植物丙酮酸磷酸双激酶（PPDK）研究进展

丙酮酸磷酸双激酶（PPDK）是C4植物和景天科酸代谢（CAM）植物光合作用的关键酶,催化形成固定CO2的初始分子受体磷酸烯醇式丙酮酸（PEP）。本文重点比较了植物的ppDK基因结构及分子进化关系,综述了PPDK在C4植物和C3植物中的功能、PPDK的调控机理、PPDK在胁迫条件下的功能以及转PPDK基因等在近年来的研究进展。     

丙酮酸磷酸双激酶及其基因结构

丙酮酸磷酸双激酶（PPDK）在植物C4光合途径中催化CO2原初受体磷酸烯醇式丙酮酸（PEP）的形成。本文介绍PPDK的类型、分布、生理功能、活性调节、基因结构和C4植物的PPDK基因转化C3植物及其与转基因植株光合作用之间关系的研究进展。     

多聚磷酸激酶及其在ATP再生体系构建中的进展

构建高效的腺嘌呤核苷三磷酸(adenosinetriphosphate,ATP)再生体系可显著提高生物催化磷酸基团转移反应的效率。多聚磷酸激酶(poly phosphate kinase, PPK)能利用来源广、廉价且稳定的多聚磷酸(polyphosphate, Poly P)盐作为磷酸基供体，能够实现单磷酸腺苷(adenosine monophosphate,AMP)、二磷酸腺苷(adenosinediphosphate,ADP)、ATP、PolyP之间磷酸基的高效定向转移，已成为构建ATP再生体系的首选。本文介绍了不同类型PPK的结构特征、相关催化机制以及不同来源的PPK在酶活、催化效率、稳定性和底物偏好性的特征差异；归纳和列举了针对野生PPK酶学性质不足进行分子改造的实例，并对PPK在ATP再生体系构建的研究进展进行了总结。     

植物丙酮酸磷酸双激酶研究进展

丙酮酸磷酸双激酶(pyruvate orthophosphate dikinase,PPDK)作为C4光合途径中一个非常重要的限速酶,其功能已经清楚,但在C3植物中以及逆境条件下的作用尚不明确。在阐述PPDK基本生物学特征的基础上,重点介绍了PPDK在C4植物和C3植物中的功能、活性调控、基因工程以及PPDK对逆境胁迫应答的研究进展,以期为植物抗逆基因挖掘及抗逆种质创制提供参考。     

籽粒苋丙酮酸磷酸双激酶(PPDK)基因的克隆及其特征分析

丙酮酸磷酸双激酶(pyruvate orthophosphate dikinase, PPDK)是C4植物在光合过程中的关键限速酶之一。本研究采用RT-PCR技术,从籽粒苋叶片中克隆到籽粒苋丙酮酸磷酸双激酶基因cDNA全长。核苷酸序列分析表明,该基因cDNA序列开放阅读框全长为2 871 bp (GenBank登录号:JF907701.1),可编码956个氨基酸,分子量为104 kD。籽粒苋丙酮酸磷酸双激酶PPDK与长寿花(Kalanchoe fedtschenko)、冰叶日中花(M.crystallinum)和板栗(Castanea mollissima)等双子叶植物PPDK的同源性较高,分别为82.5%、82.0%和81.4%;与稗草(Echinochloa crusgalli)、高粱(Sorghum bicolor)和玉米(Zea mays)等单子叶植物PPDK的序列一致性分别为74.5%、74.5%和73.9%。进化树分析表明,其与禾本科植物长寿花最为接近。半定量RT-PCR研究表明,该基因受光诱导而上调表达,且在绿色叶片中的表达水平最高。克隆籽粒苋PPDK基因将为今后作物高光效分子育种提供备选基因。     

PKM2在肿瘤形成中的作用及其活性调节

肿瘤细胞利用有氧糖酵解将葡萄糖转变为细胞代谢及增殖所需的物质,如核苷酸、氨基酸和脂质等,并产生ATP。丙酮酸激酶是糖酵解途径中的限速酶,催化磷酸烯醇式丙酮酸生成丙酮酸。其四种同工酶之一PKM2(pyruvate kinase M2),由四个亚基组成,有单体、二聚体及四聚体等多种存在形式。其中,PKM2四聚体活性最强,能促进葡萄糖通过氧化磷酸化彻底氧化分解生成ATP,而其二聚体则促进Warburg效应,即葡萄糖的有氧酵解。两者之间的平衡在肿瘤形成中起到了很重要的作用,同时也受到一系列因子的调控。该文就PKM2在肿瘤代谢中的作用及其活性调节作一介绍。     

