Functional importance of Crenarchaea-specific extra-loop revealed by an X-ray structure of a heterotetrameric crenarchaeal splicing endonuclease

Archaeal splicing endonucleases (EndAs) are currently classified into three groups. Two groups require a single subunit protein to form a homodimer or homotetramer. The third group requires two nonidentical protein components for the activity. To elucidate the molecular architecture of the two-subunit EndA system, we studied a crenarchaeal splicing endonuclease from Pyrobaculum aerophilum. In the present study, we solved a crystal structure of the enzyme at 1.7-Å resolution. The enzyme adopts a heterotetrameric form composed of two catalytic and two structural subunits. By connecting the structural and the catalytic subunits of the heterotetrameric EndA, we could convert the enzyme to a homodimer that maintains the broad substrate specificity that is one of the characteristics of heterotetrameric EndA. Meanwhile, a deletion of six amino acids in a Crenarchaea-specific loop abolished the endonuclease activity even on a substrate with canonical BHB motif. These results indicate that the subunit architecture is not a major factor responsible for the difference of substrate specificity between single- and two-subunit EndA systems. Rather, the structural basis for the broad substrate specificity is built into the crenarchaeal splicing endonuclease itself.     

X-ray structure of the fourth type of archaeal tRNA splicing endonuclease: insights into the evolution of a novel three-unit composition and a unique loop involved in broad substrate specificity

Cleavage of introns from precursor transfer RNAs (tRNAs) by tRNA splicing endonuclease (EndA) is essential for tRNA maturation in Archaea and Eukarya. In the past, archaeal EndAs were classified into three types (α′₂, α₄ and α₂β₂) according to subunit composition. Recently, we have identified a fourth type of archaeal EndA from an uncultivated archaeon Candidatus Micrarchaeum acidiphilum, referred to as ARMAN-2, which is deeply branched within Euryarchaea. The ARMAN-2 EndA forms an ε₂ homodimer and has broad substrate specificity like the α₂β₂ type EndAs found in Crenarchaea and Nanoarchaea. However, the precise architecture of ARMAN-2 EndA was unknown. Here, we report the crystal structure of the ε₂ homodimer of ARMAN-2 EndA. The structure reveals that the ε protomer is separated into three novel units (α^N, α and β^C) fused by two distinct linkers, although the overall structure of ARMAN-2 EndA is similar to those of the other three types of archaeal EndAs. Structural comparison and mutational analyses reveal that an ARMAN-2 type-specific loop (ASL) is involved in the broad substrate specificity and that K161 in the ASL functions as the RNA recognition site. These findings suggest that the broad substrate specificities of ε₂ and α₂β₂ EndAs were separately acquired through different evolutionary processes.     

Phylogenetic analysis of archaeal PCNA homologues

Proliferating cell nuclear antigen (PCNA) is an essential component of the DNA replication and repair machinery in the domain Eucarya. Eukaryotes and euryarchaeotes, which belong to one subdomain of Archaea, possess a single PCNA homologue, whereas two distinct PCNA homologues have been identified from Sulfolobus solfataricus, which belongs to the other archaeal subdomain, Crenarchaeota. We have cloned and sequenced two genes of PCNA homologues from the thermoacidophilic crenarchaeon Sulfurisphaera ohwakuensis. These genes, referred to as the Soh PCNA A gene and the Soh PCNA B gene, were found to encode 245 amino acids (aa) (27 kDa) and 248 aa (27 kDa), respectively. In deduced amino acid sequences of both PCNA homologues, the motif L/I-A-P-K/R, implicated in binding of PCNA with replication factor C (RFC), was identified. Phylogenetic analysis of all available archaeal PCNA homologues suggests that crenarchaeal homologues are divided into two groups. Group A consists of Soh PCNA A, one of the S. solfataricus PCNA homologues, and one of the Aeropyrum pernix PCNA homologues. The other crenarchaeal homologues form group B. Crenarchaeal PCNA homologues constitute a monophyletic subfamily. These results suggest that the evolution of crenarchaeal PCNA homologues has been characterized by one or two gene duplication events, which are assumed to have occurred after the split of the crenarchaeal and euryarchaeal lineages. Received: July 10, 2000 / Accepted: September 26, 2000     

