Characterization and Functional Analysis of Four Glutathione S-Transferases from the Migratory Locust,Locusta migratoria

Glutathione S-transferases (GSTs) play an important role in detoxification of xenobiotics in both prokaryotic and eukaryotic cells. In this study, four GSTs (LmGSTd1, LmGSTs5, LmGSTt1, and LmGSTu1) representing different classes were identified from the migratory locust, Locusta migratoria. These four proteins were heterologously expressed in Escherichia coli as soluble fusion proteins, purified by Ni²⁺-nitrilotriacetic acid agarose column and biochemically characterized. LmGSTd1, LmGSTs5, and LmGSTu1 showed high activities with 1-chloro-2, 4-dinitrobenzene (CDNB), detectable activity with p-nitro-benzyl chloride (p-NBC) and 1, 2-dichloro-4-nitrobenzene (DCNB), whereas LmGSTt1 showed high activity with p-NBC and detectable activity with CDNB. The optimal pH of the locust GSTs ranged between 7.0 to 9.0. Ethacrynic acid and reactive blue effectively inhibited all four GSTs. LmGSTs5 was most sensitive to heavy metals (Cu²⁺ and Cd²⁺). The maximum expression of the four GSTs was observed in Malpighian tubules and fat bodies as evaluated by western blot. The nymph mortalities after carbaryl treatment increased by 28 and 12% after LmGSTs5 and LmGSTu1 were silenced, respectively. The nymph mortalities after malathion and chlorpyrifos treatments increased by 26 and 18% after LmGSTs5 and LmGSTu1 were silenced, respectively. These results suggest that sigma GSTs in L. migratoria play a significant role in carbaryl detoxification, whereas some of other GSTs may also involve in the detoxification of carbaryl and chlorpyrifos.     

The Effects of Temperature and Body Mass on Jump Performance of the Locust Locusta migratoria

Locusts jump by rapidly releasing energy from cuticular springs built into the hind femur that deform when the femur muscle contracts. This study is the first to examine the effect of temperature on jump energy at each life stage of any orthopteran. Ballistics and high-speed cinematography were used to quantify the energy, distance, and take-off angle of the jump at 15, 25, and 35°C in the locust Locusta migratoria. Allometric analysis across the five juvenile stages at 35°C reveals that jump distance (D; m) scales with body mass (M; g) according to the power equation D = 0.35M^{0.17±0.08 (95% CI)}, jump take-off angle (A; degrees) scales as A = 52.5M^0.00±0.06, and jump energy (E; mJ per jump) scales as E = 1.91M^1.14±0.09. Temperature has no significant effect on the exponent of these relationships, and only a modest effect on the elevation, with an overall Q₁₀ of 1.08 for jump distance and 1.09 for jump energy. On average, adults jump 87% farther and with 74% more energy than predicted based on juvenile scaling data. The positive allometric scaling of jump distance and jump energy across the juvenile life stages is likely facilitated by the concomitant relative increase in the total length (L_f+t; mm) of the femur and tibia of the hind leg, L_f+t = 34.9M^0.37±0.02. The weak temperature-dependence of jump performance can be traced to the maximum tension of the hind femur muscle and the energy storage capacity of the femur''s cuticular springs. The disproportionately greater jump energy and jump distance of adults is associated with relatively longer (12%) legs and a relatively larger (11%) femur muscle cross-sectional area, which could allow more strain loading into the femur''s cuticular springs. Augmented jump performance in volant adult locusts achieves the take-off velocity required to initiate flight.     

