基于结构域活性分析的大肠杆菌T蛋白结构研究

大肠杆菌T蛋白含有三个结构域:分支酸变位酶、预苯酸脱氢酶和调节结构域。文章作者分段克隆了T蛋白的分支酸变位酶、预苯酸脱氢酶和调节结构域等片段,并对其进行了活性研究。研究发现,定位于N末端的分支酸变位酶结构域的比活性虽然不高,而稳定性较好;同时拥有调节结构域和预苯酸脱氢酶结构域的C末端片段,其预苯酸脱氢酶比活性的剩余百分率虽然高于分支酸变位酶结构域,但稳定性较差。作者进而表达了C末端切除38个氨基酸的T/1-336片段,发现预苯酸脱氢酶活性彻底丧失,而其分支酸变位酶和调节结构域的活性却基本保留。这说明T蛋白中分支酸变位酶结构域拥有一个相对独立、完整的结构,而预苯酸脱氢酶结构域和调节结构域交织共存,结构松散。     

利用蛋白质限制性水解法解析大肠杆菌T蛋白的独立结构域

为了解析分支酸变位酶和预苯酸脱氢酶在大肠杆菌T蛋白的定位,根据T蛋白限制性水解结果,分段克隆分支酸变位酶和预苯酸脱氢酶.T蛋白限制性水解结果显示,第93位氨基酸是大片段的N端,分段克隆的1～93 片段测定得到分支酸变位酶活性,96～373片段得到了预苯酸脱氢酶活性.研究表明,大肠杆菌T蛋白由两个独立结构域组成,N端93个氨基酸组成了分支酸变位酶,C端277个氨基酸组成了预苯酸脱氢酶.     

橡胶树白粉菌分支酸变位酶基因克隆及蛋白表达纯化

由橡胶树白粉菌引起的橡胶树白粉病是橡胶树的重要的叶部病害之一,严重影响橡胶的产量。然而目前对橡胶树白粉菌的致病机理研究匮乏。分支酸变位酶是莽草酸途径的关键酶,能够将分支酸转化为预苯酸,为植物提供氨基酸及大量代谢产物,在植物抗病中起到非常重要的作用。而植物病原物在致病过程当中能够分泌分支酸变位酶影响植物莽草酸途径,从而抑制植物的防卫反应。因此研究橡胶树白粉菌分支酸变位酶在其致病过程中的功能具有一定的意义。试验利用同源比对分析在橡胶树白粉菌基因组中获得一个分支酸变位酶同源蛋白,并用PCR克隆获得橡胶树白粉菌分支酸变位酶基因,命名为OHCmu。后续构建GST-OHCmu融合原核表达载体,筛选最优诱导条件,并利用GST亲和层析柱对蛋白进行纯化。结果表明橡胶树白粉菌OHCmu基因大小843 bp,具有1个内含子,编码263个氨基酸;具有d5csma_结构域,属于Chorismate mutaseⅡ蛋白家族;GST-OHCmu融合蛋白外源诱导表达在供试条件下(IPTG:0.8 mmol/L, 16℃)可以有较好的表达,获得融合蛋白大小约为56 kD。经过GST亲和层析柱纯化、切割GST标签后,顺利获得浓度较高、较纯的橡胶树白粉菌OHCmu蛋白。研究结果为后续OHCmu蛋白的特性及致病机理研究提供参考。     