Three-dimensional structure determined for a subunit of human tRNA splicing endonuclease (Sen15) reveals a novel dimeric fold

Splicing of eukaryal intron-containing tRNAs requires the action of the heterotetrameric splicing endonuclease, which is composed of two catalytic subunits, Sen34 and Sen2, and two structural subunits, Sen15 and Sen54. Here we report the solution structure of the human tRNA splicing endonuclease subunit HsSen15. To facilitate the structure determination, we removed the disordered 35 N-terminal and 14 C-terminal residues of the full-length protein to produce HsSen15(36-157). The structure of HsSen15(36-157), the first for a subunit of a eukaryal splicing endonuclease, revealed that the protein possesses a novel homodimeric fold. Each monomer consists of three alpha-helices and a mixed antiparallel/parallel beta-sheet, arranged in a topology similar to that of the C-terminal domain of Methanocaldococcus jannaschii endonuclease. The dimeric interface is dominated by a beta-barrel structure, formed by face-to-face packing of two, three-stranded beta-sheets. Each of the beta-sheets results from reciprocal parallel pairing of one beta-strand from one subunit with two other beta-strands from the symmetric subunit. The structural model provides insights into the functional assembly of the human tRNA splicing endonuclease.     

Structural Basis for Dimerization and Catalysis of a Novel Esterase from the GTSAG Motif Subfamily of the Bacterial Hormone-sensitive Lipase Family

Hormone-sensitive lipases (HSLs) are widely distributed in microorganisms, plants, and animals. Microbial HSLs are classified into two subfamilies, an unnamed new subfamily and the GDSAG motif subfamily. Due to the lack of structural information, the detailed catalytic mechanism of the new subfamily is not yet clarified. Based on sequence analysis, we propose to name the new subfamily as the GTSAG motif subfamily. We identified a novel HSL esterase E25, a member of the GTSAG motif subfamily, by functional metagenomic screening, and resolved its structure at 2.05 Å. E25 is mesophilic (optimum temperature at 50 °C), salt-tolerant, slightly alkaline (optimum pH at 8.5) for its activity, and capable of hydrolyzing short chain monoesters (C2–C10). E25 tends to form dimers both in the crystal and in solution. An E25 monomer contains an N-terminal CAP domain, and a classical α/β hydrolase-fold domain. Residues Ser¹⁸⁶, Asp²⁸², and His³¹² comprise the catalytic triad. Structural and mutational analyses indicated that E25 adopts a dimerization pattern distinct from other HSLs. E25 dimer is mainly stabilized by an N-terminal loop intersection from the CAP domains and hydrogen bonds and salt bridges involving seven highly conserved hydrophilic residues from the catalytic domains. Further analysis indicated that E25 also has some catalytic profiles different from other HSLs. Dimerization is essential for E25 to exert its catalytic activity by keeping the accurate orientation of the catalytic Asp²⁸² within the catalytic triad. Our results reveal the structural basis for dimerization and catalysis of an esterase from the GTSAG motif subfamily of the HSL family.     

Structural insights into catalytic and substrate binding mechanisms of the strategic EndA nuclease from Streptococcus pneumoniae

EndA is a sequence non-specific endonuclease that serves as a virulence factor during Streptococcus pneumoniae infection. Expression of EndA provides a strategy for evasion of the host''s neutrophil extracellular traps, digesting the DNA scaffold structure and allowing further invasion by S. pneumoniae. To define mechanisms of catalysis and substrate binding, we solved the structure of EndA at 1.75 Å resolution. The EndA structure reveals a DRGH (Asp-Arg-Gly-His) motif-containing ββα-metal finger catalytic core augmented by an interesting ‘finger-loop’ interruption of the active site α-helix. Subsequently, we delineated DNA binding versus catalytic functionality using structure-based alanine substitution mutagenesis. Three mutants, H154A, Q186A and Q192A, exhibited decreased nuclease activity that appears to be independent of substrate binding. Glu205 was found to be crucial for catalysis, while residues Arg127/Lys128 and Arg209/Lys210 contribute to substrate binding. The results presented here provide the molecular foundation for development of specific antibiotic inhibitors for EndA.     