华北2蝗区东亚飞蝗种群遗传结构的比较研究

利用水平淀粉凝胶电泳对采自天津北大港和河北黄骅两个相临蝗区的东亚飞蝗（Locusta migratoria manilensis)种群进行等位酶基因频率分析,比较了这两个种群的遗传结构,等位酶酶谱分析表明,19个基因座中4个基因座（Mdh-l,Pgm,Adk,G3pd)的等位基因频率变化很小,常见等位基因的频率均高于0．95,其他基因座有2－4个等位基因,但是两个种群的等位基因频率除两个基因座（Fbp,Got-2)外都很相似,多态位点的27个χ2检验表明,由于常见等位基因纯合子的高频率的和相应杂合子的缺乏,仅有北大港种群的2个基因座（Pgi,Got-1)符合Hardy-Weinberg平衡,在每个种群内的蝗虫存在明显的遗传变异,但在种群间遗传结构极为相似,多态位点的百分数P分别为73．7％和78．9％,每个基因座的平均等位基因数A为2．9和3．1,平均每个基因座的实际杂合度几乎相等（约为0．138）,F－统计量（FST＝0．053）也表明了两个种群间的遗传 一致性,遗传相似性系数（I）高达0．938,这些结果提示,这两个种群可能属于1 个大种群,在两个种群的一定位点上的遗传多态性和分化可能都与迁飞因素有关,因为东亚飞蝗的高度扩散能力有利于遗传结构的连续分布,高度的迁飞能力也导致个体暴露于各种不同的环境,而在种群水平上的遗传为异能增强种群在各种生态条件生存和繁殖能力,因此,迁飞有利于维持东亚飞蝗种群的遗传多态性的动态平衡。     

飞蝗热休克蛋白70cDNA片段的克隆和序列分析

采用R-PCR方法对海南、河北和辽宁3个飞蝗(Locusta migratoria L．)种群的热休克蛋白70(HSP70)基因cDNA片段进行克隆。在事先优化的条件下,通过简并性上游引物和下游引物扩增出了河北种群飞蝗HSP70基因的604bp cDNA片段(GenBank登录号为AY299637),推导的氨基酸序列包含201个氨基酸残基。分析表明,由飞蝗该片段推导的氨基酸序列与其他昆虫的同源性较高;3个种群该片段的核苷酸序列相似性更高达98．75％。由此推测,飞蝗种群间抗寒性的差异可能不是HSP70的序列变异引起的,而与HSP70的诱导表达有关。     

光照与飞蝗卵耐寒性的关系

用热电偶法测定长光照(L：D=14：10)和短光照(L：D=10：14)条件下饲养的飞蝗所产卵的过冷却点;并对长、短光照组分别设置5个温度(0、-5、-10、-15和-20℃)处理,每一温度又设置5个时间(6h,1、3、5和10d)处理,然后检查其28℃的孵化数,以此计算低温存活率和半致死温度。长、短光照组卵的过冷却点没有差异;两种光照条件下的低温存活率随着卵处理温度的降低和时间的延长而下降,在-5和-10℃时短光照组的低温存活率显著高于长光照组;卵的半致死温度随低温处理时间的延长而升高,短光照组的半致死温度明显低于长光照组。接受短光照的飞蝗母本能产出耐寒性较高的卵,暗示秋天所产的卵能更成功地越冬。     

飞蝗不同地理种群抗寒性研究

新疆和硕、哈密和天津北大港飞蝗种群的过冷却点随发育阶段而升高,在胚胎发育的各期都具有较低的过冷却点。飞蝗卵的过冷却点在这3个地理种群间无明显差异。雄性蝻和成虫双雌性的过冷却能力强,和硕飞蝗的成虫过冷点比北大港的飞蝗成虫要低。低温胁迫可诱导蝗卵以糖元为朱料合成抗冻保护剂,和硕飞蝗种群的卵主要合成甘油和山梨醇,北大港飞蝗种群的卵主要合成海藻糖,而哈密飞蝗种群的卵却可以合成甘油、山梨醇和海藻糖等抗冻保护剂。糖元磷酸化酶的总量随发育阶段而下降,但热休克和冷休克并不改变其总量,冷休克在胚胎发育的各阶段可以使其活性升高10%-40%,北大港飞蝗种群和哈密飞蝗种群的蝗卵经热休克2h处理后,糖元磷酸化酶的变化与在冷休克下的情况相似,而和硕飞蝗处群胚胎发育的Ⅱ期,热休克诱导糖元磷酸化酶活性的升高程度较低。     

The Presence of a Progesterone Binding Protein in Spermathecae of the Migratory Locust,Locusta migratoria migratorioides R. & F.

Summary

Radioreceptor assays revealed the presence of a progesterone binding protein in the cytosol of spermathecae of the migratory locust, Locusta migratoria migratorioides. The spermathecae were excised from adult females, 1 to 7 days after the final moult. The progesterone binding was characterized by a dissociation constant of 4.6 × 10^?8M and a capacity of 0.9 pmoles per milligram protein. Competitive inhibition experiments with other steroids demonstrated the high specificity of the binder for progesterone. Binding disappears in the presence of the proteinase Pronase P, suggesting that the binding factor is a protein.     

