The Yersinia Ysc-Yop 'type III' weaponry

'Type III secretion'--the mechanism by which some pathogenic bacteria inject proteins straight into the cytosol of eukaryotic cells to 'anaesthetize' or 'enslave' them--was discovered in 1994. Important progress has been made in this area during the past few years: the bacterial organelles responsible for this secretion--called 'injectisomes'--have been visualized, the structures of some of the bacterial protein 'effectors' have been determined, and considerable progress has been made in understanding the intracellular action of the effectors. Type III secretion is key to the pathogenesis of bacteria from the Yersinia genus.     

Alteration of polysaccharide size distribution of a vertebrate hyaluronan synthase by mutation

Hyaluronan (HA) is a nonsulfated glycosaminoglycan that has long been known to play structural roles in vertebrates. Recently, it has become increasingly obvious that this linear polysaccharide has many more uses than simply scaffolding or space filler. HA has been found to be involved in development, cell signaling, cell motility, and metastasis. These roles are often dictated by the length of the HA polymer, which can vary from a few to about 10,000 sugar residues in length. Three distinct isoforms of HA synthase exist in mammals. It has been shown previously by others that each isoform produces HA that differs in size distribution, but the regulatory mechanism is not yet known. Mutations have been described that alter the size distribution of the HA produced by the streptococcal HA synthases. We show that by mutating one particular amino acid residue of a vertebrate HA synthase, depending on the introduced side chain, the size of HA produced can be either reduced or increased. We postulate that several cysteine residues and a serine residue may be involved in binding directly or indirectly to the nascent HA chain. These data support the theory that the relative strength of the interaction between the catalyst and the polymer may be a major factor in HA size control.     

ATP合酶的结构与催化机理

ATP合酶 (F1Fo 复合物) 是生物体内进行氧化磷酸化和光合磷酸化的关键酶.随着核磁共振、X射线晶体衍射、遗传学、化学交联等技术在ATP合酶研究中的广泛应用,ATP合酶的整体结构及其各组成亚基结构的研究都有很大的进展.其中细菌ATP合酶结构的研究更为深入.目前对质子通过Fo的转运方式提出两种模型:单通道和双半通道模型.对扭力矩的形成以及旋转催化也有了进一步的认识.Boyer提出的结合改变机理推动了ATP合酶催化机制的研究,现在主要有两点催化机制和三点催化机制.ATP合酶的催化反应受酶的构象变化和外在条件的调节.     

Investigation of hyaluronan function in the mouse through targeted mutagenesis

It has become increasingly apparent that the high molecular mass glycosaminoglycan, hyaluronan (HA), is required for many morphogenetic processes during vertebrate development. This renewed understanding of the various developmental roles for HA, has come about largely through the advent of gene targeting approaches in the mouse. To date, mutations have been engineered in the enzymes responsible for biosynthesis and degradation and for those proteins that bind to HA within the extracellular matrix and at the cell surface. Collectively, the phenotypes resulting from these mutations demonstrate that HA is critical for normal mammalian embryogenesis and for various processes in postnatal and adult life (Table 1). In this article we will review our progress in understanding the biological functions for HA through targeted mutagenesis of the HA synthase 2 (Has2) and 3 (Has3) genes. Data that has been obtained from a conventional targeted disruption of the Has2 gene, is presented in an accompanying review by Camenisch and McDonald. More specifically, in this review we will provide an overview of the conditional gene targeting strategy being used to create tissue-specific deficiencies in Has2 function, along with our progress in understanding the role for Has3-dependent HA biosynthesis. Published in 2003.     

