Activities of Arabidopsis sinapoylglucose: malate sinapoyltransferase shed light on functional diversification of serine carboxypeptidase-like acyltransferases

Analysis of the catalytic properties of the serine carboxypeptidase-like (SCPL) 1-O-sinapoyl-beta-glucose:l-malate sinapoyltransferase (SMT) from Arabidopsis showed that the enzyme exhibits besides its primary sinapoylation of l-malate, minor hydrolytic and disproportionation activities, producing free sinapic acid and 1,2-di-O-sinapoyl-beta-glucose, respectively. The ability of the enzyme to liberate sinapic acid from the donor molecule 1-O-sinapoyl-beta-glucose indicates the existence of a short-lived acylenzyme intermediate in the proposed random sequential bi-bi mechanism of catalysis. SMT-catalyzed formation of disinapoylglucose has been corroborated by docking studies with an established homology structure model that illustrates the possible binding of two 1-O-sinapoyl-beta-glucose molecules in the active site and the intermolecular reaction of the two glucose esters. The SMT gene is embedded in a tandem cluster of five SCPL sinapoyltransferase genes, which encode enzymes with high amino acid sequence identities and partially overlapping substrate specificities. We assume that in recent duplications of genes encoding SCPL proteins, neofunctionalization of the duplicates to accept 1-O-sinapoyl-beta-glucose as acyl donor was gained first, followed by subfunctionalization leading to different acyl acceptor specificities.     

Key enzymes for biosynthesis of neutral lipid storage compounds in prokaryotes: properties, function and occurrence of wax ester synthases/acyl-CoA: diacylglycerol acyltransferases

Triacylglycerols (TAGs) and wax esters (WEs) are beside polyhydroxyalkanoates (PHAs) important storage lipids in some groups of prokaryotes. Accumulation of these lipids occurs in cells when they are cultivated under conditions of unbalanced growth in the presence of high concentrations of a suitable carbon source, which can be used for fatty acid and storage lipid biosyntheses. The key enzymes, which mediate both WE and TAG formations from long-chain acyl-coenzyme A (CoA) as acyl donor and long-chain fatty alcohols or diacylglycerols as respective acyl acceptors in bacteria, are WE synthases/acyl-CoA:diacylglycerol acyltransferases (WS/DGATs). The WS/DGATs identified so far represent rather unspecific enzymes with broad spectra of possible substrates; this makes them interesting for many biotechnological applications. This review traces the molecular structure and biochemical properties including the probable regions responsible for acyltransferase properties, enzymatic activity and substrate specifities. The phylogenetic relationships based on amino acid sequence similarities of this unique class of enzymes were revealed. Furthermore, recent advances in understanding the physiological functions of WS/DGATs in their natural hosts including pathogenic Mycobacterium tuberculosis were discussed.     

Formation of a complex pattern of sinapate esters in Brassica napus seeds, catalyzed by enzymes of a serine carboxypeptidase-like acyltransferase family?

Members of the Brassicaceae accumulate complex patterns of sinapate esters, as shown in this communication with seeds of oilseed rape (Brassica napus). Fifteen seed constituents were isolated and identified by a combination of high-field NMR spectroscopy and high resolution electrospray ionisation mass spectrometry. These include glucose, gentiobiose and kaempferol glycoside esters as well as sinapine (sinapoylcholine), sinapoylmalate and an unusual cyclic spermidine amide. One of the glucose esters (1,6-di-O-sinapoylglucose), two gentiobiose esters (1-O-caffeoylgentiobiose and 1,2,6'-tri-O-sinapoylgentiobiose) and two kaempferol conjugates [4'-(6-O-sinapoylglucoside)-3,7-di-O-glucoside and 3-O-sophoroside-7-O-(2-O-sinapoylglucoside)] seem to be new plant products. Serine carboxypeptidase-like (SCPL) acyltransferases catalyze the formation of sinapine and sinapoylmalate accepting 1-O-beta-acetal esters (1-O-beta-glucose esters) as acyl donors. To address the question whether the formation of other components of the complex pattern of the sinapate esters in B. napus seeds is catalyzed via 1-O-sinapoyl-beta-glucose, we performed a seed-specific dsRNAi-based suppression of the sinapate glucosyltransferase gene (BnSGT1) expression. In seeds of BnSGT1-suppressing plants the amount of sinapoylglucose decreased below the HPLC detection limit resulting in turn in the disappearance or marked decrease of all the other sinapate esters, indicating that formation of the complex pattern of these esters in B. napus seeds is dependent on sinapoylglucose. This gives rise to the assumption that enzymes of an SCPL acyltransferase family catalyze the appropriate transfer reactions to synthesize the accumulating esters.     