Genetic Variation and Population Structure of Oriental Migratory Locust, Locusta migratoria manilensis, in China by Allozyme, SSRP-PCR, and AFLP Markers

Allozyme analysis, microsatellite primer PCR (SSRP-PCR), and amplified fragment length polymorphism (AFLP) techniques were used to assess genetic diversity and population structure of the Chinese oriental migratory locust, Locusta migratoria manilensis. A total of 299 PCR markers (67 SSRPs and 232 AFLPs) were detected in eight populations, of which 98.7% were polymorphic markers. The proportion of polymorphic loci (95.5–98.8%) by SSRP+AFLP markers indicated no significant differences between populations, and all populations exhibited a similar level of variability; results of the allozyme analysis demonstrated that 19 loci gave rise to a lower level of polymorphism (55.6–66.7%). The genetic distances between the populations were relatively low. Shannon’s index and Nei’s gene diversity showed low differentiation among the populations. Allozyme analysis, however, reflected greater similarity and smaller differentiation between the populations than those shown by SSRP and AFLP markers. Neighbor-joining dendrograms derived from both the allozyme and SSRP+AFLP markers showed that the genetic distances among Chinese oriental migratory locust populations were not greatly influenced by geographic distance and breeding habitats.     

Functional Characterization of the Short Neuropeptide F Receptor in the Desert Locust,Schistocerca gregaria

Whereas short neuropeptide F (sNPF) has already been reported to stimulate feeding behaviour in a variety of insect species, the opposite effect was observed in the desert locust. In the present study, we cloned a G protein-coupled receptor (GPCR) cDNA from the desert locust, Schistocerca gregaria. Cell-based functional analysis of this receptor indicated that it is activated by both known isoforms of Schgr-sNPF in a concentration dependent manner, with EC₅₀ values in the nanomolar range. This Schgr-sNPF receptor constitutes the first functionally characterized peptide GPCR in locusts. The in vivo effects of the sNPF signalling pathway on the regulation of feeding in locusts were further studied by knocking down the newly identified Schgr-sNPF receptor by means of RNA interference, as well as by means of peptide injection studies. While injection of sNPF caused an inhibitory effect on food uptake in the desert locust, knocking down the corresponding peptide receptor resulted in an increase of total food uptake when compared to control animals. This is the first comprehensive study in which a clearly negative correlation is described between the sNPF signalling pathway and feeding, prompting a reconsideration of the diverse roles of sNPFs in the physiology of insects.     

东亚飞蝗病原褪色沙雷氏菌的分型分析

对东亚飞蝗的致病性菌褪色沙雷氏菌进行分型研究,为东亚飞蝗的生物学防治研究或可构建基因工程菌的候选菌株。利用血清型分型与脉冲场凝胶电泳分型方法对不同地区及个体的病死蝗虫中分离的10株细菌与模式菌株(ATCC 13880)进行了分析。结果显示,由不同地区及个体中分离得到的褪色沙雷氏菌同源性存在差异性,基因分型相似性在64%~100%之间,存在不同程度的亲缘关系。本试验丰富了褪色沙雷氏菌的生物学性状内容,也为对褪色沙雷氏菌的检验与进一步深入研究提供了具有参考价值的资料。     

Low Numbers of Sperm Retained in the Spermatheca May Explain the High Values of Sperm Precedence in the Migratory Locust, Locusta migratoria (Latr.)

Four hypotheses about the temporal variation of the number of spermatozoa in the spermatheca of once-mated females were tested in the migratory locust, Locusta migratoria. The best fit provided a regression model that assumed a sudden drop in sperm numbers being indicative of sperm ejection by females. Thereafter, one-fifth of an average ejaculate is stored. Low numbers of sperm stored do not lead to female sperm limitation, as none of the fitness parameters measured, fertilization success, hatching success, and offspring sex ratio, were correlated with the number of sperm present. Fertilization success decreased with successive egg pods. The offspring sex ratio was slightly male biased and tended to increase throughout the laying period. By applying our sperm ejection model to an independent data set of Parker and Smith (1975), we show that the low numbers of sperm retained by the female and subsequent sperm mixing rather than direct replacement may explain the high P₂ values found in this species.     