A novel ferredoxin-dependent glutamate synthase from the hydrogen-oxidizing chemoautotrophic bacterium Hydrogenobacter thermophilus TK-6

Glutamate synthases are classified according to their specificities for electron donors. Ferredoxin-dependent glutamate synthases had been found only in plants and cyanobacteria, whereas many bacteria have NADPH-dependent glutamate synthases. In this study, Hydrogenobacter thermophilus, a hydrogen-oxidizing chemoautotrophic bacterium, was shown to possess a ferredoxin-dependent glutamate synthase like those of phototrophs. This is the first observation, to our knowledge, of a ferredoxin-dependent glutamate synthase in a nonphotosynthetic organism. The purified enzyme from H. thermophilus was shown to be a monomer of a 168-kDa polypeptide homologous to ferredoxin-dependent glutamate synthases from phototrophs. In contrast to known ferredoxin-dependent glutamate synthases, the H. thermophilus glutamate synthase exhibited glutaminase activity. Furthermore, this glutamate synthase did not react with a plant-type ferredoxin (Fd3 from this bacterium) containing a [2Fe-2S] cluster but did react with bacterial ferredoxins (Fd1 and Fd2 from this bacterium) containing [4Fe-4S] clusters. Interestingly, the H. thermophilus glutamate synthase was activated by some of the organic acids in the reductive tricarboxylic acid cycle, the central carbon metabolic pathway of this organism. This type of activation has not been reported for any other glutamate synthases, and this property may enable the control of nitrogen assimilation by carbon metabolism.     

Bacterial NO synthases

Unlike mammalian NO synthases, bacterial NO synthases do not contain a reductase domain. The only exception from this rule is the NO synthase from myxobacterium Sorangium cellulosum, but its reductase domain has unusual structure and location in the enzyme molecule. Recent achievements in bacterial genome sequencing have revealed the gene coding NO synthase (represented as an oxygenase domain) in some bacteria and have advanced the study of structure and functions of bacterial NO synthases. Important features of structure, sources of reducing equivalents, evolutionary connections, and functions of bacterial NO synthases (i.e. participation in nitration of the indole ring of Trp, in reparation of UV-radiation damage, role in adaptation of bacteria to oxidative stress, participation in the synthesis of cGMP, and resistance of bacteria against antibiotics) are described.     

Structure and evolution of linalool synthase

Plant terpene synthases constitute a group of evolutionarily related enzymes. Within this group, however, enzymes that employ two different catalytic mechanisms, and their associated unique domains, are known. We investigated the structure of the gene encoding linalool synthase (LIS), an enzyme that uses geranyl pyrophosphate as a substrate and catalyzes the formation of linalool, an acyclic monoterpene found in the floral scents of many plants. Although LIS employs one catalytic mechanism (exemplified by limonene synthase [LMS]), it has sequence motifs indicative of both LMS-type synthases and the terpene synthases employing a different mechanism (exemplified by copalyl diphosphate synthase [CPS]). Here, we report that LIS genes analyzed from several species encode proteins that have overall 40%-96% identity to each other and have 11 introns in identical positions. Only the region encoding roughly the last half of the LIS gene (exons 9-12) has a gene structure similar to that of the LMS-type genes. On the other hand, in the first part of the LIS gene (exons 1-8), LIS gene structure is essentially identical to that found in the first half of the gene encoding CPS. In addition, the level of similarity in the coding information of this region between the LIS and CPS genes is also significant, whereas the second half of the LIS protein is most similar to LMS-type synthases. Thus, LIS appears to be a composite gene which might have evolved from a recombination event between two different types of terpene synthases. The combined evolutionary mechanisms of duplication followed by divergence and/or "domain swapping" may explain the extraordinarily large diversity of proteins found in the plant terpene synthase family.     

A unique reaction in a common pathway: mechanism and function of chorismate synthase in the shikimate pathway

Chorismate synthase, the seventh enzyme in the shikimate pathway, catalyzes the transformation of 5-enolpyruvylshikimate 3-phosphate to chorismate which is the last common precursor in the biosynthesis of numerous aromatic compounds in bacteria, fungi and plants. The enzyme has an absolute requirement for reduced FMN as a cofactor, although the 1,4-anti elimination of phosphate and the C(6proR)-hydrogen does not involve a net redox change. The role of the reduced FMN in catalysis has long been elusive. However, recent detailed kinetic and bioorganic approaches have fundamentally advanced our understanding of the mechanism of action, suggesting an initial electron transfer from tightly bound reduced flavin to the substrate, a process which results in C—O bond cleavage. Studies on chorismate synthases from bacteria, fungi and plants revealed that in these organisms the reduced FMN cofactor is made available in different ways to chorismate synthase: chorismate synthases in fungi – in contrast to those in bacteria and plants – carry a second enzymatic activity which enables them to reduce FMN at the expense of NADPH. Yet, as shown by the analysis of the corresponding genes, all chorismate synthases are derived from a common ancestor. However, several issues revolving around the origin of reduced FMN, as well as the possible regulation of the enzyme activity by means of the availability of reduced FMN, remain poorly understood. This review summarizes recent developments in the biochemical and genetic arena and identifies future aims in this field. Received: 22 June 1998 / Accepted: 7 August 1998     

