKADK,表达PPK-ADK融合蛋白。利用表面包裹聚胺醇(Polyurethane)的磁珠(magnetic beads),通过化学反应将腺苷酸双磷酸酶(apyrase)固定于磁珠表面,制备固相腺苷酸双磷酸酶(Beads-apyrase),用于除去与融合蛋白结合的内源性ADP,降低ATP扩增反应的背景,从而使之与生物发光反应相结合,测定微量外源ATP及细菌菌落数。【结果】表达的融合蛋白具有PPK和ADK的活性,利用Beads-apyrase可以方便而有效的去除内源性ADP,显著地降低反应背景,从而实现了利用ATP扩增反应与传统生物发光反应结合,测定了小于1 fmol的外源微量ATP,使生物发光法检测ATP及微生物的灵敏度提高至少100倍。【结论】利用Beads-apyrase能够方便、有效地降低PPK-ADK中的ADP背景,从而使PPK-ADK催化的ATP扩增反应能够与传统生物发光法相结合,极大地提高了生物发光法的灵敏度。  相似文献   

10.
Chen ZH  Xu K  Zhao WT 《生理科学进展》2004,35(4):329-331
包含P2X3亚基的受体为三磷酸腺苷 (ATP)门控的阳离子通道 ,包括P2X3亚基的同源多聚体(P2X3受体 )和异源多聚体 (P2X2 /3受体 )。大量研究表明包含P2X3亚基的受体在介导多种类型痛觉中有重要作用  相似文献   

11.
Polyphosphate kinases (PPKs) catalyse the polymerisation and degradation of polyphosphate chains. As a result of this process, PPK produces or consumes energy in the form of ATP. Polyphosphate is a linear molecule that contains tens to hundreds of phosphate residues connected by macroergic bonds, and it appears to be an easily obtainable and rich source of energy from prebiotic times to the present. Notably, polyphosphate is present in the cells of all three domains of life, but PPKs are widely distributed only in Bacteria, as Archaea and Eucarya use various unrelated or “nonhomologous” proteins for energy and metabolic balance. The present study focuses on PPK1 and PPK2 homologues, which have been described to some extent in Bacteria, and the aim was to determine which homologue group, PPK1 or PPK2, is older. Phylogenetic analyses of 109 sequence homologues of Escherichia coli PPK1 and 109 sequence homologues of Pseudomonas aeruginosa PPK2 from 109 bacterial genomes imply that polyphosphate consumption (PPK2) evolved first and that phosphate polymerisation (PPK1) evolved later. Independently, a theory of the trends in amino acid loss and gain also confirms that PPK2 is older than PPK1. According to the results of this study, we propose 68 hypothetical proteins to mark as PPK2 homologues and 3 hypothetical proteins to mark as PPK1 homologues.  相似文献   

12.
Corynebacterium glutamicum is able to accumulate up to 600 mM cytosolic phosphorus in the form of polyphosphate (poly P). Granular poly P (volutin) can make up to 37% of the internal cell volume. This bacterium lacks the classic enzyme of poly P synthesis, class I polyphosphate kinase (PPK1), but it possesses two genes, ppk2A (corresponds to NCgl0880) and ppk2B (corresponds to NCgl2620), for putative class II (PPK2) PPKs. Deletion of ppk2B decreased PPK activity and cellular poly P content, while overexpression of ppk2B increased both PPK activity and cellular poly P content. Neither deletion nor overexpression of ppk2A changed specific activity of PPK or cellular poly P content significantly. Purified PPK2B of C. glutamicum is active as a homotetramer and formed poly P with an average chain length of about 125, as determined with (31)P nuclear magnetic resonance. The catalytic efficiency of C. glutamicum PPK2B was higher in the poly P-forming direction than for nucleoside triphosphate formation from poly P. The ppk2B deletion mutant, which accumulated very little poly P and grew as C. glutamicum wild type under phosphate-sufficient conditions, showed a growth defect under phosphate-limiting conditions.  相似文献   