Structure activity studies with xenobiotic substrates using carboxylesterases isolated from Arabidopsis thaliana

Carboxylesterases (CXEs) catalyse the hydrolysis of xenobiotics and natural products radically altering their biological activities. Whereas the substrate selectivity of animal CXEs, such as porcine liver esterase (PLE) have been well studied, the respective enzymes in plants have yet to be defined and their activities determined. Using Arabidopsis thaliana (At) as a source, five representative members of the alpha/beta hydrolase AtCXE family of proteins have been cloned, expressed and the purified recombinant proteins assayed for esterase activity with xenobiotic substrates. Two members, AtCXE5 and AtCXE18 were found to be active carboxylesterases, though AtCXE5 proved to be highly unstable as a soluble protein. AtCXE18 and the previously characterised S-formylglutathione hydrolase from Arabidopsis (AtSFGH) were assayed against a series of esters based on methylumbelliferone in which the acyl moiety was varied with respect to size and conformation. The same series was used to assay crude esterase preparation from Arabidopsis plants and the results compared with those obtained with the commonly used PLE. With straight chain esters, AtCXE18 behaved like PLE, but the Arabidopsis hydrolases proved less tolerant of branched chain acyl components than the mammalian enzyme. While none of the enzyme preparations accurately reflected all the activities determined with crude Arabidopsis protein extracts, the plant enzymes proved more useful than PLE in predicting the hydrolysis of the more sterically constrained esters.     

Sub-families of alpha/beta barrel enzymes: a new adenine deaminase family

No gene coding for an adenine deaminase has been described in eukaryotes. However, physiological and genetical evidence indicates that adenine deaminases are present in the ascomycetes. We have cloned and characterised the genes coding for the adenine deaminases of Aspergillus nidulans, Saccharomyces cerevisiae and Schizosaccharomyces pombe. The A.nidulans gene was expressed in Escherichia coli and the purified enzyme shows adenine but not adenosine deaminase activity. The open reading frames coded by the three genes are very similar and obviously related to the bacterial and eukaryotic adenosine deaminases rather than to the bacterial adenine deaminases. The latter are related to allantoinases, ureases and dihydroorotases. The fungal adenine deaminases and the homologous adenosine deaminases differ in a number of residues, some of these being clearly involved in substrate specificity. Other prokaryotic enzymes in the database, while clearly related to the above, do not fit into either sub-class, and may even have a different specificity. These results imply that adenine deaminases have appeared twice in the course of evolution, from different ancestral enzymes constructed both around the alpha/beta barrel scaffold.     

Characterization of a Serine Hydrolase Targeted by Acyl-protein Thioesterase Inhibitors in Toxoplasma gondii

