Comprehensive computational analysis of Hmd enzymes and paralogs in methanogenic Archaea

<p>Abstract</p> <p>Background</p> <p>Methanogenesis is the sole means of energy production in methanogenic Archaea. H₂-forming methylenetetrahydromethanopterin dehydrogenase (Hmd) catalyzes a step in the hydrogenotrophic methanogenesis pathway in class I methanogens. At least one <it>hmd </it>paralog has been identified in nine of the eleven complete genome sequences of class I hydrogenotrophic methanogens. The products of these paralog genes have thus far eluded any detailed functional characterization.</p> <p>Results</p> <p>Here we present a thorough computational analysis of Hmd enzymes and paralogs that includes state of the art phylogenetic inference, structure prediction, and functional site prediction techniques. We determine that the Hmd enzymes are phylogenetically distinct from Hmd paralogs but share a common overall structure. We predict that the active site of the Hmd enzyme is conserved as a functional site in Hmd paralogs and use this observation to propose possible molecular functions of the paralog that are consistent with previous experimental evidence. We also identify an uncharacterized site in the N-terminal domains of both proteins that is predicted by our methods to directly impart function.</p> <p>Conclusion</p> <p>This study contributes to our understanding of the evolutionary history, structural conservation, and functional roles, of the Hmd enzymes and paralogs. The results of our phylogenetic and structural analysis constitute datasets that will aid in the future study of the Hmd protein family. Our functional site predictions generate several testable hypotheses that will guide further experimental characterization of the Hmd paralog. This work also represents a novel approach to protein function prediction in which multiple computational methods are integrated to achieve a detailed characterization of proteins that are not well understood.</p

Quod erat demonstrandum? The mystery of experimental validation of apparently erroneous computational analyses of protein sequences

BACKGROUND: Computational predictions are critical for directing the experimental study of protein functions. Therefore it is paradoxical when an apparently erroneous computational prediction seems to be supported by experiment. RESULTS: We analyzed six cases where application of novel or conventional computational methods for protein sequence and structure analysis led to non-trivial predictions that were subsequently supported by direct experiments. We show that, on all six occasions, the original prediction was unjustified, and in at least three cases, an alternative, well-supported computational prediction, incompatible with the original one, could be derived. The most unusual cases involved the identification of an archaeal cysteinyl-tRNA synthetase, a dihydropteroate synthase and a thymidylate synthase, for which experimental verifications of apparently erroneous computational predictions were reported. Using sequence-profile analysis, multiple alignment and secondary-structure prediction, we have identified the unique archaeal 'cysteinyl-tRNA synthetase' as a homolog of extracellular polygalactosaminidases, and the 'dihydropteroate synthase' as a member of the beta-lactamase-like superfamily of metal-dependent hydrolases. CONCLUSIONS: In each of the analyzed cases, the original computational predictions could be refuted and, in some instances, alternative strongly supported predictions were obtained. The nature of the experimental evidence that appears to support these predictions remains an open question. Some of these experiments might signify discovery of extremely unusual forms of the respective enzymes, whereas the results of others could be due to artifacts

GeneViTo: Visualizing gene-product functional and structural features in genomic datasets

BACKGROUND: The availability of increasing amounts of sequence data from completely sequenced genomes boosts the development of new computational methods for automated genome annotation and comparative genomics. Therefore, there is a need for tools that facilitate the visualization of raw data and results produced by bioinformatics analysis, providing new means for interactive genome exploration. Visual inspection can be used as a basis to assess the quality of various analysis algorithms and to aid in-depth genomic studies. RESULTS: GeneViTo is a JAVA-based computer application that serves as a workbench for genome-wide analysis through visual interaction. The application deals with various experimental information concerning both DNA and protein sequences (derived from public sequence databases or proprietary data sources) and meta-data obtained by various prediction algorithms, classification schemes or user-defined features. Interaction with a Graphical User Interface (GUI) allows easy extraction of genomic and proteomic data referring to the sequence itself, sequence features, or general structural and functional features. Emphasis is laid on the potential comparison between annotation and prediction data in order to offer a supplement to the provided information, especially in cases of "poor" annotation, or an evaluation of available predictions. Moreover, desired information can be output in high quality JPEG image files for further elaboration and scientific use. A compilation of properly formatted GeneViTo input data for demonstration is available to interested readers for two completely sequenced prokaryotes, Chlamydia trachomatis and Methanococcus jannaschii. CONCLUSIONS: GeneViTo offers an inspectional view of genomic functional elements, concerning data stemming both from database annotation and analysis tools for an overall analysis of existing genomes. The application is compatible with Linux or Windows ME-2000-XP operating systems, provided that the appropriate Java Runtime Environment is already installed in the system

Genetic engineering and characterization of LysR-type transcriptional regulators

Thesis (Ph.D.) University of Alaska Fairbanks, 2000This thesis describes research aimed at understanding the structure and function of LysR-type transcriptional regulators. I studied two LysR-type proteins. One from the archaeon Methanococcus jannaschii, MJ-LysR. The other is from Burkholderia cepacia, DgdR. The MJ-LysR is the first putative LysR-type transcriptional regulator found in archaea. It is surprising that a prokaryotic transcriptional regulator is present in archaea, whose basal transcription machinery and RNA polymerase are more closely related to those of eukaryotes. To elucidate the structure and function of M-LysR protein, the gene was subcloned and expressed in E. coli. The gene product was isolated and purified by heat treatment and size exclusion chromatography. An in vitro binding assay showed that the purified protein bound to the intergenic region between the lysR gene and its upstream gene specifically and selectively. The results also showed that the protein maintained its binding activity even at 94C̊. The DNA footprinting data demonstrated a 30 bp protected region. Thus, this protein probably regulates expression of its own structural gene and perhaps the adjacent upstream gene. DgdR protein from Burkholderia cepacia had been previously characterized. The previous study showed that 2-methylalanine, the inducer for the DgdR regulated dgdA gene expression, but not D or L-alanine induced the conformational changes on DNA-protein complex. To further confirm this result, eleven amino acids with structures similar to 2-methylalainine were tested for their ability on affecting the binding of the DgdR protein to its operator site. Among these amino acids tested, only 2-methylalanine, 1-aminocyclopentane-1-carboxylic acid, S-2-aminobutanoic acid, RS-isovaline, and 2-trifluoromethyl-2-aminobutanoic acid generated the measurable band shifting. D- or L-norvaline, 2,2-diethyl glycine, and 2-trifluoromethylalanine did not cause any measurable change. It was concluded that both alkyl side chain size and hydrophobicity are important for the inducer recognition and binding in this protein. To solve the problem in DgdR protein purification caused by low solubility of this protein, a dgdR fusion gene to malE gene was constructed. This fusion gene provides a useful tool to further study and crystallize the DgdR protein