细菌纤维素合酶的基本特性及作用机理

细菌纤维素是一种天然的生物质高分子聚合物。相较于植物纤维素,其具有更高的纯度和优异的力学性能。有望作为一种绿色的新型高分子材料被广泛应用。细菌纤维素合酶作为合成细菌纤维素的关键酶,其主导细菌纤维素的合成过程。因此,对其合成机理的探索有助于实现细菌纤维素大量生产和广泛应用。本文从细菌纤维素合酶的基本特性出发,综述了菌种筛选、提升产量和合酶的细胞定位等内容;围绕纤维素合酶的作用机理阐述了体外合成方法的影响因素,以及利用该方法探究各亚基相关作用的现状。以此探究细菌纤维素合酶的合成机制,并提出了当前研究中存在的问题。同时,展望了该领域未来的研究方向,以期通过对合成机理的探讨为细菌纤维素的大规模应用提供理论基础。     

Molecular cloning, heterologous expression, and functional characterisation of a malate synthase gene from Streptomyces coelicolor A3(2)

With the rapid generation of genetic information from the Streptomyces coelicolor genome project, deciphering the relevant gene products is critical for understanding the genetics of this model streptomycete. A putative malate synthase gene (aceB) from S. coelicolor A3(2) was identified by homology-based analysis, cloned by polymerase chain reaction, and fully sequenced on both strands. The putative malate synthase from S. coelicolor has an amino acid identity of 77% with the malate synthase of S. clavuligerus, and possesses an open reading frame which codes for a protein of 540 amino acids. In order to establish the identity of this gene, the putative aceB clones were subcloned into the expression vector pET24a, and heterologously expressed in Escherichia coli BL21(DE3). Soluble cell-free extracts containing the recombinant putative malate synthase exhibited a specific activity of 1623 (nmol.mg-1.min-1), which is an increment of 92-fold compared to the non-recombinant control. Thus, the gene product was confirmed to be a malate synthase. Interestingly, the specific activity of S. coelicolor malate synthase was found to be almost 8-fold higher than the specific activity of S. clavuligerus malate synthase under similar expression conditions. Furthermore, the genomic organisation of the three Streptomyces aceB genes cloned thus far is different from that of other bacterial malate synthases, and warrants further investigation.     

Terpenoid secondary metabolism in Arabidopsis thaliana: cDNA cloning, characterization, and functional expression of a myrcene/(E)-beta-ocimene synthase

The Arabidopsis genome project has recently reported sequences with similarity to members of the terpene synthase (TPS) gene family of higher plants. Surprisingly, several Arabidopsis terpene synthase-like sequences (AtTPS) share the most identity with TPS genes that participate in secondary metabolism in terpenoid-accumulating plant species. Expression of a putative Arabidopsis terpene synthase gene, designated AtTPS03, was demonstrated by amplification of a 392-bp cDNA fragment using primers designed to conserved regions of plant terpene synthases. Using the AtTPS03 fragment as a hybridization probe, a second AtTPS cDNA, designated AtTPS10, was isolated from a jasmonate-induced cDNA library. The partial AtTPS10 cDNA clone contained an open reading frame of 1665 bp encoding a protein of 555 amino acids. Functional expression of AtTPS10 in Escherichia coli yielded an active monoterpene synthase enzyme, which converted geranyl diphosphate (C(10)) into the acyclic monoterpenes beta-myrcene and (E)-beta-ocimene and small amounts of cyclic monoterpenes. Based on sequence relatedness, AtTPS10 was classified as a member of the TPSb subfamily of angiosperm monoterpene synthases. Sequence comparison of AtTPS10 with previously cloned monoterpene synthases suggests independent events of functional specialization of terpene synthases during the evolution of terpenoid secondary metabolism in gymnosperms and angiosperms. Functional characterization of the AtTPS10 gene was prompted by the availability of Arabidopsis genome sequences. Although Arabidoposis has not been reported to form terpenoid secondary metabolites, the unexpected expression of TPS genes belonging to the TPSb subfamily in this species strongly suggests that terpenoid secondary metabolism is active in the model system Arabidopsis.     