In eukaryotic organisms, cysteine palmitoylation is an important reversible modification that impacts protein targeting, folding, stability, and interactions with partners. Evidence suggests that protein palmitoylation contributes to key biological processes in Apicomplexa with the recent palmitome of the malaria parasite Plasmodium falciparum reporting over 400 substrates that are modified with palmitate by a broad range of protein S-acyl transferases. Dynamic palmitoylation cycles require the action of an acyl-protein thioesterase (APT) that cleaves palmitate from substrates and conveys reversibility to this posttranslational modification. In this work, we identified candidates for APT activity in Toxoplasma gondii. Treatment of parasites with low micromolar concentrations of β-lactone- or triazole urea-based inhibitors that target human APT1 showed varied detrimental effects at multiple steps of the parasite lytic cycle. The use of an activity-based probe in combination with these inhibitors revealed the existence of several serine hydrolases that are targeted by APT1 inhibitors. The active serine hydrolase, TgASH1, identified as the homologue closest to human APT1 and APT2, was characterized further. Biochemical analysis of TgASH1 indicated that this enzyme cleaves substrates with a specificity similar to APTs, and homology modeling points toward an APT-like enzyme. TgASH1 is dispensable for parasite survival, which indicates that the severe effects observed with the β-lactone inhibitors are caused by the inhibition of non-TgASH1 targets. Other ASH candidates for APT activity were functionally characterized, and one of them was found to be resistant to gene disruption due to the potential essential nature of the protein.     

Bacterial Serine/Threonine Protein Kinases in Host-Pathogen Interactions

In bacterial pathogenesis, monitoring and adapting to the dynamically changing environment in the host and an ability to disrupt host immune responses are critical. The virulence determinants of pathogenic bacteria include the sensor/signaling proteins of the serine/threonine protein kinase (STPK) family that have a dual role of sensing the environment and subverting specific host defense processes. STPKs can sense a wide range of signals and coordinate multiple cellular processes to mount an appropriate response. Here, we review some of the well studied bacterial STPKs that are essential virulence factors and that modify global host responses during infection.     

Serine Protease-mediated Host Invasion by the Parasitic Nematode Steinernema carpocapsae

Steinernema carpocapsae is an insect parasitic nematode used in biological control, which infects insects penetrating by mouth and anus and invading the hemocoelium through the midgut wall. Invasion has been described as a key factor in nematode virulence and suggested to be mediated by proteases. A serine protease cDNA from the parasitic stage was sequenced (sc-sp-1); the recombinant protein was produced in an Escherichia coli system, and a native protein was purified from the secreted products. Both proteins were confirmed by mass spectrometry to be encoded by the sc-sp-1 gene. Sc-SP-1 has a pI of 8.7, a molecular mass of 27.3 kDa, a catalytic efficiency of 22.2 × 10⁴ s⁻¹ m⁻¹ against N-succinyl-Ala-Ala-Pro-Phe-pNA, and is inhibited by chymostatin (IC 0.07) and PMSF (IC 0.73). Sc-SP-1 belongs to the chymotrypsin family, based on sequence and biochemical analysis. Only the nematode parasitic stage expressed sc-sp-1. These nematodes in the midgut lumen, prepared to invade the insect hemocoelium, expressed higher levels than those already in the hemocoelium. Moreover, parasitic nematode sense insect peritrophic membrane and hemolymph more quickly than they do other tissues, which initiates sc-sp-1 expression. Ex vivo, Sc-SP-1 was able to bind to insect midgut epithelium and to cause cell detachment from basal lamina. In vitro, Sc-SP-1 formed holes in an artificial membrane model (Matrigel), whereas Sc-SP-1 treated with PMSF did not, very likely because it hydrolyzes matrix glycoproteins. These findings highlight the S. carpocapsae-invasive process that is a key step in the parasitism thus opening new perspectives for improving nematode virulence to use in biological control.     