热玫瑰小双孢菌来源的丙酮酸磷酸双激酶的表达及应用

以热玫瑰小双孢菌基因组DNA为模板, 通过PCR扩增得到了编码PPDK的基因, 将此基因片段插入到表达载体pET28a(+)中构建得到了重组表达质粒pET28a(+)-PPDK, 将重组表达质粒pET28a(+)-PPDK转化到大肠杆菌BL21(DE3)中, 经过IPTG诱导, 重组菌成功表达了N端带有6-His Tag的重组PPDK。经SDS-PAGE分析, 重组PPDK单体分子量为101 kD。经过镍亲和层析和超滤后, 重组PPDK蛋白基本达到电泳纯, 并被成功应用于焦测序中。     

热玫瑰小双孢菌来源的丙酮酸磷酸双激酶的表达及应用

以热玫瑰小双孢菌基因组DNA为模板, 通过PCR扩增得到了编码PPDK的基因, 将此基因片段插入到表达载体pET28a(+)中构建得到了重组表达质粒pET28a(+)-PPDK, 将重组表达质粒pET28a(+)-PPDK转化到大肠杆菌BL21(DE3)中, 经过IPTG诱导, 重组菌成功表达了N端带有6-His Tag的重组PPDK。经SDS-PAGE分析, 重组PPDK单体分子量为101 kD。经过镍亲和层析和超滤后, 重组PPDK蛋白基本达到电泳纯, 并被成功应用于焦测序中。     

大豆叶片C4循环途径酶

以不同产量水平的大豆（Glycine max(L.)Merr.)品种“黑农40”和“黑农37”为实验材料,研究了苗期,开花期,初荚期和鼓粒期等不同发育时期的叶片中磷酸烯醇式丙酮酸羧化酶（PEPCase),NADP－苹果酸氢酶（NADPMDH）,NADP－苹果酸酶（NADP－ME）,丙酮酸磷酸双激酶（PPDK）等C4途径的主要酶和1,5－二磷酸核酮糖羧化酶（RuBPCase）的活性变化,结果表明两种大豆叶片均含有上述4种酶,而且从PEPCase/RuBPCase的比值看,C4酶在高产大豆“黑农40”叶片中表达较高,表明C4循环途径与大豆产量密切相关,这一结果还暗示有可能通过检测C4途径酶的表达水平及比例,筛选出具有高产潜力的大豆种质。     

嗜酸氧化亚铁硫杆菌ATP硫酸化酶的表达、纯化及性质鉴定

ATP硫酸化酶是一种催化ATP和SO42-反应生成腺嘌呤-5’-磷酸硫酸(APS)和焦磷酸盐(PPi)的酶,它是硫酸根同化反应第一步的关键酶。以嗜酸氧化亚铁硫杆菌(A.ferrooxidansATCC 23270)基因组为模板,用PCR扩增得到ATPS基因,并克隆到表达载体pLM1上。加入IPTG的诱导表达,用AKTA蛋白纯化仪的镍柱亲和层析纯化得到浓度和纯度都较高的ATPS蛋白。SDS-PAGE分析,证实其分子量大小为33 kD,并成功的测出了其活性,比活达3.0×103U/mg。     

An enzymatic cycling method using pyruvate orthophosphate dikinase and firefly luciferase for the simultaneous determination of ATP and AMP (RNA)

A novel bioluminescent enzymatic cycling assay for ATP and AMP with concomitant use of firefly luciferase and pyruvate orthophosphate dikinase (PPDK) was developed. In this system, AMP and pyrophosphate produced from ATP by firefly luciferase were converted back into ATP by PPDK. This resulted in constant luminescence once the stable phase had been reached. Background luminescence of the reagent was reduced with adenosine phosphate deaminase by degrading ATP and AMP in the reagent. The maximum recycling ratio calculated from the integrated luminescence value was 2.64 cycles/min. The measurable ranges for ATP and AMP were equal and were between 4 x 10(-13) and 4 x 10(-17) mol/assay. The amount of yeast RNA could be estimated in the range of 1 x 10(-8) to 1 x 10(-12) g/assay by estimating the amount of AMP resulting from the degradation of RNA with nuclease P1. Various food samples were subjected to measurement of the amount of ATP + AMP + RNA to provide an index for hygiene monitoring. For beef extract, sensitivity was improved by more than 20 million compared to the previous methods relying only on the amount of ATP as an index.     