The mechanism underlying the effects of the cell surface ATP synthase on the regulation of intracellular acidification during acidosis

The F1F0 ATP synthase has recently become the focus of anti‐cancer research. It was once thought that ATP synthases were located strictly on the inner mitochondrial membrane; however, in 1994, it was found that some ATP synthases localized to the cell surface. The cell surface ATP synthases are involved in angiogenesis, lipoprotein metabolism, innate immunity, hypertension, the regulation of food intake, and other processes. Inhibitors of this synthase have been reported to be cytotoxic and to induce intracellular acidification. However, the mechanisms by which these effects are mediated and the molecular pathways that are involved remain unclear. In this study, we aimed to determine whether the inhibition of cell proliferation and the induction of cell apoptosis that are induced by inhibitors of the cell surface ATP synthase are associated with intracellular acidification and to investigate the mechanism that underlines the effects of this inhibition, particularly in an acidic tumor environment. We demonstrated that intracellular acidification contributes to the cell proliferation inhibition that is mediated by cell surface ATP synthase inhibitors, but not to the induction of apoptosis. Intracellular acidification is only one of the mechanisms of ecto‐ATP synthase‐targeted antitumor drugs. We propose that intracellular acidification in combination with the inhibition of cell surface ATP generation induce cell apoptosis after cell surface ATP synthase blocked by its inhibitors. A better understanding of the mechanisms activated by ecto‐ATP synthase‐targeted cancer therapies may facilitate the development of potent anti‐tumor therapies, which target this enzyme and do not exhibit clinical limitations. J. Cell. Biochem. 114: 1695–1703, 2013. © 2013 Wiley Periodicals, Inc.     

Evolution of the isoprene biosynthetic pathway in kudzu

Isoprene synthase converts dimethylallyl diphosphate, derived from the methylerythritol 4-phosphate (MEP) pathway, to isoprene. Isoprene is made by some plants in substantial amounts, which affects atmospheric chemistry, while other plants make no isoprene. As part of our long-term study of isoprene synthesis, the genetics of the isoprene biosynthetic pathway of the isoprene emitter, kudzu (Pueraria montana), was compared with similar genes in Arabidopsis (Arabidopsis thaliana), which does not make isoprene. The MEP pathway genes in kudzu were similar to the corresponding Arabidopsis genes. Isoprene synthase genes of kudzu and aspen (Populus tremuloides) were cloned to compare their divergence with the divergence seen in MEP pathway genes. Phylogenetic analysis of the terpene synthase gene family indicated that isoprene synthases are either within the monoterpene synthase clade or sister to it. In Arabidopsis, the gene most similar to isoprene synthase is a myrcene/ocimene (acyclic monoterpenes) synthase. Two phenylalanine residues found exclusively in isoprene synthases make the active site smaller than other terpene synthase enzymes, possibly conferring specificity for the five-carbon substrate rather than precursors of the larger isoprenoids. Expression of the kudzu isoprene synthase gene in Arabidopsis caused Arabidopsis to emit isoprene, indicating that whether or not a plant emits isoprene depends on whether or not it has a terpene synthase capable of using dimethylallyl diphosphate.     