The formation of peptides from the 2′(3′)-glycyl ester of a nucleotide

Summary We have synthesized 2(3)-O-(glycyl)-adenosine-5-(O-methylphos-phate), an analogue of the 3-terminus of aminoacylated tRNA. A 0.4M solution of this compound maintained at pH 8.2, yields 5.5% of diglycine and 11.5% of diketopiperazine, in addition to the hydrolysis products glycine and adenosine-5-(O-methylphosphate). Under the same conditions, glycine ethyl ester reacts much more slowly, but ultimately gives similar yields of diglycine and diketopiperazine.The aminolysis of 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) by free glycine is relatively inefficient, but serine reacts 20 times more rapidly and yields up to 50% of N-glycylserine. The prebiotic significance of these reactions is discussed.Abbreviations MepA adenosine-5-(O-methylphosphate) - MepA-gly 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) - MepA-bis-gly 2,3-O-(bis-glycyl)-adenosine-5-(O-methylphosphate) - DKP diketopiperazine - gly Et glycine ethyl ester - gly-ser N-glycylserine - O-gly-ser O-glycylserine - O-(gly)-gly-ser O-(glycyl)-glycylserine - Boc-gly N-tert-butyloxycarbonylglycine - MepA-Boc-gly 2(3)-O-(Boc-glycyl)-adenosine-5-(O-methylphosphate) - MepA-bis-Boc-gly 2,3-O-(bis-Boc-glycyl)-adenosine-5(O-methylphosphate) - (gly)₂ diglycine - (gly)₃ triglycine     

蛋白质的结构转换

许多不相关的蛋白质含有相同的短肽序列却形成不同的空间构象. 结构转换广泛存在于蛋白质折叠和功能过程中, 具有重要的生物学意义. 综述了Serpin和EF-Tu的失活、血细胞凝集素的激活、蛋白酶成熟、亚基装配和蛋白质淀粉样化等过程中肽链同源肽段的结构转换模式, 并讨论了它在理解蛋白质折叠机理和“构象病”病因中的应用.     

Serine recombinases as tools for genome engineering

The serine recombinases differ mechanistically from the tyrosine recombinases and include proteins such as ?C31 integrase which, unlike Cre and Flp, promote unidirectional reactions. The serine recombinase family is large and includes many other proteins besides ?C31 integrase with the potential to be widely used in genome engineering. Here we review the details of the mechanism of the reactions promoted by the serine recombinases and discuss how these not only limit the utility of this class of recombinase but also creates opportunities for the engineering of new enzymes. We discuss the unanswered questions posed by genome engineering experiments in a variety of systems in which the serine recombinases have been used and finally describe more recently discovered serine recombinases that have the potential to be used in genome engineering.     

Attenuation of T Cell Receptor Signaling by Serine Phosphorylation-mediated Lysine 30 Ubiquitination of SLP-76 Protein

SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa) is an adaptor protein that is essential for T cell development and T cell receptor (TCR) signaling activation. Previous studies have identified an important negative feedback regulation of SLP-76 by HPK1 (hematopoietic progenitor kinase 1; MAP4K1)-induced Ser-376 phosphorylation. Ser-376 phosphorylation of SLP-76 mediates 14-3-3 binding, resulting in the attenuation of SLP-76 activation and downstream signaling; however, the underlying mechanism of this action remains unknown. Here, we report that phosphorylated SLP-76 is ubiquitinated and targeted for proteasomal degradation during TCR signaling. SLP-76 ubiquitination is mediated by Ser-376 phosphorylation. Furthermore, Lys-30 is identified as a ubiquitination site of SLP-76. Loss of Lys-30 ubiquitination of SLP-76 results in enhanced anti-CD3 antibody-induced ERK and JNK activation. These results reveal a novel regulation mechanism of SLP-76 by ubiquitination and proteasomal degradation of activated SLP-76, which is mediated by Ser-376 phosphorylation, leading to down-regulation of TCR signaling.     

食线虫真菌致病相关丝氨酸蛋白酶的研究进展

食线虫真菌是一类土壤微生物,作为线虫的天敌,它们对于维持线虫在土壤生态环境中的种群动态平衡发挥着十分重要的作用。食线虫真菌通过形成特殊的捕食器官或产生毒素等方式来捕捉和杀死线虫。丝氨酸蛋白酶是食线虫真菌侵染线虫的重要毒力因子,近年来,研究人员对不同食线虫真菌来源的致病相关丝氨酸蛋白酶进行了大量的研究,尤其在丝氨酸蛋白酶的晶体结构和分子进化方面的研究取得了较大的进展。本文对食线虫真菌致病相关丝氨酸蛋白酶的生物化学性质和功能进行了系统的总结,对丝氨酸蛋白酶的晶体结构、催化机制及分子进化等最新的进展进行了评述。     