Enumeration of bacterial cell numbers by amplified firefly bioluminescence without cultivation

We recently developed a novel bioluminescent enzymatic cycling assay for ATP and AMP with the concomitant use of firefly luciferase and pyruvate orthophosphate dikinase (PPDK), where AMP and pyrophosphate produced from ATP by firefly luciferase were converted back into ATP by PPDK. Background luminescence derived from contaminating ATP and AMP in the reagent was reduced using adenosine phosphate deaminase which degrades ATP, ADP, and AMP, resulting in constant and highly amplified bioluminescence with low background luminescence. To detect bacterial cells without cultivation, we applied the above bioluminescent enzymatic cycling reagent to rapid microbe detection system. ATP spots (0.31-5.0 amol/spot) at the level of a single bacterial cell were detected with 5 min signal integration, signifying that integrated luminescence was amplified 43 times in comparison to traditional ATP bioluminescence. Consequently, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Lactobacillus brevis in beer were detected without cultivation. Significant correlation was observed between the number of signal spots obtained using this novel system and the colony-forming units observed with the conventional colony-counting method (R(2)=0.973).     

Investigations of the partial reactions catalyzed by pyruvate phosphate dikinase

The kinetic mechanism of pyruvate phosphate dikinase (PPDK) from Bacteroides symbiosus was investigated with several different kinetic diagnostics. Initial velocity patterns were intersecting for AMP/PPi and ATP/Pi substrate pairs and parallel for all other substrate pairs. PPDK was shown to catalyze [14C]pyruvate in equilibrium phosphoenolpyruvate (PEP) exchange in the absence of cosubstrates, [14C]AMP in equilibrium ATP exchange in the presence of Pi/PPi but not in their absence, and [32P]Pi in equilibrium PPi exchange in the presence of ATP/AMP but not in their absence. The enzyme was also shown, by using [alpha beta-18O, beta, beta-18O2]ATP and [beta gamma-18O, gamma, gamma, gamma-18O3]ATP and 31P NMR techniques, to catalyze exchange in ATP between the alpha beta-bridge oxygen and the alpha-P nonbridge oxygen and also between the beta gamma-bridge oxygen and the beta-P nonbridge oxygen. The exchanges were catalyzed by PPDK in the presence of Pi but not in its absence. These results were interpreted to support a bi(ATP,Pi) bi(AMP,PPi) uni(pyruvate) uni(PEP) mechanism. AMP and Pi binding order was examined by carrying out dead-end inhibition studies. The dead-end inhibitor adenosine 5'-monophosphorothioate (AMPS) was found to be competitive vs AMP, noncompetitive vs PPi, and uncompetitive vs PEP. The dead-end inhibitor imidodiphosphate (PNP) was found to be competitive vs PPi, uncompetitive vs AMP, and uncompetitive vs PEP. These results showed that AMP binds before PPi. The ATP and Pi binding order was studied by carrying out inhibition, positional isotope exchange, and alternate substrate studies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pyruvate phosphate dikinase from a thermophilic actinomyces Microbispora rosea subsp. aerata: purification, characterization and molecular cloning of the gene.

Various thermophilic bacteria were analyzed by Southern hybridization analysis using oligonucleotide probes coding for the pyruvate phosphate dikinase (PPDK) gene from Clostridium symbiosum, and positive hybridization signals were observed in the chromosomal DNAs from Microbispora rosea subsp. aerata (IFO 14047). PPDK activity was detected in lactose induced cells and the enzyme was purified to homogeneity. The molecular mass of PPDK was estimated to be 230000 by gel filtration chromatography and 91000 by SDS-PAGE, suggesting that PPDK is a dimeric enzyme. This enzyme was specific for adenine nucleotide and the apparent Km values for AMP, PPi, and phosphoenolpyruvate were 5, 38, and 280 microM, respectively. It was stable in the pH range 6 to 11, and retained 80% activity after 60 min heat treatment at 60 degrees C. We cloned the PPDK gene from M. rosea. It consists of 878 amino acids with a molecular mass of 95514. Sequence comparison indicates around 50% similarity with other PPDKs and it has all the highly conserved regions of the related enzymes. We expressed the PPDK gene in Escherichia coli and obtained enzymatically active protein.     