Unusual evolutionary history of the tRNA splicing endonuclease EndA: relationship to the LAGLIDADG and PD-(D/E)XK deoxyribonucleases

The tRNA splicing endoribonuclease EndA from Methanococcus jannaschii is a homotetramer formed via heterologous interaction between the two pairs of homodimers. Each monomer consists of two alpha/beta domains, the N-terminal domain (NTD) and the C-terminal domain (CTD) containing the RNase A-like active site. Comparison of the EndA coordinates with the publicly available protein structure database revealed the similarity of both domains to site-specific deoxyribonucleases: the NTD to the LAGLIDADG family and the CTD to the PD-(D/E)XK family. Superposition of the NTD on the catalytic domain of LAGLIDADG homing endonucleases allowed a suggestion to be made about which amino acid residues of the tRNA splicing nuclease might participate in formation of a presumptive cryptic deoxyribonuclease active site. On the other hand, the CTD and PD-(D/E)XK endonucleases, represented by restriction enzymes and a phage lambda exonuclease, were shown to share extensive similarities of the structural framework, to which entirely different active sites might be attached in two alternative locations. These findings suggest that EndA evolved from a fusion protein with at least two distinct endonuclease activities: the ribonuclease, which made it an essential "antitoxin" for the cells whose RNA genes were interrupted by introns, and the deoxyribonuclease, which provided the means for homing-like mobility. The residues of the noncatalytic CTDs from the positions corresponding to the catalytic side chains in PD-(D/E)XK deoxyribonucleases map to the surface at the opposite side to the tRNA binding site, for which no function has been implicated. Many restriction enzymes from the PD-(D/E)XK superfamily might have the potential to maintain an additional active or binding site at the face opposite the deoxyribonuclease active site, a property that can be utilized in protein engineering.     

The Subfamily-specific Assembly of Eag and Erg K+ Channels Is Determined by Both the Amino and the Carboxyl Recognition Domains

A functional voltage-gated K⁺ (Kv) channel comprises four pore-forming α-subunits, and only members of the same Kv channel subfamily may co-assemble to form heterotetramers. The ether-à-go-go family of Kv channels (KCNH) encompasses three distinct subfamilies: Eag (Kv10), Erg (Kv11), and Elk (Kv12). Members of different ether-à-go-go subfamilies, such as Eag and Erg, fail to form heterotetramers. Although a short stretch of amino acid sequences in the distal C-terminal section has been implicated in subfamily-specific subunit assembly, it remains unclear whether this region serves as the sole and/or principal subfamily recognition domain for Eag and Erg. Here we aim to ascertain the structural basis underlying the subfamily specificity of ether-à-go-go channels by generating various chimeric constructs between rat Eag1 and human Erg subunits. Biochemical and electrophysiological characterizations of the subunit interaction properties of a series of different chimeric and truncation constructs over the C terminus suggested that the putative C-terminal recognition domain is dispensable for subfamily-specific assembly. Further chimeric analyses over the N terminus revealed that the N-terminal region may also harbor a subfamily recognition domain. Importantly, exchanging either the N-terminal or the C-terminal domain alone led to a virtual loss of the intersubfamily assembly boundary. By contrast, simultaneously swapping both recognition domains resulted in a reversal of subfamily specificity. Our observations are consistent with the notion that both the N-terminal and the C-terminal recognition domains are required to sustain the subfamily-specific assembly of rat Eag1 and human Erg.     