细菌萜类合成酶的生物信息学分析

【目的】目前对于萜类合成酶(Terpenoid synthase,TPS)的研究主要集中在植物和真菌中,而对细菌TPS的系统研究尚少。建立在大量已经被测序的细菌基因组基础上,利用生物信息学方法,对细菌TPS在全基因组范围内进行识别、分类和功能分析。【方法】利用TPS的隐马尔科夫模型(Pfam编号为PF03936)搜索自建的细菌蛋白质组数据库,预测出细菌TPS。对这些候选TPS的蛋白序列用MAFFT 7.130b进行多序列比对,并利用MEGA 6.0对多序列比对结果进行进化分析。利用MEME和PredictProtein分别进行细菌TPS的基序(Motifs)和点突变分析。【结果】建立在生物信息学分析的基础上,1 423条细菌TPS被识别,它们分布在8个门中,即放线菌门(Actinobacteria)、变形菌门(Proteobacteria)、蓝藻门(Cyanobacteria)、拟杆菌门(Bacteroidetes)、厚壁菌门(Firmicutes)、绿弯菌门(Chloroflexi)、酸杆菌门(Acidobacteria)和衣原体门(Chlamydiae)。进化分析表明细菌TPS可分为4大类,Motifs分析表明除了各类之间保守的基序(Motifs)外,还有特异的Motifs,这暗示着细菌TPS在不同类别之间的功能分化。点突变分析表明,细菌TPS不同位点的氨基酸突变对TPS功能的影响不同。【结论】细菌TPS主要分布于8个门中,其中在2个门中细菌TPS尚未见报道,即厚壁菌门(Firmicutes)与酸杆菌门(Acidobacteria)。基于进化分析,可以把细菌TPS分为4类,各类之间的差异可能是由类特异的Motifs决定的,另外细菌TPS不同氨基酸位点的突变分析为今后验证TPS的功能提供了很好的理论基础。     

Detection of two loci involved in (1-->3)-beta-glucan (curdlan) biosynthesis by Agrobacterium sp. ATCC31749, and comparative sequence analysis of the putative curdlan synthase gene

Genes essential for the production of a linear, bacterial (1-->3)-beta- glucan, curdlan, have been cloned for the first time from Agrobacterium sp. ATCC31749. The genes occurred in two, nonoverlapping, genomic fragments that complemented different sets of curdlan( crd )-deficient transposon-insertion mutations. These were detected as colonies that failed to stain with aniline blue, a (1-->3)-beta-glucan specific dye. One fragment carried a biosynthetic gene cluster (locus I) containing the putative curdlan synthase gene, crdS, and at least two other crd genes. The second fragment may contain only a single crd gene (locus II). Determination of the DNA sequence adjacent to several locus I mutations revealed homology to known sequences only in the cases of crdS mutations. Complete sequencing of the 1623 bp crdS gene revealed highest similarities between the predicted CrdS protein (540 amino acids) and glycosyl transferases with repetitive action patterns. These include bacterial cellulose synthases (and their homologs), which form (1-->4)-beta-glucans. No similarity was detected with putative (1-->3)- beta-glucan synthases from yeasts and filamentous fungi. Whatever the determinants of the linkage specificity of these beta-glucan synthases might be, these results raise the possibility that (1-->3)-beta-glucans and (1-->4)-beta-glucans are formed by related catalytic polypeptides.      

Evaluation of critical structural elements of UDP-sugar substrates and certain cysteine residues of a vertebrate hyaluronan synthase

The hyaluronan (HA) synthases catalyze the addition of two different monosaccharides from UDP-sugar substrates to the linear heteropolysaccharide chain. To accomplish this task, the HA synthases must be able to bind and to transfer from both UDP-sugar substrates. Until now, it has been impossible to distinguish between these two abilities. We have created a mutant of xlHAS1, a HA synthase from Xenopus laevis, that allows for the examination of the enzyme's ability to bind substrate only. The ability of different compounds to protect the xlHAS1(C337S) mutant enzyme from loss of activity due to treatment with N-ethylmaleimide, a cysteine-modifying reagent, yields information on the relative affinity of a variety of nucleotides and nucleotide-sugars. We have observed that the substrate binding selectivity is more relaxed than the specificity of catalytic transfer. The only attribute that appears to be absolutely required for binding is a nucleotide containing two phosphates complexed with magnesium ion. The role of certain cysteine residues in catalysis was also evaluated. Cys307 of xlHAS1 may play a role in catalysis or in maintaining structure. Mutation of Cys337 raises the UDP-GlcUA Michaelis constant (K(m)), suggesting that this residue participates in UDP-GlcUA substrate binding or in catalytic complex formation.