Ultrastructural and functional aspects of the spermatheca of the African Migratory Locust Locusta migratoria migratorioides (Reiche and Fairmaire) (Orthoptera: Acrididae)

The ultrastructure of the spermathecal epithelium of the African Migratory Locust Locusta migratoria migratorioides R. & F. (Orthoptera: Acrididae) was investigated with the aid of transmission and scanning electron microscopic methods. The unpaired spermatheca can be subdivided into a multiple coiled tube and a terminal bulb region with vestibule, small apical and extensive pre-apical diverticulum. The wall of the spermatheca consists of a chitin intima, a layer of epithelial cells with a distinct apical microvilli border and a layer of gland cells, whereby slender projections of the epithelial cells extend between the gland cells. Through extensive folding, the basal plasma membrane of the gland and epithelial cells form a huge labyrinth, which is bounded by a basal lamina. Extending into the above mentioned projections there are bundles of parallel-arrayed microtubules, which run perpendicular to the microvilli border of the epithelial cell. They end in the base region of the microvilli and in the basal labyrinth on hemidesmosomes and serve to provide a mechanically stressable anchorage for the epithelium. The gland cells show structures typical for the production of export proteins: ribosomes, rER, dictyosomes, as well as vesicles of different size and electron-density. Every gland cell contains an extracellular cavity, arising through invagination, which is coated with a microvilli border. Over an end-apparatus and a ductule joining onto it (also with chitin intima) the lumen of the extracellular cavity is connected with the spermathecal lumen. The release of secretions and other substances from the epithelium into the spermatheca lumen is as possible as the uptake of substances from the latter into the epithelium. Regional differences in the fine structure of the cuticular intima, epithelial and gland cells point to different functions of the epithelium in these regions.     

蝗虫型变的秘密

当你在路边的草丛里偶尔碰到一只探头探脑,听到动静就躲藏起来的蝗虫时,你会把它与恐怖的外太空生物“异形”联系起来吗？不但不会,也许你看到它那躲躲闪闪的样子、方头方脑的憨态、翠绿的色泽,反而产生一种爱怜的感觉,不由地会把脚步放轻,以免惊扰了它。如果真是这样,那你就错了。     

Functional Analysis of Free Methionine-R-sulfoxide Reductase from Saccharomyces cerevisiae