头状轮生链霉菌芳香族氨基酸合成途径的调节

对头状轮生链霉菌(Streptoverticillium caespitosus)芳香氨基酸合成途径的研究表明,第一个酶即3—脱氧—α—阿拉伯庚酮糖-7-磷酸(DAHP)合成酶无同工酶,不被L-色氨酸阻遏,比活力可被硝酸盐促进。L-色氨酸强烈地反馈抑制此酶,L-酪氨酸和L-苯丙氨酸无作用。L-色氨酸的反馈抑制对磷酸烯醇式丙酮酸(PEP)是非竞争性的,K_I为373μmol/L。酶对PEP和4-磷酸亦藓糖(E4P)的K_m值分别为50和100μmol/L。PEP和C0²⁺对酶有稳定作用。邻氨基苯甲酸合成酶活力可被1mmol/L L-色氨酸完全抑制,此酶也受L-色氨酸的阻遏,但是色氨酸支路上其余4个酶不被阻遏。分支酸变位酶被L-酪氨酸抑制。L-苯丙氨酸抑制预苯酸脱水酶,并更强地抑制预苯酸脱氢酶。     

大肠杆菌CM/PDT抗反馈抑制突变体的构建及其性质

为了获得具有高催化活性且抗反馈抑制的大肠杆菌分支酸变位酶 预苯酸脱水酶 (chorismatemutase prephenatedehydrataseCM PDT) [EC5 .4 .99.5 EC4 .2 .1.5 1],通过相关菌种CM PDT氨基酸序列同源比较 ,寻找高度保守位点 .用定点突变及PCR法构建突变酶M1(缺失 30 4T、30 5G、Q30 6K)、M2 (缺失W 338)、M3(缺失 30 1～ 386位氨基酸 )、M32 9(E32 9A)和M374 (C374A) ,野生型及各突变型基因与pET2 8a(+ )载体连接后 ,表达融合蛋白 .在非变性条件下 ,由TALON金属螯合亲和层析柱纯化野生型和突变体的酶蛋白 .酶活性测定表明 ,突变体M3的PDT活性下降为野生型活性的 2 9% ,但保持了CM活性 .突变体M374保持了CM ,PDT两种酶的活性 ,突变体M1、M2、M32 9的CM ,PDT活性有一定程度的提高 .酶抗反馈抑制作用检测表明 ,突变体M3、M374解除了苯丙氨酸的反馈抑制作用 ,M1、M2、M32 9部分解除了苯丙氨酸的反馈抑制作用 .与含野生型pheA基因的E .coliBL2 1菌株相比 ,含突变基因的E .coliBL2 1菌株对 10mmol L的苯丙氨酸代谢类似物具有强的抗反馈抑制作用 ,其中M1,M2 ,M3对 2 0mmol L的类似物具有抗反馈抑制作用     

谷氨酸棒杆菌苯丙氨酸生物合成有关基因克隆及在选育苯丙氨酸产生菌中的应用

谷氨酸棒杆菌K_(38)是一株抗对—氟苯丙氨酸和邻—氟苯丙氨酸的苯丙氨酸产生菌;而谷氨酸棒杆菌KY_(9456)则是一株缺失分枝酸变位酶和预苯酸脱水酶菌株。我们把K_(38)的分枝酸变位酶和预苯酸脱水酶基因克隆到KY_(9456)的pCE_(53)质粒中,构建了质粒pCmB_4。pCmB_4含有一段插入到pCE_(53)BamHI单一酶切位点上的9.4KbBamHIDNA片段。质粒pCmB_4弥补了KY_(9456)的苯丙氨酸和酪氨酸双重营养缺陷。pCmB_4质粒转入谷氨酸棒杆菌RRL_5中:结果RRL_5的分枝酸变位酶活性增加10倍左右。pCmB_4质粒转入K_(38)中,携带质粒的K_(38)菌可累积19.0mg/ml苯丙氨酸(比原K_(38)增产50%以上)。     

外源基因pheA、aroG和tyrB在苯丙氨酸合成途径中的共表达

利用基因工程技术提高了短杆菌的苯丙氨酸合成途径中关键酶活性,大幅度地增加了生物合成苯丙氨酸的产时。首先采用聚合酶链反应（ＰＣＲ）从大肠杆菌的氟代苯丙氨酸抗性变异菌株基因组中扩增到与苯丙氨酸合成相关的ａｒｏＧ,ｐｈｅＡ和ｔｙｒＢ３个基因。ａｒｏＧ编码３－脱氧－２－阿拉伯庚酮糖－７－磷酸合成酶（ＤＳ）,ｐｈｅＡ编码双功能酶蛋白－分枝酸变位酶（ＣＭ）和预苯酸脱水酶（ＰＤ）,ｔｙｒＢ编码转氨酶（ＡＴ）。设     