Crystal structure of the RluD pseudouridine synthase catalytic module, an enzyme that modifies 23S rRNA and is essential for normal cell growth of Escherichia coli

Pseudouridine (5-beta-D-ribofuranosyluracil, Psi) is the most commonly found modified base in RNA. Conversion of uridine to Psi is performed enzymatically in both prokaryotes and eukaryotes by pseudouridine synthases (EC 4.2.1.70). The Escherichia coli Psi-synthase RluD modifies uridine to Psi at positions 1911, 1915 and 1917 within 23S rRNA. RluD also possesses a second function related to proper assembly of the 50S ribosomal subunit that is independent of Psi-synthesis. Here, we report the crystal structure of the catalytic module of RluD (residues 68-326; DeltaRluD) refined at 1.8A to a final R-factor of 21.8% (R(free)=24.3%). DeltaRluD is a monomeric enzyme having an overall mixed alpha/beta fold. The DeltaRluD molecule consists of two subdomains, a catalytic subdomain and C-terminal subdomain with the RNA-binding cleft formed by loops extending from the catalytic sub-domain. The catalytic sub-domain of DeltaRluD has a similar fold as in TruA, TruB and RsuA, with the location of the RNA-binding cleft, active-site and conserved, catalytic Asp residue superposing in all four structures. Superposition of the crystal structure of TruB bound to a T-stem loop with RluD reveals that similar RNA-protein interactions for the flipped-out uridine base would exist in both structures, implying that base-flipping is necessary for catalysis. This observation also implies that the specificity determinants for site-specific RNA-binding and recognition likely reside in parts of RluD beyond the active site.     

Residues Asn214, Gln211, Glu219 and Gln221 contained in the subfamily 3 catalytic signature of the isocitrate lyase from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> are involved in its catalytic and thermal properties

Isocitrate lyase, encoded by the aceA gene, plays an important role in the ability of Pseudomonas aeruginosa to grow on fatty acids, acetate, acyclic terpenes, and amino acids. Phylogenetic analysis indicated that the ICL superfamily is divided in two families: the ICL family, which includes five subfamilies, and the 2-methylisocitrate lyase (MICL) family. ICL from P. aeruginosa (ICL-Pa) was identified in a different ICL node (subfamily 3) than other Pseudomonas ICL enzymes (grouped in subfamily 1). Analysis also showed that psychrophilic bacteria are mainly grouped in ICL subfamily 3, whose ICL proteins contain the highly conserved catalytic pattern Q_IEN_QV_SDE_KQ_CGHQD. We performed site-directed mutagenesis, enzymatic activity, and structure modeling of conserved residues in mutated ICLs by using ICL-Pa as a model. Our results indicated that the N214 residue is essential for catalytic function, while mutating the Q211, E219, and Q221 residues impairs its catalytic and thermostability properties. Our findings suggest that conserved residues in the subfamily 3 signature of ICL-Pa play important roles in catalysis and thermostability and are likely associated with the catalytic loop structural conformation.     

Crystal structure and assembly of the functional Nanoarchaeum equitans tRNA splicing endonuclease

The RNA splicing and processing endonuclease from Nanoarchaeum equitans (NEQ) belongs to the recently identified (αβ)₂ family of splicing endonucleases that require two different subunits for splicing activity. N. equitans splicing endonuclease comprises the catalytic subunit (NEQ205) and the structural subunit (NEQ261). Here, we report the crystal structure of the functional NEQ enzyme at 2.1 Å containing both subunits, as well as that of the NEQ261 subunit alone at 2.2 Å. The functional enzyme resembles previously known α₂ and α₄ endonucleases but forms a heterotetramer: a dimer of two heterodimers of the catalytic subunit (NEQ205) and the structural subunit (NEQ261). Surprisingly, NEQ261 alone forms a homodimer, similar to the previously known homodimer of the catalytic subunit. The homodimers of isolated subunits are inhibitory to heterodimerization as illustrated by a covalently linked catalytic homodimer that had no RNA cleavage activity upon mixing with the structural subunit. Detailed structural comparison reveals a more favorable hetero- than homodimerization interface, thereby suggesting a possible regulation mechanism of enzyme assembly through available subunits. Finally, the uniquely flexible active site of the NEQ endonuclease provides a possible explanation for its broader substrate specificity.     

