光合菌Rhodobacter sphaeroides 601hupT基因的克隆与功能分析

从紫色非硫光合细菌Rhodobacter sphaeroides 601的吸氢酶(hup)基因簇中,克隆了hupT基因,并对该基因进行了测序,分析了由其推测的氨基酸序列的同源性.hupT基因全长1 332 bp,编码一分子量约为48 23 kD的蛋白.将hupT基因引入大肠杆菌进行了体外表达.纯化基因产物HupT,并进行HupT的自身磷酸化分析.结果表明,HupT属于双组份调节系统中的组氧酸蛋白激酶.将hupT基因导入光合菌Rhodobacter capsulatus吸氢酶负调节基因突变株BSE8后.野生型吸氢酶的表型得以恢复,说明所克隆的R.sphaeroides 601中的hupT基因在吸氢酶的表达中起负调节作用.     

Bacillus subtilis CheN, a homolog of CheA, the central regulator of chemotaxis in Escherichia coli.

The Bacillus subtilis cheN gene was isolated, sequenced, and expressed. It encodes a large negatively charged protein with a molecular weight of approximately 74,000. The predicted protein sequence has 33 to 34% identity with the Escherichia coli and Salmonella typhimurium CheA and Myxococcus xanthus FrzE sequences. These proteins are found to autophosphorylate and are members of the same histidine kinase signal modulating family. CheN has several conserved regions (including the histidine that is phosphorylated in CheA) that coincide with other autophosphorylated signal transducers. A null mutant is defective in attractant-induced methanol formation and shows no behavioral response to chemoeffectors. These results imply that in B. subtilis the mechanism of chemotaxis involves phosphoryl transfer similar to that in E. coli. However, the CheN null mutant mostly tumbles, whereas CheA mutants swim smoothly, and only in B. subtilis does excitation lead to methyl transfer and methanol formation. Thus, the overall mechanism of chemotaxis is different in the two organisms.     

Autophosphorylation activity of the Arabidopsis ethylene receptor multigene family

Receptors for the gaseous phytohormone ethylene show sequence similarity to bacterial two-component histidine kinases. These receptors are encoded by a multigene family that can be divided into subfamilies 1 and 2. It has been previously shown that a subfamily 1 Arabidopsis thaliana ethylene receptor, ETR1, autophosphorylates in vitro on a conserved histidine residue (1). However, sequence comparisons between the five ethylene receptor family members suggest that subfamily 2 members do not have all the motifs necessary for histidine kinase activity. Further, a tobacco subfamily 2 receptor, NTHK1, autophosphorylates on serines and threonines in vitro (2). Here we show that all five Arabidopsis ethylene receptor proteins autophosphorylate in vitro. We analyzed the nature of the phosphorylated amino acids by acid/base stability and bi-dimensional thin layer electrophoresis and demonstrated that unlike ETR1 all other ethylene receptors autophosphorylate predominantly on serine residues. ERS1, the only other subfamily 1 receptor, is able to phosphorylate on both histidine and serine residues in the presence of Mn2+. However, histidine autophosphorylation is lost when ERS1 is assayed in the presence of both Mg2+ and Mn2+, suggesting that this activity may not occur in vivo. Furthermore, mutation of the histidine residue conserved in two-component systems does not abolish serine autophosphorylation, eliminating the possibility of a histidine to serine phosphotransfer. Our biochemical observations complement the recently published genetic data that histidine kinase activity is not necessary for ethylene receptor function in plants and suggest that ethylene signal transduction does not occur through a phosphorelay mechanism.     

Intra- and interprotein phosphorylation between two-hybrid histidine kinases controls Myxococcus xanthus developmental progression

Histidine-aspartate phosphorelay signaling systems are used to couple stimuli to cellular responses. A hallmark feature is the highly modular signal transmission modules that can form both simple "two-component" systems and sophisticated multicomponent systems that integrate stimuli over time and space to generate coordinated and fine-tuned responses. The deltaproteobacterium Myxococcus xanthus contains a large repertoire of signaling proteins, many of which regulate its multicellular developmental program. Here, we assign an orphan hybrid histidine protein kinase, EspC, to the Esp signaling system that negatively regulates progression through the M. xanthus developmental program. The Esp signal system consists of the hybrid histidine protein kinase, EspA, two serine/threonine protein kinases, and a putative transport protein. We demonstrate that EspC is an essential component of this system because ΔespA, ΔespC, and ΔespA ΔespC double mutants share an identical developmental phenotype. Neither substitution of the phosphoaccepting histidine residue nor deletion of the entire catalytic ATPase domain in EspC produces an in vivo mutant developmental phenotype. In contrast, substitution of the receiver phosphoaccepting residue yields the null phenotype. Although the EspC histidine kinase can efficiently autophosphorylate in vitro, it does not act as a phosphodonor to its own receiver domain. Our in vitro and in vivo analyses suggest the phosphodonor is instead the EspA histidine kinase. We propose EspA and EspC participate in a novel hybrid histidine protein kinase signaling mechanism involving both inter- and intraprotein phosphotransfer. The output of this signaling system appears to be the combined phosphorylated state of the EspA and EspC receiver modules. This system regulates the proteolytic turnover of MrpC, an important regulator of the developmental program.     