稻属中一个高度保守和组成型表达酪氨酸／丝氨酸－酸酸双特性蛋白激酶基因的克隆与分析

从水稻中克隆了一个在稻属植物中高度保守和组成型表达的丝氨酸／苏氨酸蛋白激酶基因（OsSTK）。该基因包含两个外显子和一个114bp的小内含子序列,预测编码一个419个氨基酸的蛋白质。该基因推导的氨基酸序列与其它已知序列的一致性均低于52％。利用从不同种和类型的野生稻克隆的部分该基因序列构建的系统树与野生稻的分类和进化关系相一致。OSPKN-端拥有一段富含丝氨酸、碱性氨基酸和带电荷氨基酸的特异性导肽序列,其中包含“GDGDGDGDG”短重复序列。由于该基因蛋白激酶结构域中的VIb,VIII和XI亚结构域中同时具有酪氨酸蛋白激酶和丝氨酸／苏氨酸蛋白激酶的特性,推测该基因可能同时具有催化酪氨酸和丝氨酸、苏氨酸磷酸化的双重功能。     

The formation of dipeptides from amino acids and the 2′(3′)-glycyl ester of an adenylate

Summary The yields of dipeptide obtained from the reaction of 0.2M 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) and 0.2M amino acid at pH 8.2 ranged from 0.1% to 35.5% for a group of 15 amino acids. The yields of glyser (35.3%), gly-cys (11.8%) and gly-thr (5.4%) were considerably greater than dipeptide yields obtained from any of the other 12 amino acids ( 1.7%). Aminolysis of 0.05M 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) by 0.4M serine ethyl ester yielded 53% glycylserine diketopiperazine, via N-(glycyl)-serine ethyl ester as a transient intermediate. The prebiotic significance of these reactions is discussed.Abbreviations MepA adenosine-5-(O-methylphosphate) - MepA-gly 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) - DKP diketopiperazine - serOEt serine ethyl ester - gly-serOEt N-(glycyl)-serine ethyl ester - Boc-gly N-tertbutyloxycarbonylglycine - cyclo-(gly-ser-) glycylserine diketo-piperazine - O-gly-ser O-glycylserine - O-(gly)-gly-ser O-(glycyl)-glycylserine - gly-ser N-glycylserine     

冬虫夏草菌3个细胞核蛋白编码基因的分子进化

【目的】分析3个细胞核蛋白编码基因(csp1、MAT1-1-1和MAT1-2-1)在不同冬虫夏草菌株间的分子进化。【方法】从125个冬虫夏草样品中分别扩增csp1、MAT1-1-1和MAT1-2-1基因序列,比较外显子和内含子间以及2个交配型基因间的序列变异程度,比较基于不同基因或基因区域所构建的系统发育树拓扑结构的差异,分析3个基因承受的选择压力和DNA重组情况。【结果】3个蛋白编码基因外显子区的长度在不同菌株间高度保守,具有4.5%?5.7%的变异位点;内含子区的长度在不同菌株间相同或不同,具有1.8%?22%的变异位点。对于2个交配型基因,MAT1-1-1的碱基变异率低于MAT1-2-1。基于外显子与内含子构建的系统发育树的拓扑结构,以及基于2个交配型基因外显子构建的系统发育树的拓扑结构都存在明显差异。3个蛋白编码基因都经历着净化选择作用。基因内部的不同DNA位点间有重组,但3个基因片段之间没有明显的重组发生。【结论】由于冬虫夏草菌不同基因以及基因的不同区域表现出进化上的差异,所以在开展冬虫夏草菌进化相关的研究时,应该联合使用多个不同的基因片段。     

Thrombin-mediated Proteoglycan Synthesis Utilizes Both Protein-tyrosine Kinase and Serine/Threonine Kinase Receptor Transactivation in Vascular Smooth Muscle Cells