核盘菌AROM蛋白的三级结构分析

核盘菌编码AROM蛋白的arom基因已经被克隆测序,本文根据该基因翻译的氨基酸序列用同源模建方法和从头模建方法分析了AROM蛋白各结构域的三级结构和功能位点,以及该蛋白二聚体可能的组装方式。结果表明,核盘菌AROM蛋白的脱氢奎尼酸合酶结构域进一步由N-端含有一个Rossmann折叠的α/β结构域和C-端的α螺旋结构域组成;5-烯醇丙酮酰莽草酸-3-磷酸合酶结构域则由两个相似结构域组成,每个结构域含有不同拷贝数的β折叠和α螺旋;莽草酸激酶结构域的N-端由三个β折叠组成;脱氢奎尼酸酶结构域为(α2β2)3多肽,在N-端有一对反平行的β链,在C-端有loop环;莽草酸脱氢酶结构域含有一个由α/β组成的催化结构域和一个含有Rossmann折叠的NADPH结合结构域。     

RGS蛋白在G蛋白信号转导中的作用与调节

RGS蛋白是近年来不断发现的新的蛋白家族,它们的结构中都包含一个高度保守的RGS结构域。目前从RGS结构域的结构及其同源性出发,对RGS蛋白与Gα亚单位及Gβγ二聚体的相互作用、RGS蛋白的调节活性及其动力学过程、RGS蛋白调节作用的分子机制及其生物学效应等进行了广泛探讨。研究发现,由于高度保守的RGS结构域的存在,几乎所有的RGS有GAP活性,并对G蛋白信号转导发挥负性调节作用。G蛋白信号转导是很多胞外信号引发细胞生理功能改变的共同途径,RGS蛋白的深入研究对于充分阐明该信号转导体系的构成及其调节机制具有深刻意义。     

芳香族氨基酸羟化酶家族结构与催化功能

芳香族氨基酸羟化酶(AAAH）家族是一类单加氢酶,包括苯丙氨酸羟化酶(PAH)、酪氨酸羟化酶(TH)和色氨酸羟化酶(TPH). 在辅因子四氢生物蝶呤、铁原子及氧存在下,分别催化苯丙氨酸、酪氨酸、色氨酸的羟化反应. 多种疾病如苯丙酮尿症、帕金森氏病以及神经相关疾病的发病机制均与这类酶有关. 本文综述近年来对芳香族氨基酸羟化酶家族蛋白结构功能、底物特异性、催化机制等方面的研究进展,为该类酶的定向进化及功能应用提供新思路.     

Mapping of chorismate mutase and prephenate dehydrogenase domains in the Escherichia coli T-protein.

The Escherichia coli bifunctional T-protein transforms chorismic acid to p-hydroxyphenylpyruvic acid in the l-tyrosine biosynthetic pathway. The 373 amino acid T-protein is a homodimer that exhibits chorismate mutase (CM) and prephenate dehydrogenase (PDH) activities, both of which are feedback-inhibited by tyrosine. Fifteen genes coding for the T-protein and various fragments thereof were constructed and successfully expressed in order to characterize the CM, PDH and regulatory domains. Residues 1-88 constituted a functional CM domain, which was also dimeric. Both the PDH and the feedback-inhibition activities were localized in residues 94-373, but could not be separated into discrete domains. The activities of cloned CM and PDH domains were comparatively low, suggesting some cooperative interactions in the native state. Activity data further indicate that the PDH domain, in which NAD, prephenate and tyrosine binding sites were present, was more unstable than the CM domain.     

