Recombinant DNA technologies enable the direct isolation and expression of novel genes from biotopes containing complex consortia of uncultured microorganisms. In this study, genomic libraries were constructed from microbial DNA isolated from insect intestinal tracts from the orders Isoptera (termites) and Lepidoptera (moths). Using a targeted functional assay, these environmental DNA libraries were screened for genes that encode proteins with xylanase activity. Several novel xylanase enzymes with unusual primary sequences and novel domains of unknown function were discovered. Phylogenetic analysis demonstrated remarkable distance between the sequences of these enzymes and other known xylanases. Biochemical analysis confirmed that these enzymes are true xylanases, which catalyze the hydrolysis of a variety of substituted -1,4-linked xylose oligomeric and polymeric substrates and produce unique hydrolysis products. From detailed polyacrylamide carbohydrate electrophoresis analysis of substrate cleavage patterns, the xylan polymer binding sites of these enzymes are proposed.The arthropod gut is a differentiated organ harboring a complex biotope comprising both resident and transient members from protozoal, bacterial, and archaeal genera. Many of these organisms are symbionts that contribute in a concerted way to a complex chemical cycle sustaining the metabolic competence of the host. These symbiotic relationships help to define the metabolic traits of the insect, contributing to efficient sharing of the derived nutrients. In addition to carbon metabolism, data have demonstrated that the microbial consortia contribute to nitrogen cycling, methano-and acetogenesis, and prevention of foreign microbial pathogenesis (29).A complete understanding of the complexities of the gut biotopes is lacking, however, due to the difficulties encountered in culturing the myriad of contributing microbes and in describing the physiologies of the individual species. Studies aimed at a description of microbial complexity have recently been undertaken in termites by using 16S ribosomal DNA analysis and have shown that a number of unique lineages of microorganism inhabit the hindgut (28). These data support the idea that the insect gut represents a contained biome wherein unique species and chemistries evolve.Polysaccharide hydrolysis is a key element in insect nutrition. Since the diet of most arthropods comprises primarily plant matter, digestion of the structural polysaccharides cellulose and hemicellulose is essential for energy metabolism and the ability to obtain carbon from these sources contributes materially to the success of the order. These polysaccharides are resistant to degradation, and the insects themselves do not secrete all of the digestive enzymes to hydrolyze -linkages in the polymer. Rather, much of the hydrolysis of these polysaccharides is carried out by enzymes produced by the microbial symbionts (4,5,36,41).Hemicellulose consists primarily of xylan and it is the second most abundant polymer type in plant materi...
We present a phylogenetic analysis to determine whether a given tRNA molecule was established in evolution before its cognate aminoacyl-tRNA synthetase. The earlier appearance of tRNA versus their metabolically related enzymes is a prediction of the RNA world theory, but the available synthetase and tRNA sequences previously had not allowed a formal comparison of their relative time of appearance. Using data recently obtained from the emerging genome projects, our analysis points to the extant forms of lysyl-tRNA synthetase being preceded in evolution by the establishment of the identity of lysine tRNA.The hypothesis of an RNA world postulates that selfreplicating RNA molecules preceded the use of DNA and proteins, and that this world existed before the appearance of the universal ancestor of the extant tree of life (1). The existence of an RNA world has been supported by the biochemical characterization of catalytic RNA molecules, either from contemporary metabolic pathways or after in vitro selection of RNA ribozymes (2-6). Viral RNA genomes and the role or tRNA-like structures in viral replication are also indicative of the ancestral existence of an RNA world (7). A more direct proof of an RNA world could come from the direct comparison of the evolutionary time of appearance of protein and RNA molecules involved in a universal metabolic pathway. If this analysis was possible, then the RNA world theory would predict that the moment of appearance of the RNA component would precede the appearance of the protein elements involved in the same reaction. Here we present a phylogenetic analysis that suggests that, in an RNA-protein interaction essential for the elucidation of the genetic code, the RNA molecule is ancestral to its associated enzyme.Aminoacyl-tRNA synthetases (aaRSs) evolved as two distinct classes (I and II), each containing 10 enzymes (8-14). Each aaRS is responsible for establishing the genetic code by specifically aminoacylating only its cognate tRNA isoacceptors, thereby linking an amino acid with its corresponding anticodon triplets. Because the aminoacylation of tRNA establishes the genetic code, a strong coevolution exists between the enzymes and their cognate tRNAs (15). The aminoacylation reaction precedes the first split of the tree of life, resulting in almost invariable conservation of aaRSs and their cognate tRNAs in all living organisms (16).The strict conservation of aaRS and tRNA sequences across the whole phylogenetic tree prevented the analysis of initial events in the evolution of the system, because no sequences exist from precursors of the extant aaRSs. Without this kind of sequence information, the relative age of the duplications that gave rise to the current set of aaRSs could not be calculated. Moreover, the relative time of appearance of aaRSs and tRNAs could not be analyzed, because no extant organism are known presently where earlier, simpler sets of aaRS or tRNAs are used. As a result, it has not been possible to calculate whether the final evolutionary events that...
