Crystal Structures of Aspergillus japonicus Fructosyltransferase Complex with Donor/Acceptor Substrates Reveal Complete Subsites in the Active Site for Catalysis

Fructosyltransferases catalyze the transfer of a fructose unit from one sucrose/fructan to another and are engaged in the production of fructooligosaccharide/fructan. The enzymes belong to the glycoside hydrolase family 32 (GH32) with a retaining catalytic mechanism. Here we describe the crystal structures of recombinant fructosyltransferase (AjFT) from Aspergillus japonicus CB05 and its mutant D191A complexes with various donor/acceptor substrates, including sucrose, 1-kestose, nystose, and raffinose. This is the first structure of fructosyltransferase of the GH32 with a high transfructosylation activity. The structure of AjFT comprises two domains with an N-terminal catalytic domain containing a five-blade β-propeller fold linked to a C-terminal β-sandwich domain. Structures of various mutant AjFT-substrate complexes reveal complete four substrate-binding subsites (−1 to +3) in the catalytic pocket with shapes and characters distinct from those of clan GH-J enzymes. Residues Asp-60, Asp-191, and Glu-292 that are proposed for nucleophile, transition-state stabilizer, and general acid/base catalyst, respectively, govern the binding of the terminal fructose at the −1 subsite and the catalytic reaction. Mutants D60A, D191A, and E292A completely lost their activities. Residues Ile-143, Arg-190, Glu-292, Glu-318, and His-332 combine the hydrophobic Phe-118 and Tyr-369 to define the +1 subsite for its preference of fructosyl and glucosyl moieties. Ile-143 and Gln-327 define the +2 subsite for raffinose, whereas Tyr-404 and Glu-405 define the +2 and +3 subsites for inulin-type substrates with higher structural flexibilities. Structural geometries of 1-kestose, nystose and raffinose are different from previous data. All results shed light on the catalytic mechanism and substrate recognition of AjFT and other clan GH-J fructosyltransferases.     

Crystal Structures of E. coli Native MenH and Two Active Site Mutants

Recent revision of the biosynthetic pathway for menaquinone has led to the discovery of a previously unrecognized enzyme 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase, also known as MenH. This enzyme has an α/β hydrolase fold with a catalytic triad comprising Ser86, His232, and Asp210. Mutational studies identified a number of conserved residues of importance to activity, and modeling further implicated the side chains of Tyr85 and Trp147 in formation of a non-standard oxyanion hole. We have solved the structure of E. coli MenH (EcMenH) at 2.75 Å resolution, together with the structures of the active site mutant proteins Tyr85Phe and Arg124Ala, both at 2.5 Å resolution. EcMenH has the predicted α/β hydrolase fold with its core α/β domain capped by a helical lid. The active site, a long groove beneath the cap, contains a number of conserved basic residues and is found to bind exogeneous anions, modeled as sulfate and chloride, in all three crystal structures. Docking studies with the MenH substrate and a transition state model indicate that the bound anions mark the binding sites for anionic groups on the substrate. The docking studies, and careful consideration of the active site geometry, further suggest that the oxyanion hole is of a conventional nature, involving peptide NH groups, rather than the proposed site involving Tyr85 and Trp147. This is in accord with conclusions from the structure of S. aureus MenH. Comparisons with the latter do, however, indicate differences in the periphery of the active site that could be of relevance to selective inhibition of MenH enzymes.     

Expression Pattern Diversity and Functional Conservation Between Retroposed PRAT Genes from Drosophila melanogaster and Drosophila virilis

Gene duplication by retrotransposition duplicates only the coding and untranslated regions of a gene and, thus, biases retroduplicated genes toward having different expression patterns from their parental genes. As such, genes duplicated by retrotransposition are more likely to develop novel expression domains. To explore this idea further, we used the Prat/Prat2 gene duplication in Drosophila as a case study to examine the aftermath of a retrotransposition event that resulted in both the parent and the child gene becoming essential for survival. We used the Gal4-UAS transgene system with EGFP as a reporter to determine the developmental expression patterns of Prat and Prat2 from D. melanogaster (DmPrat and DmPrat2) and Prat from D. virilis (DvPrat). We also tested the functional equivalence of the protein products of DmPrat and DmPrat2. We found that each of the proteins could rescue DmPrat mutations, showing that the requirement for both Prat and Prat2 in Drosophila is not simply due to differences in protein function. In contrast, we found that the DmPrat and DmPrat2 genes have developed nonoverlapping patterns of expression, which correlate with their respective loss-of-function phenotypes. We further found that DvPrat expression is similar to DmPrat during development but differs in adult gonads. Thus, the function of the Prat retrogene has not diverged in the D. melanogaster and D. virilis lineages, while some aspects of its expression pattern have evolved. Finally, we have identified promoter elements, conserved upstream of DmPrat and DvPrat, that this retrogene has acquired to drive its expression.     