Structural characterization of the catalytic subunit of a novel RNA splicing endonuclease

The RNA splicing endonuclease is responsible for recognition and excision of nuclear tRNA and all archaeal introns. Despite the conserved RNA cleavage chemistry and a similar enzyme assembly, currently known splicing endonuclease families have limited RNA specificity. Different from previously characterized splicing endonucleases in Archaea, the splicing endonuclease from archaeum Sulfolobus solfataricus was found to contain two different subunits and accept a broader range of substrates. Here, we report a crystal structure of the catalytic subunit of the S.solfataricus endonuclease at 3.1 angstroms resolution. The structure, together with analytical ultracentrifugation analysis, identifies the catalytic subunit as an inactive but stable homodimer, thus suggesting the possibility of two modes of functional assembly for the active enzyme.     

Higher-order structure in the 3′-terminal domain VI of the 23 S ribosomal RNAs from Escherichia coli and Bacillus stearothermophilus

An experimental approach was used to determine, and compare, the higher-order structure within domain VI of the 23 S ribosomal RNAs from Escherichia coli and Bacillus stearothermophilus. This domain, which encompasses approximately 300 nucleotides at the 3′ end of the RNAs, consists of two large subdomains. The 5′ subdomain has been conserved during evolution and appears to be functionally important for the binding of the EF-1 · GTP · aminoacyl-tRNA complex in eukaryotes. The 3′ subdomain has diverged widely between eubacteria and eukaryotes and has produced the 4.5 S RNA in the chloroplast ribosomes of flowering plants.The structure of domain VI within the eubacterial RNAs was probed with chemical reagents in order to establish the degree of stacking and/or accessibility of each adenosine, cytidine and guanosine residue; the double-helical segments were localized with the cobra venom ribonuclease from Naja naja oxiana, and the relatively unstructured and accessible sequences were detected with the single-strand-specific ribonucleases A, T₁ and T₂. The data enabled the three secondary structural models, proposed for the E. coli 23 S RNAs, to be examined critically and it was concluded that many of their structural features are correct. Various differences between the models were considered and evidence is provided for additional structuring in the RNA including the stacking of juxtaposed purines into double helices. The 5′ subdomain constitutes a compact and resistant structure whereas the 3′ subdomain is relatively accessible and contains most of the potential protein binding sites. Moreover, comparison of our results with the published results on 4.5 S RNA suggests that the latter forms essentially the same structure as the 3′ subdomain, in contrast to earlier conclusions.A high level of structural conservation has occurred throughout the RNA domain during the evolution of the Gram negative and Gram positive bacteria although the thermophile was generally more stable at base-pairs adjacent to the terminal loops.     

The human mitochondrial transport protein family: Identification and protein regions significant for transport function and substrate specificity

Protein sequence similarities and predicted structures identified 75 mitochondrial transport proteins (37 subfamilies) from among the 28,994 human RefSeq (NCBI) protein sequences. All, except two, have an E-value of less than 4e−05 with respect to the structure of the single subunit bovine ADP/ATP carrier/carboxyatractyloside complex (bAAC/CAT) (mGenThreader program). The two 30-kDa exceptions have E-values of 0.003 and 0.005. 21 have been functionally identified and belong to 14 subfamilies. A subset of subfamilies with sequence similarities for each of 12 different protein regions was identified. Many of the 12 protein regions for each tested protein yielded different size subsets. The sum of subfamilies in the 12 subsets was lowest for the phosphate transport protein (PTP) and highest for aralar 1. Transmembrane sequences are most unique. Sequence similarities are highest near the membrane center and matrix. They are highest for the region of transmembrane helices H1, H2 and connecting matrix loop 12 and smallest for transmembrane helices H3, H4 and loop 34. These sequence similarities and the predicted high similarities to the bAAC/CAT structure point to common structural/functional elements that could include subunit/subunit contact sites as they have been identified for PTP and AAC. The four residues protein segment (SerLysGlnIle) of loop 12 is the only segment projecting into the center of the funnel-like structure of the bAAC/CAT. It is present in its entirety only in the AACs and with some replacements in the large Ca2+-modulated aspartate/glutamate transporters. Other transporters have deletions and replacements in this region of loop 12. This protein segment with its central location and variation in size and composition likely contributes to the substrate specificity of the transporters.     