pheA (Rv3838c) of Mycobacterium tuberculosis encodes an allosterically regulated monofunctional prephenate dehydratase that requires both catalytic and regulatory domains for optimum activity

Prephenate dehydratase (PDT) is a key regulatory enzyme in l-phenylalanine biosynthesis. In Mycobacterium tuberculosis, expression of pheA, the gene encoding PDT, has been earlier reported to be iron-dependent (1, 2). We report that M. tuberculosis pheA is also regulated at the protein level by aromatic amino acids. All of the three aromatic amino acids (phenylalanine, tyrosine, and tryptophan) are potent allosteric activators of M. tuberculosis PDT. We also provide in vitro evidence that M. tuberculosis PDT does not possess any chorismate mutase activity, which suggests that, unlike many other enteric bacteria (where PDT exists as a fusion protein with chorismate mutase), M. tuberculosis PDT is a monofunctional and a non-fusion protein. Finally, the biochemical and biophysical properties of the catalytic and regulatory domains (ACT domain) of M. tuberculosis PDT were studied to observe that, in the absence of the ACT domain, the enzyme not only loses its regulatory activity but also its catalytic activity. These novel results provide evidence for a monofunctional prephenate dehydratase enzyme from a pathogenic bacterium that exhibits extensive allosteric activation by aromatic amino acids and is absolutely dependent upon the presence of catalytic as well as the regulatory domains for optimum enzyme activity.     

Regulation of phenylalanine biosynthesis. Studies on the mechanism of phenylalanine binding and feedback inhibition in the Escherichia coli P-protein.

Isothermal titration calorimetry (ITC) and site-directed mutagenesis were used to study the interaction of Phe with (a) the Escherichia coli P-protein, a bifunctional chorismate mutase/prephenate dehydratase that is feedback inhibited by Phe, (b) PDT32, a 32 kDa P-protein fragment (residues 101-386) containing the prephenate dehydratase and regulatory domains, and (c) R12, a C-terminal 12 kDa P-protein fragment (residues 286-386) containing the regulatory domain. DeltaH(total) values for PDT32, which included the heats of Phe binding, conformational change, and dimerization, established that in developing a mechanism for end product feedback inhibition, the P-protein has evolved a ligand recognition domain that exhibits Phe-binding enthalpies comparable to those reported for other full-fledged amino acid receptor proteins. Sequence alignments of R12 with other Phe-binding enzymes identified two highly conserved regions, GALV (residues 309-312) and ESRP (residues 329-332). Site-directed mutagenesis and ITC established that changes in the GALV and ESRP regions affected Phe binding and feedback inhibition to different extents. Mutagenesis further showed that C374 was essential for feedback inhibition, but not for Phe binding, while W338 was involved in Phe binding, but not in the Phe-induced conformational change required for feedback inhibition.     

Structural analysis of a 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase with an N-terminal chorismate mutase-like regulatory domain

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) catalyzes the first step in the biosynthesis of a number of aromatic metabolites. Likely because this reaction is situated at a pivotal biosynthetic gateway, several DAHPS classes distinguished by distinct mechanisms of allosteric regulation have independently evolved. One class of DAHPSs contains a regulatory domain with sequence homology to chorismate mutase-an enzyme further downstream of DAHPS that catalyzes the first committed step in tyrosine/phenylalanine biosynthesis-and is inhibited by chorismate mutase substrate (chorismate) and product (prephenate). Described in this work, structures of the Listeria monocytogenes chorismate/prephenate regulated DAHPS in complex with Mn(2+) and Mn(2+) + phosphoenolpyruvate reveal an unusual quaternary architecture: DAHPS domains assemble as a tetramer, from either side of which chorismate mutase-like (CML) regulatory domains asymmetrically emerge to form a pair of dimers. This domain organization suggests that chorismate/prephenate binding promotes a stable interaction between the discrete regulatory and catalytic domains and supports a mechanism of allosteric inhibition similar to tyrosine/phenylalanine control of a related DAHPS class. We argue that the structural similarity of chorismate mutase enzyme and CML regulatory domain provides a unique opportunity for the design of a multitarget antibacterial.     