Methionine sulfoxide reductases (Msrs) are oxidoreductases that catalyze thiol-dependent reduction of oxidized methionines. MsrA and MsrB are the best known Msrs that repair methionine-S-sulfoxide (Met-S-SO) and methionine-R-sulfoxide (Met-R-SO) residues in proteins, respectively. In addition, an Escherichia coli enzyme specific for free Met-R-SO, designated fRMsr, was recently discovered. In this work, we carried out comparative genomic and experimental analyses to examine occurrence, evolution, and function of fRMsr. This protein is present in single copies and two mutually exclusive subtypes in about half of prokaryotes and unicellular eukaryotes but is missing in higher plants and animals. A Saccharomyces cerevisiae fRMsr homolog was found to reduce free Met-R-SO but not free Met-S-SO or dabsyl-Met-R-SO. fRMsr was responsible for growth of yeast cells on Met-R-SO, and the double fRMsr/MsrA mutant could not grow on a mixture of methionine sulfoxides. However, in the presence of methionine, even the triple fRMsr/MsrA/MsrB mutant was viable. In addition, fRMsr deletion strain showed an increased sensitivity to oxidative stress and a decreased life span, whereas overexpression of fRMsr conferred higher resistance to oxidants. Molecular modeling and cysteine residue targeting by thioredoxin pointed to Cys¹⁰¹ as catalytic and Cys¹²⁵ as resolving residues in yeast fRMsr. These residues as well as a third Cys, resolving Cys⁹¹, clustered in the structure, and each was required for the catalytic activity of the enzyme. The data show that fRMsr is the main enzyme responsible for the reduction of free Met-R-SO in S. cerevisiae.Among the 20 common amino acids in proteins, Met and Cys are the residues most susceptible to oxidation by reactive oxygen species (ROS).³ Upon oxidation, Met forms a diastereomeric mixture of methionine-S-sulfoxide (Met-S-SO) and methionine-R-sulfoxide (Met-R-SO). Met-S-SO and Met-R-SO can be reduced back to Met by MsrA (Met-S-SO reductase) and MsrB (Met-R-SO reductase), respectively (1). These enzymes have been reported to play important roles in the protection of cells and proteins against oxidative stress (2–8). Reversible Met oxidation has also been proposed to scavenge ROS, thereby protecting cells from oxidative damage (9–11). Increased expression of MsrA and MsrB can extend the life span of yeast cells and fruit flies, whereas deletion of the MsrA gene leads to the reduction in life span in mice and yeast (12–14).Previously, three MsrB isozymes and a single MsrA were found in mammals. MsrB1 (also known as SelR or SelX) is a selenoprotein, which contains selenocysteine (Sec) in the active site and is localized to cytosol and nucleus. MsrB2 and MsrB3 are Cys-containing homologs of MsrB1. MsrB2 resides in mitochondria, whereas human MsrB3 has two alternative splice forms, wherein MsrB3A localizes to the endoplasmic reticulum and MsrB3B is targeted to mitochondria (15).The catalytic mechanism of MsrA involves a sulfenic acid intermediate at the catalytic Cys followed by the formation of a disulfide bond between the catalytic and resolving Cys. A third Cys may then form a disulfide with the resolving Cys (16, 17). The resulting disulfide is reduced by thioredoxin or other oxidoreductases, generating the initial, reduced form of the protein. X-ray structures of MsrAs from several organisms have been solved (17, 18).Cys-containing MsrBs (e.g. mammalian MsrB2 and MsrB3) follow the same mechanism, although the two Msr types have no homology and are characterized by different structural folds (19–21). Sec-containing mammalian MsrB1 has also been characterized and compared with Cys-containing MsrBs (20). Interestingly, Cys-containing MsrBs share some active site features (e.g. conserved residues His⁷⁷, Val⁸¹, and Asn⁹⁷, numbering based on mouse MsrB1 sequence), which are absent in selenoprotein MsrB1s. When these three residues were introduced into the Sec-containing MsrB1, the enzyme was inactive. However, when the three residues were introduced into the Cys mutant form of MsrB1, the activity was partially recovered (20). This evidence supports the idea that catalytic Cys and Sec require different active site features.In addition to MsrA and MsrB functions, previous studies suggested the presence of additional Msr activities in Escherichia coli and yeast cells, which were especially evident in cells deficient in both enzymes (14, 21–23). Recently, Lowther and colleagues (24) discovered a new enzyme, designated fRMsr (free Met-R-SO reductase), which catalyzes the reduction of free Met-R-SO in E. coli. They showed that this activity is associated with a GAF-like-domain-containing protein. Homologs of this enzyme were found in other bacteria as well as in eukaryotes, suggesting that these proteins also could function as fRMsrs. However, none of these other proteins have been functionally characterized.In this work, we cloned a yeast homolog of bacterial fRMsr and functionally characterized it with regard to the in vivo function and catalytic mechanism. In addition, we carried out comparative genomic analyses to examine evolution of this protein family. The data show that fRMsr is the main enzyme responsible for the reduction of free Met-R-SO in both prokaryotes and unicellular eukaryotes.     

六六六对于飞蝗蝻期的熏蒸作用

一、前言 六六六不但是一种强有力的胃毒兼接触剂,并且具有相当的熏蒸作用,过去从事研究六六六熏蒸作用的为人不多,Srivastava和Wilson(1947)曾用德国蟑螂,东方天幕毛虫和五种仓库害虫装入金属纱笼里边,在容积二五二.二八立方尺的减压瓶里,分四种方法研究。(1)测定六六六蒸气对以上几种昆虫的熏蒸作用,认为六六六蒸气对於不同种类的昆虫和时期有不同程度的作用。(2)研究六六六     