13.
Inorganic polyphosphate (polyP) is obtained by the polymerization of the terminal phosphate of ATP through the action of the enzyme polyphosphate kinase (PPK). Despite the presence of polyP in every living cell, a gene homologous to that of known PPKs is missing from the currently sequenced genomes of Eukarya, Archaea, and several bacteria. To further study the metabolism of polyP in Archaea, we followed the previously published purification procedure for a glycogen-bound protein of 57 kDa with PPK as well as glycosyl transferase (GT) activities from Sulfolobus acidocaldarius (R. Skórko, J. Osipiuk, and K. O. Stetter, J. Bacteriol. 171:5162-5164, 1989). In spite of using recently developed specific enzymatic methods to analyze polyP, we could not reproduce the reported PPK activity for the 57-kDa protein and the polyP presumed to be the product of the reaction most likely corresponded to glycogen-bound ATP under our experimental conditions. Furthermore, no PPK activity was found associated to any of the proteins bound to the glycogen-protein complex. We cloned the gene corresponding to the 57-kDa protein by using reverse genetics and functionally characterized it. The predicted product of the gene did not show similarity to any described PPK but to archaeal and bacterial glycogen synthases instead. In agreement with these results, the recombinant protein showed only GT activity. Interestingly, the GT from S. acidocaldarius was phosphorylated in vivo. In conclusion, our results convincingly demonstrate that the glycogen-protein complex of S. acidocaldarius does not contain a PPK activity and that what was previously reported as being glycogen-bound PPK is a bacterial enzyme-like thermostable glycogen synthase.  相似文献   

14.
We have cloned the gene for polyphosphate:AMP phosphotransferase (PAP), the enzyme that catalyzes phosphorylation of AMP to ADP at the expense of polyphosphate [poly(P)] in Acinetobacter johnsonii 210A. A genomic DNA library was constructed in Escherichia coli, and crude lysates of about 6,000 clones were screened for PAP activity. PAP activity was evaluated by measuring ATP produced by the coupled reactions of PAP and purified E. coli poly(P) kinases (PPKs). In this coupled reaction, PAP produces ADP from poly(P) and AMP, and the resulting ADP is converted to ATP by PPK. The isolated pap gene (1,428 bp) encodes a protein of 475 amino acids with a molecular mass of 55.8 kDa. The C-terminal region of PAP is highly homologous with PPK2 homologs isolated from Pseudomonas aeruginosa PAO1. Two putative phosphate-binding motifs (P-loops) were also identified. The purified PAP enzyme had not only strong PAP activity but also poly(P)-dependent nucleoside monophosphate kinase activity, by which it converted ribonucleoside monophosphates and deoxyribonucleoside monophosphates to ribonucleoside diphosphates and deoxyribonucleoside diphosphates, respectively. The activity for AMP was about 10 times greater than that for GMP and 770 and about 1,100 times greater than that for UMP and CMP.  相似文献   

15.
Inorganic polyphosphate (polyP) is obtained by the polymerization of the terminal phosphate of ATP through the action of the enzyme polyphosphate kinase (PPK). Despite the presence of polyP in every living cell, a gene homologous to that of known PPKs is missing from the currently sequenced genomes of Eukarya, Archaea, and several bacteria. To further study the metabolism of polyP in Archaea, we followed the previously published purification procedure for a glycogen-bound protein of 57 kDa with PPK as well as glycosyl transferase (GT) activities from Sulfolobus acidocaldarius (R. Skórko, J. Osipiuk, and K. O. Stetter, J. Bacteriol. 171:5162–5164, 1989). In spite of using recently developed specific enzymatic methods to analyze polyP, we could not reproduce the reported PPK activity for the 57-kDa protein and the polyP presumed to be the product of the reaction most likely corresponded to glycogen-bound ATP under our experimental conditions. Furthermore, no PPK activity was found associated to any of the proteins bound to the glycogen-protein complex. We cloned the gene corresponding to the 57-kDa protein by using reverse genetics and functionally characterized it. The predicted product of the gene did not show similarity to any described PPK but to archaeal and bacterial glycogen synthases instead. In agreement with these results, the recombinant protein showed only GT activity. Interestingly, the GT from S. acidocaldarius was phosphorylated in vivo. In conclusion, our results convincingly demonstrate that the glycogen-protein complex of S. acidocaldarius does not contain a PPK activity and that what was previously reported as being glycogen-bound PPK is a bacterial enzyme-like thermostable glycogen synthase.  相似文献   

16.
Catalytic properties of Escherichia coli polyphosphate kinase (EC 2.7.4.1), a promising enzyme for use in ATP regeneration (Hoffman, et al., 1988, Biotechnol. Appl. Biochem. 10, 107-117), are reported here. E. coli polyphosphate kinase (PPK) is broadly active in the pH range 5.5 to 8.5, having an optimal Vmax at pH 7.2. The Km values for the substrates, ADP and polyphosphate (Pn), change little in the same pH range. The optimal concentration range for the Mg2+ activator is 1-20 mM, with an activity maximum at 10 mM Mg2+. In addition to Mg2+, Mn2+ and Co2+ can serve as activators of E. coli PPK, whereas Zn2+ and Cu2+ are highly inhibitory. E. coli PPK is most active with Pn substrates of chain length greater than 132 phosphoryl units. The enzyme activity decreases with decreasing Pn chain length and approaches zero (less than 1%) at a chain length less than or equal to 5. Equilibrium yields of ATP of greater than 85% are readily attained at substrate concentrations below 1 mM. An operational equilibrium constant for the PPK reaction, defined as [ATP]/[ADP][Pn], was determined to be 7.5 (+/- 3.4) x 10(5) M-1. The data presented here serve as a base of information from which assessments of the suitability of E. coli PPK for specific ATP regeneration applications can be made.  相似文献   