The small size of the archaebacterial Methanococcus jannaschii tyrosyl-tRNA synthetase may give insights into the historical development of tRNAs and tRNA synthetases. The L-shaped tRNA has two major arms-the acceptor⅐TC minihelix with the amino acid attachment site and the anticodon-containing arm. The structural organization of the tRNA synthetases parallels that of tRNAs. The more ancient synthetase domain contains the active site and insertions that interact with the minihelix portion of the tRNA. A second, presumably more recent, domain interacts with the anticodon-containing section of tRNA. The small size of the M. jannaschii enzyme is due to the absence of most of the second domain, including a segment thought to bind to the anticodon. Consistent with the absence of an anticodonbinding motif, a mutation of the central base of the anticodon had a relatively small effect on the aminoacylation efficiency of the M. jannaschii enzyme. In contrast, others showed earlier that the same mutation severely reduced charging by a normal-sized bacterial enzyme that has the aforementioned anticodon-binding motif. However, the M. jannaschii enzyme has a peptide insertion into its catalytic domain. This insertion is shared with all other tyrosyl-tRNA synthetases and is needed for a critical minihelix interaction. We show that the M. jannaschii enzyme is active on minihelix substrates over a wide temperature range and has preserved the same peptide-dependent minihelix specificity seen in other tyrosine enzymes. These findings are consistent with the concept that anticodon interactions of tRNA synthetases were later adaptations to the emerging synthetase-tRNA complex that was originally framed around the minihelix.The tRNA secondary structure consists of two domains: the acceptor⅐TC stem-loop and the anticodon⅐D stem-loop ( Fig. 1) (1-3). This secondary structure forms an L-shaped tertiary fold where one domain (the acceptor⅐TC minihelix) is formed by the coaxial stacking of the 5-bp 1 TC stem onto the 7-bp acceptor helix (1, 4, 5). It contains the amino acid attachment site at the 3Ј-end. For many aminoacyl-tRNA synthetases (aaRSs), the acceptor⅐TC minihelix alone is a substrate for specific aminoacylation where sequence and structural elements in the acceptor helix confer specific recognition by an aaRS (6 -14). The second domain contains the anticodon triplet of the genetic code and is formed by the stacking of the anticodon stem onto the D stem. The anticodon domain serves as the template (mRNA) reading head.Aminoacyl-tRNA synthetases are universal proteins believed to have arisen early during the development of the genetic code. They are organized into two classes designated as class I and class .This classification is based on the sequences and structures of their active site domains. Like tRNAs, aaRSs are comprised of two major domainsϪa conserved class-defining catalytic domain and a second domain that is not conserved even for synthetases in the same class (20 -23). These two domains of the synthetase interact wit...
The soluble colicin E1 channel peptide has a roughly spherical, highly alpha-helical, compact structure. The structural unfolding properties of the colicin E1 channel peptide were analyzed using fluorescence techniques. The guanidine hydrochloride-induced unfolding pattern of the wild-type channel peptide was examined by monitoring intrinsic tryptophan fluorescence. Additionally, peptide unfolding was examined with the fluorophore, 1-anilinonaphthalene-8-sulfonic acid. In order to probe the unfolding of local segments, single-tryptophan channel peptides were constructed by site-directed mutagenesis. Shifts in fluorescence emission maxima of the single tryptophan residues were used to monitor site-specific unfolding events, in the presence of guanidine hydrochloride. The unfolding patterns reported by tryptophans in different regions of the peptide were diverse. The concentration of guanidine hydrochloride at the unfolding transition midpoint for each mutant peptide and the free energy of unfolding were calculated in order to estimate local segment stabilities. Also, secondary structure unfolding was monitored using circular dichroism spectroscopy. The results of unfolding analysis showed that the channel peptide's unfolding mechanism involves an intermediate structure stabilized by the C-terminal hydrophobic core of the peptide. Knowledge of the unfolding pattern of the soluble channel peptide will aid in the understanding of the secondary and tertiary structural interactions within the channel peptide and the mechanism of colicin E1 activation.
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