Browsing by Author "Dharmarajan, Lakshmi"

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    The complete genome sequence of Staphylothermus marinus reveals differences in sulfur metabolism among heterotrophic Crenarchaeota
    Anderson, Iain J.; Dharmarajan, Lakshmi; Rodriguez, Jason; Hooper, Sean; Porat, Iris; Ulrich, Luke E.; Elkins, James G.; Mavromatis, Kostas; Sun, Hui; Land, Miriam; Lapidus, Alla; Lucas, Susan; Barry, Kerrie W.; Huber, Harald; Zhulin, Igor B.; Whitman, William B.; Mukhopadhyay, Biswarup; Woese, Carl; Bristow, James; Kyrpides, Nikos C. (2009-04-02)
    Background Staphylothermus marinus is an anaerobic, sulfur-reducing peptide fermenter of the archaeal phylum Crenarchaeota. It is the third heterotrophic, obligate sulfur reducing crenarchaeote to be sequenced and provides an opportunity for comparative analysis of the three genomes. Results The 1.57 Mbp genome of the hyperthermophilic crenarchaeote Staphylothermus marinus has been completely sequenced. The main energy generating pathways likely involve 2-oxoacid:ferredoxin oxidoreductases and ADP-forming acetyl-CoA synthases. S. marinus possesses several enzymes not present in other crenarchaeotes including a sodium ion-translocating decarboxylase likely to be involved in amino acid degradation. S. marinus lacks sulfur-reducing enzymes present in the other two sulfur-reducing crenarchaeotes that have been sequenced - Thermofilum pendens and Hyperthermus butylicus. Instead it has three operons similar to the mbh and mbx operons of Pyrococcus furiosus, which may play a role in sulfur reduction and/or hydrogen production. The two marine organisms, S. marinus and H. butylicus, possess more sodium-dependent transporters than T. pendens and use symporters for potassium uptake while T. pendens uses an ATP-dependent potassium transporter. T. pendens has adapted to a nutrient-rich environment while H. butylicus is adapted to a nutrient-poor environment, and S. marinus lies between these two extremes. Conclusion The three heterotrophic sulfur-reducing crenarchaeotes have adapted to their habitats, terrestrial vs. marine, via their transporter content, and they have also adapted to environments with differing levels of nutrients. Despite the fact that they all use sulfur as an electron acceptor, they are likely to have different pathways for sulfur reduction.
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    Genomic characterization of methanomicrobiales reveals three classes of methanogens
    Anderson, Iain J.; Ulrich, Luke E.; Lupa, Boguslaw; Susanti, Dwi; Porat, Iris; Hooper, Sean D.; Lykidi, Athanasios; Sieprawska-Lupa, Magdalena; Dharmarajan, Lakshmi; Goltsman, Eugene; Lapidus, Alla; Saunders, Elizabeth; Han, Cliff; Land,Miriam; Lucas, Susan; Mukhopadhyay, Biswariup; Whitman, William B.; Woese, Carl; Bristow, James; Kyrpides, Nikos (Public Library of Science, 2009-06-04)
    Background: Methanomicrobiales is the least studied order of methanogens. While these organisms appear to be more closely related to the Methanosarcinales in ribosomal-based phylogenetic analyses, they are metabolically more similar to Class I methanogens. Methodology/Principal Findings: In order to improve our understanding of this lineage, we have completely sequenced the genomes of two members of this order, Methanocorpusculum labreanum Z and Methanoculleus marisnigri JR1, and compared them with the genome of a third, Methanospirillum hungatei JF-1. Similar to Class I methanogens, Methanomicrobiales use a partial reductive citric acid cycle for 2-oxoglutarate biosynthesis, and they have the Eha energy-converting hydrogenase. In common with Methanosarcinales, Methanomicrobiales possess the Ech hydrogenase and at least some of them may couple formylmethanofuran formation and heterodisulfide reduction to transmembrane ion gradients. Uniquely, M. labreanum and M. hungatei contain hydrogenases similar to the Pyrococcus furiosus Mbh hydrogenase, and all three Methanomicrobiales have anti-sigma factor and anti-anti-sigma factor regulatory proteins not found in other methanogens. Phylogenetic analysis based on seven core proteins of methanogenesis and cofactor biosynthesis places the Methanomicrobiales equidistant from Class I methanogens and Methanosarcinales. Conclusions/Significance: Our results indicate that Methanomicrobiales, rather than being similar to Class I methanogens or Methanomicrobiales, share some features of both and have some unique properties. We find that there are three distinct classes of methanogens: the Class I methanogens, the Methanomicrobiales (Class II), and the Methanosarcinales (Class III).
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    Structure-Function Studies on Two Phosphoenolpyruvate Carboxylases
    Dharmarajan, Lakshmi (Virginia Tech, 2010-10-29)
    Phosphoenolpyruvate carboxykinase (PEPCK) and phosphoenolpyruvate carboxylase (Pepc) are two important CO₂-fixation enzymes which share a similar reaction mechanism. Both operate through a lid-gated active site and have a hypothesized enol-pyruvate intermediate in their catalytic pathway. While PEPCK is an important metabolic enzyme in animals and plays a broad role in cataplerosis, gluconeogenesis and glyceroneogenesis, Pepc reaction in plants catalyzes the first committed step in CO₂ fixation in CAM and C₄ plants via Rubisco. We are studying the structure-function aspects of both enzymes, with a goal of discovering new elements in these enzymes which can modulate catalysis. We have undertaken an interdisciplinary approach for this work and have shown that a combination of experimental and computational techniques can be complementary and can provide novel information. We have determined that in human PEPCK, Tyr235 forms an anion-quadrupole interaction with the carboxylate of PEP and thus positions the latter with respect to the enzyme-bound Mn²+ for optimal phosphoryl transfer and catalysis. We have also identified Pro82 as a catalytically influential residue in this enzyme. Using molecular dynamics simulations we have noted that absence of ligands induces active-site lid opening in GTP-PEPCKS and we have made the first observation of the intermediary structures of the lid opening event, the dynamics of which is an important element that controls GTP-PEPCK catalysis. We have determined the first three-dimensional crystal structure of an archaeal-type Pepc, i.e. C. perfringens PepcA. Our experimental data also provide information about the oligomerization of PepcAs and reveal that aspartate inhibits the C. perfringens enzyme competitively compared to the allosteric inhibition in Pepcs. Structure-based modeling has led to the identification of putative aspartate- and bicarbonate-binding residues in C. perfringens PepcA, of which Arg82, His11, Ser201, Arg390, Lys340, Arg342 and Arg344 probably play an important role.