Functional reconstitution of a crenarchaeal splicing endonuclease in vitro

Sulfolobus tokodaii strain 7 is one of Crenarchaea whose entire genome has been sequenced. The genome sequence revealed that it possesses two open reading frames (ORFs) that are homologous to EndA, a protein responsible for splicing endonuclease activity in Archaea. Interestingly, one of the two ORFs lacks a putative catalytic amino acid residue for the nuclease activity. To investigate their functions, the two ORF products were individually expressed in Escherichia coli, partially purified, and tested for their nuclease activities in vitro. Using in vitro transcribed tRNA precursor as a substrate, we found that the two ORF products are concurrently required to cleave exon-intron junctions. Our finding implies that the splicing endonuclease for the organism is a multi-subunit complex composed of the two endA gene products.     

A single amino acid substitution in the DNA-binding domain of <Emphasis Type="Italic">Aeropyrum pernix</Emphasis> DNA ligase impairs its interaction with proliferating cell nuclear antigen

Proliferating cell nuclear antigen (PCNA) is known as a DNA sliding clamp that acts as a platform for the assembly of enzymes involved in DNA replication and repair. Previously, it was reported that a crenarchaeal PCNA formed a heterotrimeric structure, and that each PCNA subunit has distinct binding specificity to PCNA-binding proteins. Here we describe the PCNA-binding properties of a DNA ligase from the hyperthermophilic crenarchaeon Aeropyrum pernix K1. Based on our findings on the Pyrococcus furiosus DNA ligase–PCNA interaction, we predicted that the aromatic residue, Phe132, in the DNA-binding domain of A. pernix DNA ligase (ApeLig) would play a critical role in binding to A. pernix PCNA (ApePCNA). Surface plasmon resonance analyses revealed that the ApeLig F132A mutant does not interact with an immobilized subunit of ApePCNA. Furthermore, we could not detect any stimulation of the ligation activity of the ApeLig F132A protein by ApePCNA in vitro. These results indicated that the phenylalanine, which is located in our predicted PCNA-binding region in ApeLig, has a critical role for the physical and functional interaction with ApePCNA.     

Conserved Negative Charges in the N-terminal Tetramerization Domain Mediate Efficient Assembly of Kv2.1 and Kv2.1/Kv6.4 Channels

Voltage-gated potassium (Kv) channels are transmembrane tetramers of individual α-subunits. Eight different Shaker-related Kv subfamilies have been identified in which the tetramerization domain T1, located on the intracellular N terminus, facilitates and controls the assembly of both homo- and heterotetrameric channels. Only the Kv2 α-subunits are able to form heterotetramers with members of the silent Kv subfamilies (Kv5, Kv6, Kv8, and Kv9). The T1 domain contains two subdomains, A and B box, which presumably determine subfamily specificity by preventing incompatible subunits to assemble. In contrast, little is known about the involvement of the A/B linker sequence. Both Kv2 and silent Kv subfamilies contain a fully conserved and negatively charged sequence (CDD) in this linker that is lacking in the other subfamilies. Neutralizing these aspartates in Kv2.1 by mutating them to alanines did not affect the gating properties, but reduced the current density moderately. However, charge reversal arginine substitutions strongly reduced the current density of these homotetrameric mutant Kv2.1 channels and immunocytochemistry confirmed the reduced expression at the plasma membrane. Förster resonance energy transfer measurements using confocal microscopy showed that the latter was not due to impaired trafficking, but to a failure to assemble the tetramer. This was further confirmed with co-immunoprecipitation experiments. The corresponding arginine substitution in Kv6.4 prevented its heterotetrameric interaction with Kv2.1. These results indicate that these aspartates (especially the first one) in the A/B box linker of the T1 domain are required for efficient assembly of both homotetrameric Kv2.1 and heterotetrameric Kv2.1/silent Kv6.4 channels.     