Conservation of gene order, structure and sequence between three closely linked genes in Drosophila melanogaster and Drosophila virilis

In Drosophila melanogaster, the apparently unrelated genes anon-66Da, RpL14, and anon-66Db (from telomere to centromere) are located on a 5547 bp genomic fragment on chromosome arm 3L at cytological position 66D8. The three genes are tightly linked, and flanked by two relatively large genes with unknown function. We have taken a comparative genomic approach to investigate the evolutionary history of the three genes. To this end we isolated a Drosophila virilis 7.3 kb genomic fragment which is homologous to a 5.5 kb genomic region of D. melanogaster. Both fragments map to Muller's element D, namely to section 66D in D. melanogaster and to section 32E in D. virilis, and harbor the genes anon-66Da, RpL14, and anon-66Db. We demonstrate that the three genes exhibit a high conservation of gene topography in general and in detail. While most introns and intergenic regions reveal sequence divergences, there are, however, a number of interspersed conserved sequence motifs. In particular, two introns of the RpL14 gene contain a short, highly conserved 60 nt long sequence located at corresponding positions. This sequence represents a novel Drosophila small nucleolar RNA, which is homologous to human U49. Whereas DNA flanking the three genes shows no significant interspecies homologies, the 3'-flanking region in D. virilis contains sequences from the transposable element Penelope. The Penelope family of transposable elements has been shown to promote chromosomal rearrangements in the D. virilis species group. The presence of Penelope sequences in the D. virilis 7.3 kb genomic fragment may be indicative for a transposon-induced event of transposition which did not yet scramble the order of the three genes but led to the breakdown of sequence identity of the flanking DNA.     

Crystal structure of the Rasputin NTF2-like domain from Drosophila melanogaster

The crystal structure of the NTF2-like domain of the Drosophila homolog of Ras GTPase SH3 Binding Protein (G3BP), Rasputin, was determined at 2.7 Å resolution. The overall structure is highly similar to nuclear transport factor 2: It is a homodimer comprised of a β-sheet and three α-helices forming a cone-like shape. However, known binding sites for RanGDP and FxFG containing peptides show electrostatic and steric differences compared to nuclear transport factor 2. A HEPES molecule bound in the structure suggests a new, and possibly physiologically relevant, ligand binding site.     

果蝇miRNA的结构与功能及研究策略

miRNA是一类在动植物基因组中广泛存在的小分子非编码RNA, 作为真核细胞转录后水平上基因表达的关键调控者, 它通过与靶基因mRNA的特定位点结合, 抑制mRNA的翻译或诱导mRNA的降解, 从而介导生物体的许多重要生理活动。本文简要总结了模式昆虫黑腹果蝇Drosophila melanogaster miRNA的鉴定情况, 综述了miRNA的结构特征、生物合成途径和作用机制。miRNA可能同时调节成百上千个靶标, 其生物功能主要体现为: 调节细胞分化与凋亡, 调节器官和神经系统的发育, 控制肌肉分化, 保持能量动态平衡, 调节昆虫变态或综合调节作用。miRNA具有“低丰度、短序列、难富集”的特点, miRNA基因的获得和功能鉴定研究的基本策略是实验生物学和生物信息学方法的有机结合。鉴定新miRNA及其靶标, 深入研究其生物功能和基因进化等可能成为今后一段时间昆虫miRNA研究的重要内容。     

Characterisation of a bis(5'-nucleosyl)-tetraphosphatase (asymmetrical) from Drosophila melanogaster