Nano‐sized bacterial magnetic particles displaying pyruvate phosphate dikinase for pyrosequencing

There is a high demand for inexpensive and high‐throughput DNA sequencing technologies in molecular biology and applied biosciences. In this study, novel nano‐sized magnetic particles displaying enzymes for pyrosequencing, a rather novel bioluminometric DNA sequencing method based on the sequencing‐by‐synthesis principle by employing a cascade of several enzymatic reactions, was developed. A highly thermostable enzyme, pyruvate phosphate dikinase (PPDK) which converts PPi to ATP was successfully expressed onto bacterial magnetic particles (BacMPs) using a novel protein display system of Magnetospirillum magneticum AMB‐1. The enzymatic stability of BacMPs displaying PPDK (PPDK‐BacMPs) to pH and temperature was evaluated and its broad range of properties was shown. Subsequently, PPDK‐BacMPs were applied in pyrosequencing and a target oligonucleotide was successfully sequenced. The PPDK enzyme displayed on BacMPs was shown to be recyclable in each sequence reaction as they can be manipulated by magnetic force. It was concluded that nano‐sized PPDK‐BacMPs are useful for the scale down of pyrosequencing reaction volumes, thus, permitting high‐throughput. The recycling of enzymes was also shown to be promising and applicable for the development of an inexpensive DNA sequencing at a low running cost. Biotechnol. Bioeng. 2009;103: 130–137. © 2008 Wiley Periodicals, Inc.     

Examination of the structure, stability, and catalytic potential in the engineered phosphoryl carrier domain of pyruvate phosphate dikinase

Pyruvate phosphate dikinase (PPDK) is a multidomain protein that catalyzes the interconversion of ATP, pyruvate, and phosphate with AMP, phosphoenolpyruvate (PEP), and pyrophosphate using its central domain to transport phosphoryl groups between two distant active sites. In this study, the mechanism by which the central domain moves between the two catalytic sites located on the N-terminal and C-terminal domains was probed by expressing this domain as an independent protein and measuring its structure, stability, and ability to catalyze the ATP/phosphate partial reaction in conjunction with the engineered N-terminal domain protein (residues 1-340 of the native PPDK). The encoding gene was engineered to express the central domain as residues 381-512 of the native PPDK. The central domain was purified and shown to be soluble, monomeric (13,438 Da), and stable (deltaG = 4.3 kcal/mol for unfolding in buffer at pH 7.0, 25 degrees C) and to possess native structure, as determined by multidimensional heteronuclear NMR analysis. The main chain structure of the central domain in solution aligns closely with that of the X-ray structure of native PPDK (the root-mean-square deviation is 2.2 A). Single turnover reactions of [14C]ATP and phosphate, carried out in the presence of equal concentrations of central domain and the N-terminal domain protein, did not produce the expected products, in contrast to efficient product formation observed for the N-terminal central domain construct (residues 1-553 of the native PPDK). These results are interpreted as evidence that the central domain, although solvent-compatible, must be tethered by the flexible linkers to the N-terminal domain for the productive domain-domain docking required for efficient catalysis.     

Enzymatic method for continuous monitoring of inorganic pyrophosphate synthesis

A sensitive method for the analysis of inorganic pyrophosphate (PPi) which utilizes the enzymes ATP sulfurylase and firefly luciferase is described. The assay is based on continuous monitoring of the ATP formed in the ATP sulfurylase reaction using purified firefly luciferase. The assay can be completed in less than 2 s and is not affected by inorganic phosphate. The method has been used for continuous monitoring of formation of PPi in Rhodospirillum rubrum chromatophores. The assay is extremely sensitive, the linear range of the assay being 1 X 10(-9) - 5 X 10(-7) M PPi. It is suitable for routine applications. It is also possible to use the method for determination of low amounts of adenosine 5'-phosphosulfate.     

Real-time detection and quantification of adenosine triphosphate sulfurylase activity by a bioluminometric approach.

A real-time, sensitive, and simple assay for detection and quantification of adenosine triphosphate sulfurylase (ATP:sulfate adenylytransferase, EC 2.7.7.4) activity has been developed. The method is based on detection of ATP generated in the ATP sulfurylase reaction between APS and PPi by the firefly luciferase system. For the Saccharomyces cerevisiae ATP sulfurylase, the concentrations of APS and PPi at the half-maximal rate were found to be about 0.5 and 7 microM, respectively. The assay is sensitive and yields linear response between 0.1 microU and 50 mU. The method can be used for monitoring and quantification of recombinant ATP sulfurylase activity in Escherichia coli lysate, as well as for detection of the activity during different purification procedures.