Enlarging the gas access channel to the active site renders the regulatory hydrogenase HupUV of Rhodobacter capsulatus O2 sensitive without affecting its transductory activity

In the photosynthetic bacterium Rhodobacter capsulatus, the synthesis of the energy-producing hydrogenase, HupSL, is regulated by the substrate H2, which is detected by a regulatory hydrogenase, HupUV. The HupUV protein exhibits typical features of [NiFe] hydrogenases but, interestingly, is resistant to inactivation by O2. Understanding the O2 resistance of HupUV will help in the design of hydrogenases with high potential for biotechnological applications. To test whether this property results from O2 inaccessibility to the active site, we introduced two mutations in order to enlarge the gas access channel in the HupUV protein. We showed that such mutations (Ile65-->Val and Phe113-->Leu in HupV) rendered HupUV sensitive to O2 inactivation. Also, in contrast with the wild-type protein, the mutated protein exhibited an increase in hydrogenase activity after reductive activation in the presence of reduced methyl viologen (up to 30% of the activity of the wild-type). The H2-sensing HupUV protein is the first component of the H2-transduction cascade, which, together with the two-component system HupT/HupR, regulates HupSL synthesis in response to H2 availability. In vitro, the purified mutant HupUV protein was able to interact with the histidine kinase HupT. In vivo, the mutant protein exhibited the same hydrogenase activity as the wild-type enzyme and was equally able to repress HupSL synthesis in the absence of H2.     

烟草乙烯受体类似物NTHK2全长基因的克隆及其激酶结构域生化特性

应用5＇-RACE方法克隆到烟草NTHK2的全长cDNA.其全长cDNA共有3 216bp,其中5'非编码区为509bp,3＇非编码区为427bp,编码区为2 280bp,编码产物为760个氨基酸.NTHK2氨基酸序列与植物中的许多杂合型的两组分乙烯受体基因有较高的同源性,具有推测的组氨酸激酶结构域和接受域;但是,在激酶结构域中没有保守的组氨酸,而是被一个天冬氨酸残基所替代.为了研究其生化特性,在酵母中以融合蛋白的形式表达了激酶结构域.体外激酶分析表明,当有Mg2+存在的情况下NTHK2能够自我磷酸化.进一步的研究应阐明NTHK2在植物体内是否能够作为乙烯受体,参与乙烯的信号传导过程.     

烟草乙烯受体类似物NTHK2全长基因的克隆及其激酶结构域生化特性

应用5′-ARCE方法克隆到烟草NTHK2的全长cDNA。其全长cDNA共有3216bp,其中5′非编码区为509bp,3′非编码区为427bp,编码区为2280bp,编码产物为760个氨基酸。NTHK2氨基酸序列与植物中的许多杂合型的两组分乙烯受体基因有较高的同源性,具有推测的组氨酸激酶结构域和接受域。但是,在激酶结构域中没有保守的组氨酸,而是被一个天冬氨酸残基所替代。为了研究其生化特性,在酵母中以融合蛋白的形式表达了激酶结构域,体外激酶分析表明,当有Mg^2 存在的情况下NTHK2能够自我磷酸化。进一步的研究应阐明NTHK2在植物体内是否能够作为乙烯受体。参与乙烯的信号传导过程。     

The quorum-sensing hybrid histidine kinase LuxN of Vibrio harveyi contains a periplasmically located N terminus

Hydropathy profile analyses of the amino acid sequence of the quorum-sensing hybrid histidine kinase LuxN of Vibrio harveyi predict a periplasmic location of the N terminus. To test this, two-hybrid proteins consisting of LuxN and an N-terminally fused maltose-binding protein with or without a leader sequence were analyzed with regard to the enzymatic activities of LuxN, protease accessibility, and complementation of an Escherichia coli malE mutant. The results strongly support a periplasmic location of the N terminus, implying that LuxN is anchored with nine transmembrane domains in the cytoplasmic membrane.     