Functional Analysis of the VirSR Phosphorelay from Clostridium perfringens

Toxin production in Clostridium perfringens is controlled by the VirSR two-component signal transduction system, which comprises the VirS sensor histidine kinase and the VirR response regulator. Other studies have concentrated on the elucidation of the genes controlled by this network; there is little information regarding the phosphorelay cascade that is the hallmark of such regulatory systems. In this study, we have examined each step in this cascade, beginning with autophosphorylation of VirS, followed by phosphotransfer from VirS to VirR. We also have studied the effects of gene dosage and phosphorylation in vivo. We have used random and site-directed mutagenesis to identify residues in VirS that are important for its function and have identified a region in the putative sensory domain of VirS that appeared to be essential for function. In vitro phosphorylation studies showed that VirSc, a truncated VirS protein that lacked the N-terminal sensory domain, was capable of autophosphorylation and could subsequently act as a phosphodonor for its cognate response regulator, VirR. Conserved residues of both VirS and VirR, including the D57 residue of VirR, were shown to be essential for this process. By use of Targetron technology, we were able to introduce a single copy of virR or virR_D57N onto the chromosome of a virR mutant of C. perfringens. The results showed that in vivo, when virR was present in single copy, the production of wild-type levels of perfringolysin O was dependent on the presence of virS and an unaltered D57 residue in VirR. These results provide good evidence that phosphorylation is critical for VirR function.     

Structure and Functional Analysis of RifR, the Type II Thioesterase from the Rifamycin Biosynthetic Pathway