The intracellular functions of diadenosine polyphosphates are still poorly defined. To understand these better, we have expressed and characterized a heat stable, 16.6kDa Nudix hydrolase (Apf) that specifically metabolizes these nucleotides from a Drosophila melanogaster cDNA. Apf always produces an NTP product, with substrate preference depending on pH and divalent ion (Zn(2+) or Mg(2+)). For example, diadenosine tetraphosphate is hydrolysed to ATP and AMP with K(m), k(cat) and k(cat)/K(m) values 9microM, 43s(-1) and 4.8microM(-1)s(-1) (pH 6.5, 0.1mMZn(2+)) and 12microM, 13s(-1) and 1.1microM(-1)s(-1) (pH 7.5, 20mMMg(2+)), respectively. However, diadenosine hexaphosphate is efficiently hydrolysed to ATP only at pH 7.5 with 20mMMg(2+) (K(m), k(cat) and k(cat)/K(m) values of 15microM 4.0s(-1), and 0.27microM(-1)s(-1)). Fluoride potently inhibits diadenosine tetraphosphate hydrolysis in the presence of Mg(2+) (IC(50)=20microM), whereas it is ineffective in the presence of Zn(2+), supporting the view that inhibition involves a specific, MgF(3)(-)-containing transition state analogue complex. Patterns of Apf expression in Drosophila tissues show Apf mRNA levels to be highest in embryos and adult females. Subcellular localization with Apf-EGFP fusion constructs reveals Apf to be predominantly nuclear, having an apparent preferential association with euchromatin and facultative heterochromatin. This supports a nuclear function for diadenosine tetraphosphate. Our results show Apf to be a fairly typical member of the bis (5'-nucleosyl)-tetraphosphatase subfamily of Nudix hydrolases with features that distinguish it from a previously reported bis (5'-nucleosyl)-tetraphosphatase hydrolase activity from Drosophila embryos.     

Crystal Structure and Functional Analysis of the Glutaminyl Cyclase from Xanthomonas campestris

Glutaminyl cyclases (QCs) (EC 2.3.2.5) catalyze the formation of pyroglutamate (pGlu) at the N-terminus of many proteins and peptides, a critical step for the maturation of these bioactive molecules. Proteins having QC activity have been identified in animals and plants, but not in bacteria. Here, we report the first bacterial QC from the plant pathogen Xanthomonas campestris (Xc). The crystal structure of the enzyme was solved and refined to 1.44-Å resolution. The structure shows a five-bladed β-propeller and exhibits a scaffold similar to that of papaya QC (pQC), but with some sequence deletions and conformational changes. In contrast to the pQC structure, the active site of XcQC has a wider substrate-binding pocket, but its accessibility is modulated by a protruding loop acting as a flap. Enzyme activity analyses showed that the wild-type XcQC possesses only 3% QC activity compared to that of pQC. Superposition of those two structures revealed that an active-site glutamine residue in pQC is substituted by a glutamate (Glu⁴⁵) in XcQC, although position 45 is a glutamine in most bacterial QC sequences. The E45Q mutation increased the QC activity by an order of magnitude, but the mutation E45A led to a drop in the enzyme activity, indicating the critical catalytic role of this residue. Further mutagenesis studies support the catalytic role of Glu⁸⁹ as proposed previously and confirm the importance of several conserved amino acids around the substrate-binding pocket. XcQC was shown to be weakly resistant to guanidine hydrochloride, extreme pH, and heat denaturations, in contrast to the extremely high stability of pQC, despite their similar scaffold. On the basis of structure comparison, the low stability of XcQC may be attributed to the absence of both a disulfide linkage and some hydrogen bonds in the closure of β-propeller structure. These results significantly improve our understanding of the catalytic mechanism and extreme stability of type I QCs, which will be useful in further applications of QC enzymes.     

Mutational Analysis of Cysteine Residues of the Insect Odorant Co-receptor (Orco) from Drosophila melanogaster Reveals Differential Effects on Agonist- and Odorant-tuning Receptor-dependent Activation

Insect odorant receptors are heteromeric odorant-gated cation channels comprising a conventional odorant-sensitive tuning receptor (ORx) and a highly conserved co-receptor known as Orco. Orco is found only in insects, and very little is known about its structure and the mechanism leading to channel activation. In the absence of an ORx, Orco forms homomeric channels that can be activated by a synthetic agonist, VUAA1. Drosophila melanogaster Orco (DmelOrco) contains eight cysteine amino acid residues, six of which are highly conserved. In this study, we replaced individual cysteine residues with serine or alanine and expressed Orco mutants in Flp-In 293 T-Rex cells. Changes in intracellular Ca²⁺ levels were used to determine responses to VUAA1. Replacement of two cysteines (Cys-429 and Cys-449) in a predicted intracellular loop (ICL3), individually or together, gave variants that all showed similar increases in the rate of response and sensitivity to VUAA1 compared with wild-type DmelOrco. Kinetic modeling indicated that the response of the Orco mutants to VUAA1 was faster than wild-type Orco. The enhanced sensitivity and faster response of the Cys mutants was confirmed by whole-cell voltage clamp electrophysiology. In contrast to the results from direct agonist activation of Orco, the two cysteine replacement mutants when co-expressed with a tuning receptor (DmelOR22a) showed an ∼10-fold decrease in potency for activation by 2-methyl hexanoate. Our work has shown that intracellular loop 3 is important for Orco channel activation. Importantly, this study also suggests differences in the structural requirements for the activation of homomeric and heteromeric Orco channel complexes.     