Modulation of the catalytic activity of the Src family tyrosine kinase Hck by autophosphorylation at a novel site in the unique domain

Autophosphorylation is a key event in the activation of protein kinases. In this study, we demonstrate that autophosphorylation of the recombinant Src family kinase Hck leads to a 20-fold increase in its specific enzymatic activity. Hck was found to autophosphorylate readily to a stoichiometry of 1.3 mol of phosphate per mol of enzyme, indicating that the kinase autophosphorylated at more than one site. Solid phase sequencing and two-dimensional mapping of the phosphopeptide fragments derived from the autophosphorylated enzyme revealed that the kinase can undergo autophosphorylation at the following two sites: (i) Tyr-388, which is located to the consensus autophosphorylation site commonly found in the activation loop of many protein kinases, and (ii) Tyr-29, which is located in the unique domain of Hck. Hck purified from mouse bone marrow-derived macrophages could also autophosphorylate in vitro at both Tyr-388 and Tyr-29, indicating that naturally occurring Hck can also autophosphorylate at Tyr-29. Furthermore, Hck transiently expressed in human embryonic kidney 293T cells was found to be phosphorylated at Tyr-29 and Tyr-388, proving that Hck can also undergo autophosphorylation at both sites in vivo. The recombinant enzyme carrying the mutation of Tyr-388 to Phe was also able to autophosphorylate at Tyr-29, albeit at a significantly slower rate. A 2-fold increase in the specific enzymatic activity was seen with this mutant despite the stoichiometry of autophosphorylation only approaching 0.2 mol of phosphate per mol of enzyme. This indicates that autophosphorylation of Tyr-29 contributes significantly to the activation of Hck. Regulation of the catalytic activity by phosphorylation of Tyr-29 in the unique domain may represent a new mechanism of regulation of Src family tyrosine kinases.     

Are the phytochromes protein kinases?

Summary The biochemical mechanism of phytochrome action is unknown. We have examined the proposal, based on sequence similarities to the sensor histidine kinase components of bacterial two-component signaling systems, that the phytochromes may be functional homologs of these kinases. Four amino acids, three highly conserved between the phytochrome and bacterial kinase molecules and the other, the histidine residue putatively the target of autophosphorylation, were changed singly in the oat phytochrome A sequence by in vitro site-directed mutagenesis, and the resultant mutant photo-receptor molecules were assayed for activity by overexpression in transgenic Arabidopsis. Three of the four mutant molecules retained activity equivalent to that of the unmutagenized parent sequence, whereas the fourth mutant could not be evaluated because of low expression. The data show that the former three mutagenized residues are not essential for phytochrome A function in transgenic Arabidopsis, but, because of the negative nature of the results, the possibility cannot be precluded that the photoreceptor functions as a protein kinase independent of these residues.Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

Genetic and biochemical characterization of MbeA,the relaxase involved in plasmid ColE1 conjugative mobilization

MbeA is a 60 kDa protein encoded by plasmid ColE1. It plays a key role in conjugative mobilization. MbeA*, a slightly truncated version of MbeA, was purified for in vitro analysis. MbeA* catalysed DNA cleavage and strand-transfer reactions using oligonucleotides embracing the ColE1 nic site, which was mapped to 5'-(1469)CTGG/CTTA(1462)-3'. Thus MbeA is the relaxase for ColE1 conjugal mobilization, in spite of the fact that it lacks a three histidine motif considered the invariant signature of conjugative relaxases. Amino acid sequence comparisons suggest MbeA is nevertheless related to the common relaxase protein family. For instance, MbeA residue Y19 could correspond to the invariant tyrosine in Motif I, whereas H97, E104 and N106 may constitute the equivalent residues to the histidine triad in Motif III. This hypothesis was tested by site-directed mutagenesis. MbeA amino acid residues Y19, H97, E104 and N106 were changed to alanine. MbeA mutant N106A showed reduced oligonucleotide cleavage and strand-transfer activities, whereas mutation in the other three residues resulted in proteins without detectable activity, suggesting they are directly implicated in catalysis of DNA-cleavage and strand-transfer reactions. A double substitution of E104 and N106 by histidines, therefore reconstituting the canonical histidine triad, restored relaxase activities to 1% of wild type. Thus, MbeA is a variant of the common relaxase theme with a HEN signature motif, which has to be added to the canonical three histidine motif of previously reported relaxases.     