G protein-coupled receptor signaling is mediated by three main mechanisms of action; these are the classical pathway, β-arrestin scaffold signaling, and the transactivation of protein-tyrosine kinase receptors such as those for EGF and PDGF. Recently, it has been demonstrated that G protein-coupled receptors can also mediate signals via transactivation of serine/threonine kinase receptors, most notably the transforming growth factor-β receptor family. Atherosclerosis is characterized by the development of lipid-laden plaques in blood vessel walls. Initiation of plaque development occurs via low density lipoprotein retention in the neointima of vessels due to binding with modified proteoglycans secreted by vascular smooth muscle cells. Here we show that transactivation of protein-tyrosine kinase receptors is mediated by matrix metalloproteinase triple membrane bypass signaling. In contrast, serine/threonine kinase receptor transactivation is mediated by a cytoskeletal rearrangement-Rho kinase-integrin system, and both protein-tyrosine kinase and serine/threonine kinase receptor transactivation concomitantly account for the total proteoglycan synthesis stimulated by thrombin in vascular smooth muscle. This work provides evidence of thrombin-mediated proteoglycan synthesis and paves the way for a potential therapeutic target for plaque development and atherosclerosis.     

Molecular modeling of diastereoisomeric aggregates of <Emphasis Type="SmallCaps">L</Emphasis>/<Emphasis Type="SmallCaps">D</Emphasis> ser/histamine amide with 5′-TpTpdC-3′

Novel nonpeptide serine/histamine amides (1: l-Ser-Hism,2: d-Ser-Hism) with potent DNA cleavage activity were designed. Conformational analysis and docking study were carried out in an attempt to understand the DNA cleavage mechanism of the designed enantiomeric nonpeptides. First, the most stable conformers of the designed amides were obtained from the conformational analysis by random search. Next, the three-dimensional structures of l-Ser-Hism.5'-TpTpdC-3' and d-Ser-Hism.5'-TpTpdC-3' complexes were built using molecular docking techniques. The docked diastereoisomeric aggregates show that both l-Ser-Hism and d-Ser-Hism bind to two neighboring phosphates in the 5'-TpTpdC-3' backbone through H-bonds. This binding mode suggests a possible phosphodiester bond hydrolysis mechanism. In addition, the binding energies of two constructed complexes were also calculated with the Tripos force field. It indicates that the binding ability between l-Ser-Hism and 5'-TpTpdC-3' is stronger than that of d-Ser-Hism, suggesting a stronger DNA cleavage activity of l-Ser-Hism than that of d-Ser-Hism. The results agree with our experimental DNA cleavage assays. Supplementary material is available for this article if you access the article at http://dx.doi.org/10.1007/s00894-002-0114-9. A link in the frame on the left on that page takes you directly to the supplementary material. Figure Docking structures of1 and2 binding with oligonucleotide: l-Ser-Hism.5'-TpTpdC-3'(left), d-Ser-Hism.5'-TpTpdC-3'(right). Hydrogen bonds are shown in dotted lines. Only one strand of the oligonucleotide is shown for clarity     

The crystal structure of a (-) gamma-lactamase from an Aureobacterium species reveals a tetrahedral intermediate in the active site

The structure of the recombinant (-) gamma-lactamase from an Aureobacterium species has been solved at 1.73A resolution in the cubic space group F23 with unit cell parameters a=b=c=240.6A. The trimeric enzyme has an alpha/beta hydrolase fold and closely resembles the cofactor free haloperoxidases. The structure has been solved in complex with a covalently bound ligand originating from the host cell and also in the unligated form. The associated density in the former structure has been interpreted as the two-ring ligand (3aR,7aS)-3a,4,7,7a-tetrahydro-benzo [1,3] dioxol-2-one which forms a tetrahedral complex with OG of the catalytic Ser98. Soaks of these crystals with the industrial substrate gamma-lactam or its structural analogue, norcamphor, result in the displacement of the ligand from the enzyme active site, thereby allowing determination of the unligated structure. The presence of the ligand in the active site protects the enzyme from serine hydrolase inhibitors. Cyclic ethylene carbonate, the first ring of the ligand, was shown to be a substrate of the enzyme.