Reproductive isolation in natural populations of Drosophila melanogaster from Brazzaville (Congo)

The aim of this work is to analyze the homogamy previously detected between two natural populations of Drosophila melanogaster from Brazzaville. It is shown that mating isolation was still maintained under laboratory conditions 10 years after the populations samples were trapped. Isolation seemed to be due mainly to pre-mating isolation and we checked for any suggestion of post-mating mortality of hybrids. Pre-mating isolation was not symmetrical, and significant ² values were found in 3/4 possible 3-way mating choice experiments. The only exception involved a male from the countryside and two females (one from each population) for which no significant mating preference was detected. Mortality of hybrids was intermediate between those of the parental strains showing a clear maternal effect and the existence of partial dominance. Major differences in the cuticular hydrocarbons were also found and they could account for the isolation. These findings in populations from African breweries indicate that they are closely related to European ones, suggesting that this phenomenon is not a case of sympatric speciation, but probably attributable to the reintroduction of an allopatric population.     

Amino acid sequence of an invertebrate FBP aldolase (from Drosophila melanogaster)

The complete amino acid sequence of FBP aldolase from Drosophila melanogaster has been determined. The enzyme contains four identical subunits of 360 amino acid residues. The primary structure of the monomer was established using automated Edman degradation on fragments prepared by CNBr-cleavage, by partial acid cleavage at the unique Asp-Pro bond and by oxidative cleavage at the three tryptophan residues. Manual Edman-Chang degradation was used on smaller peptides obtained by digestion with Staphylococcus aureus V8 protease, trypsin or chymotrypsin. The primary structure of Drosophila aldolase exhibits very extensive homology with the sequence of rabbit muscle aldolase (71% identity), thus explaining the early observation that Drosophila and mammalian aldolases form active interspecies hybrid quaternary structures (Brenner-Holzach, O. and Leuthardt, F., Eur. J. Biochem. (1972) 31, 423-426).     

Isolation of Protease-Free Alcohol Dehydrogenase (ADH) from Drosophila simulans and Several Homozygous and Heterozygous Drosophila melanogaster Variants

The enzyme alcohol dehydrogenase (ADH) fromseveral naturally occurring ADH variants ofDrosophila melanogaster and Drosophilasimulans was isolated. Affinity chromatography withthe ligand Cibacron Blue and elution with NAD⁺ showed similarbehavior for D. melanogaster ADH-FF, ADH-71k,and D. simulans ADH. Introduction of a secondCibacron Blue affinity chromatography step, withgradient elution with NAD⁺, resulted in pure and stable enzymes. D.melanogaster ADH-SS cannot be eluted from theaffinity chromatography column at a high concentrationof NAD⁺ and required a pH gradient for itspurification, preceded by a wash step with a high concentration ofNAD⁺. Hybrid Drosophila melanogasteralcohol dehydrogenase FS has been isolated fromheterozygous flies, using affinity chromatography withfirst elution at a high concentration NAD⁺, directlyfollowed by affinity chromatography elution with a pHgradient. Incubation of equal amounts of pure homodimersof Drosophila melanogaster ADH-FF and ADH-SS,in the presence of 3 M urea at pH 8.6, for 30 min at roomtemperature, followed by reassociation yielded activeDrosophila melanogaster ADH-FS heterodimers. Noproteolytic degradation was found after incubation ofpurified enzyme preparations in the absence or presenceof SDS, except for some degradation of ADH-SS after verylong incubation times. The thermostabilities of D.melanogaster ADH-71k and ADH-SS were almostidentical and were higher than those of D.melanogaster ADH-FF and D. simulans ADH. Thethermostability of D. melanogaster ADH-FS waslower than those of D. melanogaster ADH-FF andADH-SS. D. melanogaster ADH-FF and ADH-71k have identical inhibition constantswith the ligand Cibacron Blue at pH 8.6, which are twotimes higher at pH 9.5. The K_i values forD. simulans ADH are three times lower at bothpH values. D. melanogaster ADH-SS and ADH-FS havesimilar K_i values, which are lower than thosefor D. melanogaster ADH-FF at pH 8.6. But at pH9.5 the K_i value for ADH-FS is the same as atpH 8.6, while that of ADH-SS is seven times higher. Kinetic parameters ofDrosophila melanogaster ADH-FF, ADH-SS, andADH-71k and Drosophila simulans ADH, at pH 8.6and 9.5, showed little or no variation inK_m ^eth values. TheK_m ^NAD values measured at pH 9.5for Drosophila alcohol dehydrogenases are alllower than those measured at pH 8.6. The rate constants(k_cat) determined for all fourDrosophila alcohol dehydrogenases are higher at pH 9.5 than at pH 8.6. D.melanogaster ADH-FS showed nonlinear kinetics.     