Two thioesterases are commonly found in natural product biosynthetic clusters, a type I thioesterase that is responsible for removing the final product from the biosynthetic complex and a type II thioesterase that is believed to perform housekeeping functions such as removing aberrant units from carrier domains. We present the crystal structure and the kinetic analysis of RifR, a type II thioesterase from the hybrid nonribosomal peptide synthetases/polyketide synthase rifamycin biosynthetic cluster of Amycolatopsis mediterranei. Steady-state kinetics show that RifR has a preference for the hydrolysis of acyl units from the phosphopantetheinyl arm of the acyl carrier domain over the hydrolysis of acyl units from the phosphopantetheinyl arm of acyl-CoAs as well as a modest preference for the decarboxylated substrate mimics acetyl-CoA and propionyl-CoA over malonyl-CoA and methylmalonyl-CoA. Multiple RifR conformations and structural similarities to other thioesterases suggest that movement of a helical lid controls access of substrates to the active site of RifR.Assembly line complexes, which include modular polyketide synthases (PKS)³ and nonribosomal peptide synthetases (NRPS), are multifunctional proteins composed of modules that work in succession to synthesize secondary metabolites, many of which are precursors of potent antibiotics, immunosuppressants, anti-tumor agents, and other bioactive compounds. Rifamycin, the precursor to the anti-tuberculosis drug rifampicin, is produced by the rifamycin assembly line complex, which is an NRPS/PKS hybrid system composed of one NRPS-like and 10 PKS modules (1). Each module in an assembly line complex extends and modifies the intermediate compound before passing it on to the next module in the series (Fig. 1A). The intermediate compounds are covalently attached through a thioester linkage to the phosphopantetheine arm (Ppant) of carrier domains, one associated with each module, until they are released from the synthase, usually by a type I thioesterase (TEI) (2, 3).Open in a separate windowFIGURE 1.Proposed functions of thioesterase II proteins. A, chain elongation by a PKS module. The chain elongation intermediate is transferred from the ACP of the upstream module to the ketosynthase (KS) domain. The acyltransferase (AT) domain transfers an acyl group building block from CoA to the ACP within the module. The KS domain catalyzes condensation of the new building block with the intermediate, releasing CO₂. B, production of a decarboxylated acyl unit by the ketosynthase domain and the subsequent hydrolysis by a TEII. C, mispriming of a PKS by transfer of an acyl-phosphopantetheine arm by a promiscuous phosphopantetheinyl transferase (Pptase) and the subsequent hydrolysis by a TEII. D, hydrolysis of an amino acid derivative by a TEII from an NRPS module comprising an adenylation domain (A) and a peptide carrier protein (PCP) domain.TEIs are usually integrated into the final module of the assembly line complex and remove the final product through macrocyclization or hydrolysis. Occasionally, tandem type I thioesterases are integrated at the C terminus of the final module of NRPS pathways (4).Although TEIs are covalently attached to the terminal module and generally process only the final product of an assembly line complex, type II thioesterases (TEIIs) are discrete proteins that can remove intermediates from any module in the complex. A variety of functions have been attributed to TEIIs, the most prevalent of which is a “housekeeping function,” the removal of aberrant acyl units from carrier domains. These aberrant acyl units may be due to premature decarboxylation by a PKS ketosynthase domain (5) (Fig. 1B) or to mispriming of the carrier domain by a promiscuous phosphopantetheinyl transferase (6–8) (Fig. 1C). Other proposed functions for TEIIs include the removal of intermediates from the synthase as in the case of the mammary gland rat fatty acid synthase (FAS) TEII in lactating rats, which removes medium chain C₈-C₁₂ fatty acids from the ACP domain (9) and the removal of amino acid derivatives from a carrier domain (10–13), allowing these derivatives to be incorporated into the natural product by a later module in the assembly line complex (Fig. 1D).Disruption of the TEI function results in a complete loss of product, whereas disruption of TEII function results in a significant decrease in product yield (30–95%) (4, 14–24). Removal of the TEII from the rifamycin assembly line resulted in a 60% decrease in product yield (25). Neither TEIs nor TEIIs may rescue the disrupted function of the other (6), but a TEII from another pathway may rescue the function of a disrupted TEII (26).Two models have been proposed for the TEII housekeeping function (5). In the high specificity model, the TEII scans the complex and efficiently removes only aberrant acyl units. In the low specificity model, the TEII removes both correct and incorrect acyl units from the Ppant arm at an inefficient rate. Correct acyl units are quickly incorporated into the growing intermediate compound. In contrast, incorrect acyl units stall the assembly line, providing a longer window of opportunity for removal by a TEII. Thus a slow, low specificity enzyme can be effective.TEIIs from different pathways have differing specificities, but general trends include a preference for decarboxylated acyl units over carboxylated acyl units (5, 6, 27), substrates linked to a carrier domain over substrates linked to CoA or the phosphopantetheine mimic N-acetylcysteamine (7, 28), and single amino acids over di- or tri-peptides (6, 7). TEIIs are able to hydrolyze substrates attached to carrier domains from their native pathway as well as other pathways (6, 20, 28).PKS/NRPS/FAS thioesterases belong to the α/β hydrolase family. Structures are reported for seven PKS/NRPS/FAS thioesterases: crystal structures for the TEIs from the pikromycin (PikTE) PKS (29), 6-deoxyerythronolide B (DEBSTE) PKS (30), surfactin NRPS (SrfTE) (31), fengycin NRPS (FenTE) (32), and human fatty acid synthase (hFasTE) (33) systems, and NMR structures for enterobactin TEI (34), and surfactin TEII (35). Like PKS modules, PKS TEIs are dimers. The dimer interface comprises two N-terminal helices that are unique to the PKS TEIs. NRPS TEIs are monomeric, like NRPS modules. The NRPS TEII of surfactin is also monomeric (31). Although the FAS complex is dimeric, the FAS TEI is a monomer (33). All of the TEs have an α-helical insertion after strand β5 that forms a lid over the active site. Additionally, in the PKS TEIs, the N-terminal dimer-forming helices contribute to the lid structure, forming a fixed channel that runs the length of the TE and contains the active site. In contrast, the active site pocket of monomeric NRPS TEIs and TEIIs is flexible; two conformations of the lid and active site pocket were observed in the surfactin TEI (SrfTEI) crystal structure (7), and chemical shift observations suggested greater flexibility for residues of the lid region in the surfactin TEII (SrfTEII) solution structure (35). These movements seem to be of functional importance, because a movement of a linker peptide in SrfTEI determines the shape of the active site pocket and a movement of the first lid helix appears to modulate access to the active site (31).We report the structure and activity of recombinant RifR, the TEII of the rifamycin biosynthetic cluster. Steady-state kinetic analysis of the hydrolytic activity of RifR on a wide range of acyl-CoA and acyl-ACP substrates demonstrates that acyl-ACP substrates are preferred over the acyl-CoAs. Aberrant, decarboxylated acyl units are processed more efficiently than are the natural rifamycin building blocks. We report the crystal structure of RifR, the first for any hybrid PKS/NRPS TEII. The size and shape of the substrate chamber are variable, because one of the elements forming the chamber, an extended linker segment, is highly flexible, and different crystal forms reveal different shapes for the substrate binding site. Access to the active site is severely restricted, and structural comparisons with other thioesterases suggest that a conformational change in the lid and the flexible linker region is required for access to the substrate pocket.