An exploration of phosphorylases for the synthesis of carbohydrate polymers

Phosphorylases are interesting enzymes with regard to both their role in metabolism and their use in the in vitro synthesis of carbohydrates. The disaccharide phosphorylases have attracted attention because of their strict stereo- and regiospecificity and their tractability. The polymerising phosphorylases have received less attention due to heterogeneous product formation, requiring more complex analyses. In this work three polymerising carbohydrate phosphorylases have been studied. The plant α-1,4-glucan phosphorylase PHS2 is closely related to the well characterised mammalian glycogen phosphorylase. We present the first crystal structures of the plant enzyme which reveals a unique surface binding site. PHS2 allowed the production of novel starch like surface, both in two and three dimensions, which show some of the same properties as a native starch granule. This can now be used to study starch-active enzymes on an insoluble glucan surface which is analogous to the native starch granule. The bacterial β-1,4-glucan phosphorylase CDP is involved in degradation of cellulose. In the reverse direction this enzyme allows the rapid synthesis of cellulose polymers in solution and also allows the synthesis of hemicellulose-like materials. The substrate specificity can in part be probed in the crystal structure presented here, which represents the first structure of a polymerising, inverting phosphorylase. Together these data provide the foundation for further work with this enzyme in the synthesis of plant cell wall related glycans. The third enzyme studied was the β-1,3-glucan from the unsequenced alga Euglena gracilis, which was used for the facile enzymatic synthesis of β-glucosyl glycerols. In order to identify the sequence of this enzyme we obtained de novo transcriptome sequencing data from this alga, which has revealed unexpected metabolic diversity. Aside from complex carbohydrate metabolism, there are also many surprising features, including novel enzyme architectures, antioxidants only previously noted in human parasites and complex natural product synthases

Aerospace medicine and biology: A continuing bibliography with indexes (supplement 400)

This bibliography lists 397 reports, articles and other documents introduced into the NASA Scientific and Technical Information System during April 1995. Subject coverage includes: aerospace medicine and physiology, life support systems and man/system technology, protective clothing, exobiology and extraterrestrial life, planetary biology, and flight crew behavior and performance

Origin of the cell nucleus, mitosis and sex: roles of intracellular coevolution

BACKGROUND: The transition from prokaryotes to eukaryotes was the most radical change in cell organisation since life began, with the largest ever burst of gene duplication and novelty. According to the coevolutionary theory of eukaryote origins, the fundamental innovations were the concerted origins of the endomembrane system and cytoskeleton, subsequently recruited to form the cell nucleus and coevolving mitotic apparatus, with numerous genetic eukaryotic novelties inevitable consequences of this compartmentation and novel DNA segregation mechanism. Physical and mutational mechanisms of origin of the nucleus are seldom considered beyond the long-standing assumption that it involved wrapping pre-existing endomembranes around chromatin. Discussions on the origin of sex typically overlook its association with protozoan entry into dormant walled cysts and the likely simultaneous coevolutionary, not sequential, origin of mitosis and meiosis. RESULTS: I elucidate nuclear and mitotic coevolution, explaining the origins of dicer and small centromeric RNAs for positionally controlling centromeric heterochromatin, and how 27 major features of the cell nucleus evolved in four logical stages, making both mechanisms and selective advantages explicit: two initial stages (origin of 30 nm chromatin fibres, enabling DNA compaction; and firmer attachment of endomembranes to heterochromatin) protected DNA and nascent RNA from shearing by novel molecular motors mediating vesicle transport, division, and cytoplasmic motility. Then octagonal nuclear pore complexes (NPCs) arguably evolved from COPII coated vesicle proteins trapped in clumps by Ran GTPase-mediated cisternal fusion that generated the fenestrated nuclear envelope, preventing lethal complete cisternal fusion, and allowing passive protein and RNA exchange. Finally, plugging NPC lumens by an FG-nucleoporin meshwork and adopting karyopherins for nucleocytoplasmic exchange conferred compartmentation advantages. These successive changes took place in naked growing cells, probably as indirect consequences of the origin of phagotrophy. The first eukaryote had 1-2 cilia and also walled resting cysts; I outline how encystation may have promoted the origin of meiotic sex. I also explain why many alternative ideas are inadequate. CONCLUSION: Nuclear pore complexes are evolutionary chimaeras of endomembrane- and mitosis-related chromatin-associated proteins. The keys to understanding eukaryogenesis are a proper phylogenetic context and understanding organelle coevolution: how innovations in one cell component caused repercussions on others. REVIEWERS: This article was reviewed by Anthony Poole, Gáspár Jékely and Eugene Koonin