Probing the catalytic mechanism of prephenate dehydratase by site-directed mutagenesis of the Escherichia coli P-protein dehydratase domain

The Escherichia coli bifunctional P-protein, which plays a central role in L-phenylalanine (Phe) biosynthesis, contains distinct chorismate mutase (CM) and prephenate dehydratase (PDT) domains as well as a regulatory (R) domain for feedback control by Phe. To elucidate the catalytic mechanism of PDT in the P-protein, 24 mutations of 15 conserved residues in the PDT domain were created, expressed in the pheA(-)E. coli strain NK6024, and studied for their effect on PDT activity. Fourteen mutant enzymes were purified to homogeneity, tested for feedback inhibition by Phe, and characterized by kinetic analysis and circular dichroism spectroscopy. Selected mutant enzymes were further studied by gel filtration, fluorescence emission, and microcalorimetry. In addition, a monofunctional PDT domain (PDT20, residues 101-285) was cloned and overexpressed in plasmid pET with expression levels up to 200-250 mg/L. PDT20 retained full PDT activity, lacked CM activity, and was insensitive to feedback inhibition by Phe. Four residues (T278, N160, Q215, and S208) were shown to be important for PDT catalysis. The values of k(cat)/K(m) for the S208A/C and T278S mutant enzymes were 100-fold lower, and 500-fold lower for the N160A and Q215A mutant enzymes than the wild-type (WT) protein. The T278A and T278V mutant enzymes displayed no measurable catalytic activity, yet bound both prephenate and a competitive inhibitor (S-DNBA) comparably to the WT protein. These data, taken together with the normal CD spectra of the mutant enzymes, strongly suggested that T278 was involved in the catalytic mechanism. To establish whether acidic residues were involved in catalysis, all the conserved Glu and Asp residues in the PDT domain were mutated to Ala. None of these mutations significantly reduced PDT activity, indicating that the acidic residues of the PDT domain are not directly involved in catalysis. However, two mutant enzymes (E159A and E232A) displayed higher levels of PDT activity (2.2- and 3.5-fold, respectively), which was due to enhanced substrate binding. For the double mutant enzyme (E159A-E232A), k(cat)/K(m) was ca. 7-fold higher than for the WT enzyme, while its K(m) was 4.6-fold lower.     

Interconvertible molecular-weight forms of the bifunctional chorismate mutase-prephenate dehydratase from Acinetobacter calcoaceticus

Acinetobacter calcoaceticus belongs to a large phylogenetic cluster of gram-negative procaryotes that all utilize a bifunctional P-protein (chorismate mutase-prephenate dehydratase) [EC 5.4.99.5-4.2.1.51] for phenylalanine biosynthesis. These two enzyme activities from Ac. calcoaceticus were inseparable by gel-filtration or DEAE-cellulose chromatography. The molecular weight of the P-protein in the absence of effectors was 65,000. In the presence of L-tyrosine (dehydratase activator) or L-phenylalanine (inhibitor of both P-protein activities), the molecular weight increased to 122,000. Maximal activation (23-fold) of prephenate dehydratase was achieved at 0.85 mM L-tyrosine. Under these conditions, dehydratase activity exhibited a hysteretic response to increasing protein concentration. Substrate saturation curves for prephenate dehydratase were hyperbolic at L-tyrosine concentrations sufficient to give maximal activation (yielding a Km,app of 0.52 mM for prephenate), whereas at lower L-tyrosine concentrations the curves were sigmoidal. Dehydratase activity was inhibited by L-phenylalanine, and exhibited cooperative interactions for inhibitor binding. A Hill plot yielded an n' value of 3.1. Double-reciprocal plots of substrate saturation data obtained in the presence of L-phenylalanine indicated cooperative interactions for prephenate in the presence of inhibitor. The n values obtained were 1.4 and 3.0 in the absence or presence of 0.3 mM L-phenylalanine, respectively. The hysteretic response of chorismate mutase activity to increasing enzyme concentration was less dramatic than that of prephenate dehydratase. A Km,app for chorismate of 0.63 mM was obtained. L-Tyrosine did not affect chorismate mutase activity, but mutase activity was inhibited both by L-phenylalanine and by prephenate. Interpretations are given about the physiological significance of the overall pattern of allosteric control of the P-protein, and the relationship between this control and the effector-induced molecular-weight transitions. The properties of the P-protein in Acinetobacter are considered within the context of the ubiquity of the P-protein within the phylogenetic cluster to which this genus belongs.     