Phosphorylation activity of the response regulator of the two-component signal transduction system AtoS-AtoC in E. coli

Antizyme, long known to be a non-competitive inhibitor of ornithine decarboxylase, is encoded by the atoC gene in Escherichia coli. The present study reveals another role for AtoC, that of a response regulator of the AtoS-AtoC two component system regulating the expression of the atoDAEB operon upon acetoacetate induction. This operon encodes enzymes involved in short-chain fatty acid catabolism in E. coli. Evidence is presented to show that AtoS is a sensor kinase that together with AtoC constitutes a two-component signal transduction system. AtoS is a membrane protein which can autophosphorylate and then transfer that phosphoryl group to AtoC. This process can also be reproduced in vitro. AtoC contains in its amino acid sequence a conserved aspartic acid (D55), which is the putative phosphorylation site, as well as an unexpected "H box" consensus sequence (SHETRTPV), common to histidine kinases, with the histidine contained therein (H73) being a second potential target for phosphorylation. Substitution of either D55 or H73 in His10-AtoC diminished but did not abrogate AtoC phosphorylation suggesting that either both residues can be phosphorylated independently or that the phosphate group can be transferred between them. However, the D55 mutation in comparison to H73 had a more pronounced effect in vivo, on the activation of atoDAEB promoter after acetoacetate induction, although it was the presence of both mutations that rendered AtoC totally unresponsive to induction. These data provide evidence that the gene products of atoS and atoC constitute a two-component signal transduction system, with some unusual properties, involved in the regulation of the atoDAEB operon.     

The atypical two-component sensor kinase Lpl0330 from Legionella pneumophila controls the bifunctional diguanylate cyclase-phosphodiesterase Lpl0329 to modulate bis-(3'-5')-cyclic dimeric GMP synthesis

A significant part of bacterial two-component system response regulators contains effector domains predicted to be involved in metabolism of bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), a second messenger that plays a key role in many physiological processes. The intracellular level of c-di-GMP is controlled by diguanylate cyclase and phosphodiesterases activities associated with GGDEF and EAL domains, respectively. The Legionella pneumophila Lens genome displays 22 GGDEF/EAL domain-encoding genes. One of them, lpl0329, encodes a protein containing a two-component system receiver domain and both GGDEF and EAL domains. Here, we demonstrated that the GGDEF and EAL domains of Lpl0329 are both functional and lead to simultaneous synthesis and hydrolysis of c-di-GMP. Moreover, these two opposite activities are finely regulated by Lpl0329 phosphorylation due to the atypical histidine kinase Lpl0330. Indeed, Lpl0330 was found to autophosphorylate on a histidine residue in an atypical H box, which is conserved in various bacteria species and thus defines a new histidine kinase subfamily. Lpl0330 also catalyzes the phosphotransferase to Lpl0329, which results in a diguanylate cyclase activity decrease whereas phosphodiesterase activity remains efficient. Altogether, these data present (i) a new histidine kinase subfamily based on the conservation of an original H box that we named HGN H box, and (ii) the first example of a bifunctional enzyme that modulates synthesis and turnover of c-di-GMP in response to phosphorylation of its receiver domain.     

Histidine decarboxylase of Lactobacillus 30a. Comparative sequences of the beta chain from wild type and mutant enzymes

Manual and automated sequencing of peptides isolated from tryptic, chymotryptic, and Staphylococcus aureus V8 protease digests of the beta chain of histidine decarboxylase from Lactobacillus 30a have established the following sequence for the wild type protein: NH2-Ser-Gly-Leu-Asp-Ala-Lys-Leu-Asn-Lys-Leu-Gly-Val-Asp-Arg-Ile-Ala-Ile-Ser-Pro -Tyr-Lys-Gln-Trp-Thr-Arg-Gly-Tyr-Met-Glu-Pro-Gly-Asn-Ile-Gly-Asn-Gly-Tyr-Val-Thr-Gly-Leu-Lys-Val-Asp-Ala-Gly-Val-Arg-Asp-Lys-Ser-Asp-Asp-Asp-Val-Leu-Asp-Gly-I le-Val-Ser-Tyr-Asp-Arg-Ala-Glu-Thr-Lys-Asn-Ala-Tyr-Ile-Gly-Gln-Ile-Asn-Met-Thr- Thr-Ala-Ser-COOH The beta chain from mutant 3 of this organism shows two amino acid replacements: Ser-51 is replaced by Ala and Gly-58 by Asp. These amino acid replacements result in a significant increase in the predicted alpha-helical content and a significant decrease in the isoelectric point of the mutant beta chain and are consistent with changes in physical and catalytic properties of the mutant histidine decarboxylase. In addition, about 15% of the mutant chains contain Met-Ser at the NH2 terminus rather than Ser. Asn-35 is partially deamidated in both proteins. Structural comparisons show that the histidine decarboxylase from Lactobacillus 30a and a similar pyruvoyl enzyme from Micrococcus sp. n. have evolved from a common ancestral protein.     

Transforming mutant v-mos protein kinases that are deficient in in vitro autophosphorylation.

We investigated the importance of specific serine residues for autophosphorylation and transformation by serine-threonine protein kinase p37mos. When either serine 326 or 358 was replaced with alanine, the resulting mutant protein retained the ability to transform NIH 3T3 cells but failed to autophosphorylate in vitro. These studies represent the first functional uncoupling of these two activities for p37mos.