Membrane bioenergetics at major transitions in evolution

This thesis presents theoretical and computational studies of four major evolutionary transitions in which cellular membranes and their embedded proteins played crucial roles: 1)The divergence of archaea and bacteria Archaea and bacteria are the basal domains of life, so it is important to understand how they came to diverge. They share several core traits, such as transcription, translation, and the genetic code. They also share the chemiosmotic exploitation of ion gradients across membranes, yet they do not share the membranes themselves. Notably, the phospholipid backbone is sn-glycerol-1-phosphate in archaea but the enantiomer sn-glycerol-3-phosphate in bacteria. The synthesising enzymes are unrelated. I used mathematical modelling to propose an explanation for this divergence in the context of natural proton gradients in alkaline hydrothermal vents, plausible scenarios for an autotrophic origin of life. Results show that early membranes had to be leaky, so both pumping and glycerol-phosphate backbones (which drastically decrease permeability) evolved later, and independently, in archaea and bacteria. 2)The evolution of Homochirality The “dual homochirality” of lipids suggests that the stereospecificity of bioorganic catalysis itself, not prebiotic physics or chemistry, is behind the origin of handedness in life’s molecules (e.g. L-amino acids and D-sugars). 3)The evolution of membrane proteins I report that membrane proteins are less shared across the tree of life. Faster evolution of outside-facing regions and true gene losses point to a common cause: as cells adapt to new environments selective pressure is stronger on the outside, while the inside, subject to strong homeostasis, evolves more slowly. 4)The bacterial nature of eukaryotic membranes Eukaryotes arose from a merger of a bacterium into an archaeon, so the first eukaryote must have had an archaeal plasma membrane and bacterial (proto)mitochondrial membranes; yet all modern eukaryotes have exclusively bacterial membranes. I suggest that archaeal membranes were lost and bacterial ones kept because of the bioenergetic adaptation of mitochondrial proteins to the bacterial membrane

DNA typing of the human small intestinal protozoan parasite Giardia lamblia

PhDAt present there is no satisfactory means of typing strains of Giardia lamblia which can explain the broad range of clinical symptoms seen in giardiasis or which can identify genotypes in epidemiological studies. This thesis attempts to address these problems by developing DNA based typing systems sensitive enough to be able to identify many different Giardia genotypes and which may be applied to the organisms found in clinical samples. Four different techniques were assessed for their ability to identify multiple polymorphic loci in the Giardia genome which may be used to genotype and identify isolates of Giardia and upon which the future development of PCR-typing protocols may be based. These techniques included RFLP analysis, random amplified polymorphic DNA (RAPD) analysis, M13 DNA fingerprinting and minisatellite DNA fingerprinting. Minisatellite DNA fingerprinting proved to be the most discriminatory, recognising many hypervariable loci within the Giardia genome which proved useful for in vitro studies on genotypic heterogeneity within Giardia isolates. This approach would require further development in order to be used on in vivo infections where it could directly assess the relationship between genotype and pathogenicity. Therefore the variable repeats recognised on Giardia fingerprints were sought by constructing and screening a Giardia genomic DNA cosmid library. Once cloned these repeats would form the basis of sensitive and specific PCR-based fingerprinting protocols ideal for typing large numbers of infections. The repeat sequences cloned in this way turned out to be Giardia variable surface protein genes with short, imperfect tandem repeats in their 3' flanking DNA. This work has important implications for the future development and use of fingerprinting techniques on Giardia and may be useful in the study of chromosome rearrangement in Giardia which is likely to be involved in surface antigen switching

Mycobacterial fatty acid metabolism: identification of novel drug targets and chemotherapeutics.

Tuberculosis has been a deadly human pathogen for thousands of years and is as prevalent and lethal now as it was in the pre-antimicrobials era. With new challenges continually being presented in the form of multidrug resistant strains evolving and the implications of the HIV epidemic, it is imperative that every effort is made to understand the causative agent, Mycobacterium tuberculosis, and develop new effective and affordable drugs to treat the disease. With this in mind, the first part of this project tests novel drugs that have been identified using different approaches. The desired targets for all the compounds were the fatty acid and mycolic acid biosynthesis systems in M. tuberculosis, Mycobacterium bovis BCG and Mycobacterium smegmatis. Some promising compounds were identified, which inhibited enzymes of the prokaryotic FAS-II system, whilst not affecting the mammalian FAS-I system. As well as identifying new drugs, it is equally important to recognise the essential genes of M. tuberculosis, which could be novel drug targets. Whilst the fatty acid biosynthesis pathway has been well studied, a lot less is known about fatty acid degradation. M. tuberculosis has an abundance of fad genes, yet it is only recently that they have started to be explored. Here, the functions and roles of the fadB genes in M. tuberculosis, M. bovis BCG and M. smegmatis have been explored. By producing purified recombinant protein and generating gene deletion mutants, it has been possible to fully characterise Mt-FadB2 and provide preliminary information regarding fadB3, fadB4 and fadB5