Crystal Structures of Glycosyltransferase UGT78G1 Reveal the Molecular Basis for Glycosylation and Deglycosylation of (Iso)flavonoids

The glycosyltransferase UGT78G1 from Medicago truncatula catalyzes the glycosylation of various (iso)flavonoids such as the flavonols kaempferol and myricetin, the isoflavone formononetin, and the anthocyanidins pelargonidin and cyanidin. It also catalyzes a reverse reaction to remove the sugar moiety from glycosides. The structures of UGT78G1 bound with uridine diphosphate or with both uridine diphosphate and myricetin were determined at 2.1 Å resolution, revealing detailed interactions between the enzyme and substrates/products and suggesting a distinct binding mode for the acceptor/product. Comparative structural analysis and mutagenesis identify glutamate 192 as a key amino acid for the reverse reaction. This information provides a basis for enzyme engineering to manipulate substrate specificity and to design effective biocatalysts with glycosylation and/or deglycosylation activity.     

Characterization of the EstP Protein in Drosophila melanogaster and Its Conservation in Drosophilids

The -esterase cluster of D. melanogaster comprises two tandemly duplicated genes. Est6 encodes the well-characterized 5 gene, but the product of the second gene, denoted EstP, had not previously been identified. Here we show that the EstP gene encodes the carboxylesterase EST7. Expression of EstP using the Baculovirus system led to production of a carboxylesterase biochemically indistinguishable from EST7. Furthermore, a naturally occurring EstP variant produces greatly reduced amounts of EstP mRNA and no detectable EST7 protein. Finally, introduction of a wild-type copy of EstP by germline transformation into the variant strain confers the wild-type EST7 phenotype. We show that EST7 differs from EST6 in its substrate and inhibitor specificities and tissue distribution. Germline transformation experiments show that EstP expression is controlled by sequences located between 192 bp 5 and 609 bp 3 of the EstP coding region. Data comparisons with other drosophilid esterases suggest that the site of expression, and hence the function, of EST7 has been conserved across lineages in both the subgenera Drosophila and Sophophora.     

Current status of the Drosophila melanogaster species-group (Diptera)

Abstract. The Drosphila melanogaster species-group, established by Sturtevant (1942) for fourteen species, is now known to contain 115 described species here divided into twelve named subgroups (including one newly proposed), as well as further undescribed species. Three of the species, melanogaster, simulans and ananassae , are cosmopolitan; two others, kikkawai and malerkotliana , are widespread in the southern hemisphere, the latter apparently a recent introduction to South America. The greatest numbers of species otherwise occur in the Oriental region with smaller numbers in the Ethiopian, eastern Palaearctic and Australian regions and in several islands of the South Pacific. D.rajasekari and D.raychaudhurii are synonymized with D.biamipes;also D.andamanensis Parshad & Singh is synonymized with D.andamanensis Gupta & Raychaudhuri.     

Development of Anncaliia algerae (Microsporidia) in Drosophila melanogaster

Representatives of the genus Anncaliia are known as natural parasites of dipteran and coleopteran insects, amphipod crustaceans, but also humans, primarily with immunodeficiency. Anncaliia algerae‐caused fatal myositis is considered as an emergent infectious disease in humans. A. (=Nosema, Brachiola) algerae, the best studied species of the genus, demonstrates the broadest among microsporidia range of natural and experimental hosts, but it has never been propagated in Drosophila. We present ultrastructural analysis of development of A. algerae in visceral muscles and adipocytes of Drosophila melanogaster 2 wk after per oral experimental infection. We observed typical to Anncaliia spp. features of ultrastructure and cell pathology including spore morphology, characteristic extensions of the plasma membrane, and presence of “ridges” and appendages of tubular material at proliferative stages. Anncaliia algerae development in D. melanogaster was particularly similar to one of A. algerae and A.(Brachiola) vesicularum in humans with acute myositis. Given D. melanogaster is currently the most established genetic model, with a fully sequenced genome and easily available transgenic forms and genomic markers, a novel host–parasite system might provide new genetic tools to investigate host–pathogen interactions of A. algerae, as well to test antimicrosporidia drugs.