17.
Polyphosphate kinase (PPK), the principal enzyme required for the synthesis of inorganic polyphosphate (polyP) from ATP, also exhibits other enzymatic activities, which differ significantly in their biochemical optima and responses to chemical agents. These several activities include: polyP synthesis (forward reaction), nATP --> polyP(n) + nADP (Equation 1); ATP synthesis from polyP (reverse reaction), ADP + polyP(n) --> ATP + polyP(n - 1) (Equation 2); general nucleoside-diphosphate kinase, GDP + polyP(n) --> GTP + polyP(n - 1) (Equation 3); linear guanosine 5'-tetraphosphate (ppppG) synthesis, GDP + polyP(n) --> ppppG + polyP(n - 2) (Equation 4); and autophosphorylation, PPK + ATP --> PPK-P + ADP (Equation 5). The Mg(2+) optima are 5, 2, 1, and 0.2 mM, respectively, for the activities in Equations 1, 2, 3, and 4. Inorganic pyrophosphate inhibits the activities in Equations 1 and 3 but stimulates that in Equation 4. The kinetics of the activities in Equations 1, 2, and 3 are highly processive, whereas the transfer of a pyrophosphoryl group from polyP to GDP (Equation 4) is distributive and demonstrates a rapid equilibrium, random Bi-Bi catalytic mechanism. Radiation target analysis revealed that the principal functional unit of the homotetrameric PPK is a dimer. Exceptions are a trimer for the synthesis of ppppG (Equation 4) and a tetrameric state for the autophosphorylation of PPK (Equation 5) at low ATP concentrations. Thus, the diverse functions of this enzyme involve different subunit organizations and conformations. The highly conserved homology of PPK among 18 microorganisms was used to determine important residues and conserved regions by alanine substitution, by site-directed mutagenesis, and by deletion mutagenesis. Of 46 single-site mutants, seven exhibit none of the five enzymatic activities; in one mutant, ATP synthesis from polyP is reduced relative to GTP synthesis. Among deletion mutants, some lost all five PPK activities, but others retained partial activity for some reactions but not for others.  相似文献   

18.
Polyphosphate (polyP) is a ubiquitous biopolymer whose function and metabolism are incompletely understood. The polyphosphate kinase (PPK) of Acinetobacter sp. strain ADP1, an organism that accumulates large amounts of polyP, was purified to homogeneity and characterized. This enzyme, which adds the terminal phosphate from ATP to a growing chain of polyP, is a 79-kDa monomer. PPK is sensitive to magnesium concentrations, and optimum activity occurs in the presence of 3 mM MgCl(2). The optimum pH was between pH 7 and 8, and significant reductions in activity occurred at lower pH values. The greatest activity occurred at 40 degrees C. The half-saturation ATP concentration for PPK was 1 mM, and the maximum PPK activity was 28 nmol of polyP monomers per microg of protein per min. PPK was the primary, although not the sole, enzyme responsible for the production of polyP in Acinetobacter sp. strain ADP1. Under low-phosphate (P(i)) conditions, despite strong induction of the ppk gene, there was a decline in net polyP synthesis activity and there were near-zero levels of polyP in Acinetobacter sp. strain ADP1. Once excess phosphate was added to the P(i)-starved culture, both the polyP synthesis activity and the levels of polyP rose sharply. Increases in polyP-degrading activity, which appeared to be mainly due to a polyphosphatase and not to PPK working in reverse, were detected in cultures grown under low-P(i) conditions. This activity declined when phosphate was added.  相似文献   

19.
Vibrio cholerae O1, biotype El Tor, accumulates inorganic polyphosphate (poly P) principally as large clusters of granules. Poly P kinase (PPK), the enzyme that synthesizes poly P from ATP, is encoded by the ppk gene, which has been cloned from V. cholerae, overexpressed, and knocked out by insertion-deletion mutagenesis. The predicted amino acid sequence of PPK is 701 residues (81.6 kDa), with 64% identity to that of Escherichia coli, which it resembles biochemically. As in E. coli, ppk is part of an operon with ppx, the gene that encodes exopolyphosphatase (PPX). However, unlike in E. coli, PPX activity was not detected in cell extracts of wild-type V. cholerae. The ppk null mutant of V. cholerae has diminished adaptation to high concentrations of calcium in the medium as well as motility and abiotic surface attachment.  相似文献   

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