Cloning, sequencing, and expression of the P-protein gene (pheA) of Pseudomonas stutzeri in Escherichia coli: implications for evolutionary relationships in phenylalanine biosynthesis

The pheA gene encoding the bifunctional P-protein (chorismate mutase:prephenate dehydratase) was cloned from Pseudomonas stutzeri and sequenced. This is the first gene of phenylalanine biosynthesis to be cloned and sequenced from Pseudomonas. The pheA gene was expressed in Escherichia coli, allowing complementation of an E. coli pheA auxotroph. The enzymic and physical properties of the P-protein from a recombinant E. coli auxotroph expressing the pheA gene were identical to those of the native enzyme from P. stutzeri. The nucleotide sequence of the P. stutzeri pheA gene was 1095 base pairs in length, predicting a 365-residue protein product with an Mr of 40,844. Codon usage in the P. stutzeri pheA gene was similar to that of Pseudomonas aeruginosa but unusual in that cytosine and guanine were used at nearly equal frequencies in the third codon position. The deduced P-protein product showed sequence homology with peptide sequences of the E. coli P-protein, the N-terminal portion of the E. coli T-protein (chorismate mutase:prephenate dehydrogenase), and the monofunctional prephenate dehydratases of Bacillus subtilis and Corynebacterium glutamicum. A narrow range of values (26-35%) for amino acid matches revealed by pairwise alignments of monofunctional and bifunctional proteins possessing activity for prephenate dehydratase suggests that extensive divergence has occurred between even the nearest phylogenetic lineages.     

Two components of chorismate mutase in Brevibacterium flavum.

Chorismate mutase of Brevibacterium flavum, a common enzyme in phenylalanine and tyrosine biosynthesis, was separted into two different component, A and B, with molecular weights of 250,000 and 25,000, respectively, by ammonium sulfate fractionation or gel-filtration. Both components were essential for the enzymatic activity. In the presence of the reaction substrate, chorismate, the two components associated reversibly to give an active enzyme complex with a molecular weight of 320,000. Binding sites of the feedback inhibitors, phenylalanine and tyrosine, on the enzyme were localized on component A as determined by hybridization experiments with the wild-type and mutant components. Tyrosine repressed the synthesis of component B much more strongly than that of component A, while phenylalanine did not show any significant repressive effect on either component. The wild-type strain No. 2247 had four times more component A than component B. Elution patterns in gel, DEAE-cellulose or hydroxyapatite column chromatography as well as the disc-gel electrophoretic pattern of chorismate mutase component A and 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthetase activities completely overlapped, suggesting the presence of a bifunctional protein having the two activities. In accord with this suggestion, chorismate mutase as well as DAHP synthetase was insensitive to feedback inhibition by phenylalanine and tyrosine in all the 3-fluorophenylalanine-resistant mutants tested that excreted both phenylalanine and tyrosine. All the phenylalanine and tyrosine double auxotrophs defective in chorismate mutase lacked component B but not A.