Comparative Mitogenomic Analysis of Damsel Bugs Representing Three Tribes in the Family Nabidae (Insecta: Hemiptera)

Background

Nabidae, a family of predatory heteropterans, includes two subfamilies and five tribes. We previously reported the complete mitogenome of Alloeorhynchus bakeri, a representative of the tribe Prostemmatini in the subfamily Prostemmatinae. To gain a better understanding of architecture and evolution of mitogenome in Nabidae, mitogenomes of five species representing two tribes (Gorpini and Nabini) in the subfamily Nabinae were sequenced, and a comparative mitogenomic analysis of three nabid tribes in two subfamilies was carried out.

Methodology/Principal Findings

Nabid mitogenomes share a similar nucleotide composition and base bias, except for the control region, where differences are observed at the subfamily level. In addition, the pattern of codon usage is influenced by the GC content and consistent with the standard invertebrate mitochondrial genetic code and the preference for A+T-rich codons. The comparison among orthologous protein-coding genes shows that different genes have been subject to different rates of molecular evolution correlated with the GC content. The stems and anticodon loops of tRNAs are extremely conserved, and the nucleotide substitutions are largely restricted to TψC and DHU loops and extra arms, with insertion-deletion polymorphisms. Comparative analysis shows similar rates of substitution between the two rRNAs. Long non-coding regions are observed in most Gorpini and Nabini mtDNAs in-between trnI-trnQ and/or trnS2-nad1. The lone exception, Nabis apicalis, however, has lost three tRNAs. Overall, phylogenetic analysis using mitogenomic data is consistent with phylogenies constructed mainly form morphological traits.

Conclusions/Significance

This comparative mitogenomic analysis sheds light on the architecture and evolution of mitogenomes in the family Nabidae. Nucleotide diversity and mitogenomic traits are phylogenetically informative at subfamily level. Furthermore, inclusion of a broader range of samples representing various taxonomic levels is critical for the understanding of mitogenomic evolution in damsel bugs.     

Mutational and biochemical analysis of the DNA-entry nuclease EndA from Streptococcus pneumoniae

EndA is a membrane-attached surface-exposed DNA-entry nuclease previously known to be required for genetic transformation of Streptococcus pneumoniae. More recent studies have shown that the enzyme also plays an important role during the establishment of invasive infections by degrading extracellular chromatin in the form of neutrophil extracellular traps (NETs), enabling streptococci to overcome the innate immune system in mammals. As a virulence factor, EndA has become an interesting target for future drug design. Here we present the first mutational and biochemical analysis of recombinant forms of EndA produced either in a cell-free expression system or in Escherichia coli. We identify His160 and Asn191 to be essential for catalysis and Asn182 to be required for stability of EndA. The role of His160 as the putative general base in the catalytic mechanism is supported by chemical rescue of the H160A variant of EndA with imidazole added in excess. Our study paves the way for the identification and development of protein or low-molecular-weight inhibitors for EndA in future high-throughput screening assays.     

Direct appraisal of the potato tuber ADP-glucose pyrophosphorylase large subunit in enzyme function by study of a novel mutant form

The higher plant ADP-glucose pyrophosphorylase is a heterotetramer consisting of two subunit types, which have evolved at different rates from a common ancestral gene. The potato tuber small subunit (SS) displays both catalytic and regulatory properties, whereas the exact role of the large subunit (LS), which contains substrate and effector binding sites, remains unresolved. We identified a mutation, S302N, which increased the solubility of the recombinant potato tuber LS and, in turn, enabling it to form a homotetrameric structure. The LS302N homotetramer possesses very little enzyme activity at a level 100-fold less than that seen for the unactivated SS homotetramer. Unlike the SS enzyme, however, the LS302N homotetramer enzyme is neither activated by the effector 3-phosphoglycerate nor inhibited by P(i). When combined with the catalytically silenced SS, S D143N, however, the LS302N-containing enzyme shows significantly enhanced catalytic activity and restored 3-PGA activation. This unmasking of catalytic and regulatory potential of the LS is conspicuously evident when the activities of the resurrected L(K41R.T51K.S302N) homotetramer are compared with its heterotetrameric form assembled with S D143N. Overall, these results indicate that the LS possesses catalytic and regulatory properties only when assembled with SS and that the net properties of the heterotetrameric enzyme is a product of subunit synergy.