36 research outputs found
Experimental validation of in silico model-predicted isocitrate dehydrogenase and phosphomannose isomerase from Dehalococcoides mccartyi
Gene sequences annotated as proteins of unknown or non-specific function and hypothetical proteins account for a large fraction of most genomes. In the strictly anaerobic and organohalide respiring Dehalococcoides mccartyi, this lack of annotation plagues almost half the genome. Using a combination of bioinformatics analyses and genome-wide metabolic modelling, new or more specific annotations were proposed for about 80 of these poorly annotated genes in previous investigations of D. mccartyi metabolism. Herein, we report the experimental validation of the proposed reannotations for two such genes (KB1_0495 and KB1_0553) from D. mccartyi strains in the KB-1 community. KB1_0495 or DmIDH was originally annotated as an NAD[superscript +]-dependent isocitrate dehydrogenase, but biochemical assays revealed its activity primarily with NADP[superscript +] as a cofactor. KB1_0553, also denoted as DmPMI, was originally annotated as a hypothetical protein/sugar isomerase domain protein. We previously proposed that it was a bifunctional phosphoglucose isomerase/phosphomannose isomerase, but only phosphomannose isomerase activity was identified and confirmed experimentally. Further bioinformatics analyses of these two protein sequences suggest their affiliation to potentially novel enzyme families within their respective larger enzyme super families.University of TorontoNatural Sciences and Engineering Research Council of CanadaGenome Canada (Firm) (Ontario Genomics Institute 2009-OGI-ABC-1405)United States. Dept. of Defense. Strategic Environmental Research and Development Progra
Experimental validation of in silico model-predicted isocitrate dehydrogenase and phosphomannose isomerase from Dehalococcoides mccartyi
Gene sequences annotated as proteins of unknown or non-specific function and hypothetical proteins account for a large fraction of most genomes. In the strictly anaerobic and organohalide respiring Dehalococcoides mccartyi, this lack of annotation plagues almost half the genome. Using a combination of bioinformatics analyses and genome-wide metabolic modelling, new or more specific annotations were proposed for about 80 of these poorly annotated genes in previous investigations of D. mccartyi metabolism. Herein, we report the experimental validation of the proposed reannotations for two such genes (KB1_0495 and KB1_0553) from D. mccartyi strains in the KB-1 community. KB1_0495 or DmIDH was originally annotated as an NAD+-dependent isocitrate dehydrogenase, but biochemical assays revealed its activity primarily with NADP+ as a cofactor. KB1_0553, also denoted as DmPMI, was originally annotated as a hypothetical protein/sugar isomerase domain protein. We previously proposed that it was a bifunctional phosphoglucose isomerase/phosphomannose isomerase, but only phosphomannose isomerase activity was identified and confirmed experimentally. Further bioinformatics analyses of these two protein sequences suggest their affiliation to potentially novel enzyme families within their respective larger enzyme super families
Biochemical and structural insights into enzymatic depolymerization of polylactic acid and other polyesters by microbial carboxylesterases
Polylactic
acid (PLA) is a biodegradable polyester derived from
renewable resources, which is a leading candidate for the replacement
of traditional petroleum-based polymers. Since the global production
of PLA is quickly growing, there is an urgent need for the development
of efficient recycling technologies, which will produce lactic acid
instead of CO<sub>2</sub> as the final product. After screening 90
purified microbial α/β-hydrolases, we identified hydrolytic
activity against emulsified PLA in two uncharacterized proteins, ABO2449
from <i>Alcanivorax borkumensis</i> and RPA1511 from <i>Rhodopseudomonas palustris</i>. Both enzymes were also active
against emulsified polycaprolactone and other polyesters as well as
against soluble α-naphthyl and <i>p</i>-nitrophenyl
monoesters. In addition, both ABO2449 and RPA1511 catalyzed complete
or extensive hydrolysis of solid PLA with the production of lactic
acid monomers, dimers, and larger oligomers as products. The crystal
structure of RPA1511 was determined at 2.2 Å resolution and revealed
a classical α/β-hydrolase fold with a wide-open active
site containing a molecule of polyethylene glycol bound near the catalytic
triad Ser114-His270-Asp242. Site-directed mutagenesis of both proteins
demonstrated that the catalytic triad residues are important for the
hydrolysis of both monoester and polyester substrates. We also identified
several residues in RPA1511 (Gln172, Leu212, Met215, Trp218, and Leu220)
and ABO2449 (Phe38 and Leu152), which were not essential for activity
against soluble monoesters but were found to be critical for the hydrolysis
of PLA. Our results indicate that microbial carboxyl esterases can
efficiently hydrolyze various polyesters making them attractive biocatalysts
for plastics depolymerization and recycling
Screening and Characterization of Novel Polyesterases from Environmental Metagenomes with High Hydrolytic Activity against Synthetic Polyesters
International audienc
Activity screening of environmental metagenomic libraries reveals novel carboxylesterase families
Metagenomics has made accessible an enormous reserve of global biochemical diversity. To tap into this vast resource of novel enzymes, we have screened over one million clones from metagenome DNA libraries derived from sixteen different environments for carboxylesterase activity and identified 714 positive hits. We have validated the esterase activity of 80 selected genes, which belong to 17 different protein families including unknown and cyclase-like proteins. Three metagenomic enzymes exhibited lipase activity, and seven proteins showed polyester depolymerization activity against polylactic acid and polycaprolactone. Detailed biochemical characterization of four new enzymes revealed their substrate preference, whereas their catalytic residues were identified using site-directed mutagenesis. The crystal structure of the metal-ion dependent esterase MGS0169 from the amidohydrolase superfamily revealed a novel active site with a bound unknown ligand. Thus, activity-centered metagenomics has revealed diverse enzymes and novel families of microbial carboxylesterases, whose activity could not have been predicted using bioinformatics tools
The bacterial stressosome:a modular system that has been adapted to control secondary messenger signaling
SummaryThe stressosome complex regulates downstream effectors in response to environmental signals. In Bacillus subtilis, it activates the alternative sigma factor σB, leading to the upregulation of the general stress regulon. Herein, we characterize a stressosome-regulated biochemical pathway in Moorella thermoacetica. We show that the presumed sensor, MtR, and the scaffold, MtS, form a pseudo-icosahedral structure like that observed in B. subtilis. The N-terminal domain of MtR is structurally homologous to B. subtilis RsbR, despite low sequence identity. The affinity of the switch kinase, MtT, for MtS decreases following MtS phosphorylation and not because of structural reorganization. Dephosphorylation of MtS by the PP2C type phosphatase MtX permits the switch kinase to rebind the stressosome to reset the response. We also show that MtT regulates cyclic di-GMP biosynthesis through inhibition of a GG(D/E)EF-type diguanylate cyclase, demonstrating that secondary messenger levels are regulated by the stressosome
Dimerisation induced formation of the active site and the identification of three metal sites in EAL-phosphodiesterases
The bacterial second messenger cyclic di-3′,5′-guanosine monophosphate (c-di-GMP) is a key regulator of bacterial motility and virulence. As high levels of c-di-GMP are associated with the biofilm lifestyle, c-di-GMP hydrolysing phosphodiesterases (PDEs) have been identified as key targets to aid development of novel strategies to treat chronic infection by exploiting biofilm dispersal. We have studied the EAL signature motif-containing phosphodiesterase domains from the Pseudomonas aeruginosa proteins PA3825 (PA3825EAL) and PA1727 (MucREAL). Different dimerisation interfaces allow us to identify interface independent principles of enzyme regulation. Unlike previously characterised two-metal binding EAL-phosphodiesterases, PA3825EAL in complex with pGpG provides a model for a third metal site. The third metal is positioned to stabilise the negative charge of the 5′-phosphate, and thus three metals could be required for catalysis in analogy to other nucleases. This newly uncovered variation in metal coordination may provide a further level of bacterial PDE regulation
Structural Basis for c-di-GMP-Mediated Inside-Out Signaling Controlling Periplasmic Proteolysis
X-ray crystallographic structural analyses of the bacterial transmembrane receptor LapD identify conserved molecular mechanisms that control biofilm formation in response to changes in the intracellular levels of the second messenger c-di-GMP
The COMBREX Project: Design, Methodology, and Initial Results
© 2013 Brian P. et al.Prior to the “genomic era,” when the acquisition of DNA sequence involved significant labor and expense, the sequencing of genes was strongly linked to the experimental characterization of their products. Sequencing at that time directly resulted from the need to understand an experimentally determined phenotype or biochemical activity. Now that DNA sequencing has become orders of magnitude faster and less expensive, focus has shifted to sequencing entire genomes. Since biochemistry and genetics have not, by and large, enjoyed the same improvement of scale, public sequence repositories now predominantly contain putative protein sequences for which there is no direct experimental evidence of function. Computational approaches attempt to leverage evidence associated with the ever-smaller fraction of experimentally analyzed proteins to predict function for these putative proteins. Maximizing our understanding of function over the universe of proteins in toto requires not only robust computational methods of inference but also a judicious allocation of experimental resources, focusing on proteins whose experimental characterization will maximize the number and accuracy of follow-on predictions.COMBREX is funded by a GO grant from the National Institute of General Medical Sciences (NIGMS) (1RC2GM092602-01).Peer Reviewe