Clostridium tetanin todennäköisesti aiheuttama jäykkäkouristus nautaeläimellä : kaksi tapausselostusta

Summary: Bovine tetany probably caused by Clostridium tetani - two case reports

Clostridium perfringens -bakteeri kuokkavieraana pitopöydässä

Summary: Clostridium perfringens, an uninvited visitor of a banquet

Klostridien ja Clostridium perfringensin esiintyminen terveen hevosen ulostenäytteessä sisä- ja ulkoruokintakaudella : menetelmävertailu

Summary: Occurence of clostridia and Clostridium perfringens in the faecal samples of healthy horses during indoor and outdoor feeding seasons : comparison of methods

The Structural Basis for inorganic Pyrophosphatase Catalysis and Regulation

Inorganic pyrophosphatases (PPases) are essential enzymes for every living cell. PPases provide the necessary thermodynamic pull for many biosynthetic reactions by hydrolyzing pyrophosphate. There are two types of PPases: integral membrane-bound and soluble enzymes. The latter type is divided into two non-homologous protein families, I and II. Family I PPases are present in all kingdoms of life, whereas family II PPases are only found in prokaryotes, including archae. Family I PPases, particularly that from Saccharomyces cerevisiae, are among the most extensively characterized phosphoryl transfer enzymes. In the present study, we have solved the structures of wild-type and seven active site variants of S. cerevisiae PPase bound to its natural metal cofactor, magnesium ion. These structures have facilitated derivation of the complete enzyme reaction scheme for PPase, fulfilling structures of all the reaction intermediates. The main focus in this study was on a novel subfamily of family II PPases (CBSPPase) containing a large insert formed by two CBS domains and a DRTGG domain within the catalytic domain. The CBS domain (named after cystathionine beta-synthase in which it was initially identified) usually occurs as tandem pairs with two or four copies in many proteins in all kingdoms of life. The structure formed by a pair of CBS domains is also known as a Bateman domain. CBS domains function as regulatory units, with adenylate ligands as the main effectors. The DRTGG domain (designated based on its most conserved residues) occurs less frequently and only in prokaryotes. Often, the domain co-exists with CBS domains, but its function remains unknown. The key objective of the current study was to explore the structural rearrangements in the CBS domains induced by regulatory adenylate ligands and their functional consequences. Two CBS-PPases were investigated, one from Clostridium perfringens (cpCBS-PPase) containing both CBS and DRTGG domains in its regulatory region and the other from Moorella thermoacetica (mt CBS-PPase) lacking the DRTGG domain. We additionally constructed a separate regulatory region of cpCBS-PPase (cpCBS). Both full-length enzymes and cpCBS formed homodimers. Two structures of the regulatory region of cpCBS-PPase complexed with the inhibitor, AMP, and activator, diadenosine tetraphosphate, were solved. The structures were significantly different, providing information on the structural pathway from bound adenylates to the interface between the regulatory and catalytic parts. To our knowledge, these are the first reported structures of a regulated CBS enzyme, which reveal large conformational changes upon regulator binding. The activator-bound structure was more open, consistent with the different thermostabilities of the activator- and inhibitor-bound forms of cpCBS-PPase. The results of the functional studies on wild-type and variant CBS-PPases provide support for inferences made on the basis of structural analyses. Moreover, these findings indicate that CBS-PPase activity is highly sensitive to adenine nucleotide distribution between AMP, ADP and ATP, and hence to the energy level of the cell. CBS-PPase activity is markedly inhibited at low energy levels, allowing PPi energy to be used for cell survival instead of being converted into heat.

Functional studies on bacterial nucleotide-regulated inorganic pyrophosphatases

CBS domains are ~60 amino acid tandemly repeated regulatory modules forming a widely distributed domain superfamily. Found in thousands of proteins from all kingdoms of life, CBS domains have adopted a variety of functions during evolution, one of which is regulation of enzyme activity through binding of adenylate-containing compounds in a hydrophobic cavity. Mutations in human CBS domain-containing proteins cause hereditary diseases. Inorganic pyrophosphatases (PPases) are ubiquitous enzymes, which pull pyrophosphate (PPi) producing reactions forward by hydrolyzing PPi into phosphate. Of the two nonhomologous soluble PPases, dimeric family II PPases, belonging to the DHH family of phosphoesterases, require a transition metal and magnesium for maximal activity. A quarter of the almost 500 family II PPases, found in bacteria and archaea, contain a 120-250 amino acid N-terminal insertion, comprised of two CBS domains separated in sequence by a DRTGG domain. These enzymes are thus named CBS-PPases. The function of the DRTGG domain in proteins is unknown. The aim of this PhD thesis was to elucidate the structural and functional differences of CBS-PPases in comparison to family II PPases lacking the regulatory insert. To this end, we expressed, purified and characterized the CBS-PPases from Clostridium perfringens (cpCBS-PPase) and Moorella thermoacetica (mtCBS-PPase), the latter lacking a DRTGG domain. Both enzymes are homodimers in solution and display maximal activity against PPi in the presence of Co2+ and Mg2+. Uniquely, the DRTGG domain was found to enable tripolyphosphate hydrolysis at rates similar to that of PPi. Additionally, we found that AMP and ADP inhibit, while ATP and AP4A activate CBSPPases, thus enabling regulation in response to changes in cellular energy status. We then observed substrate- and nucleotide-induced conformational transitions in mtCBS-PPase and found that the enzyme exists in two differentially active conformations, interconverted through substrate binding and resulting in a 2.5-fold enzyme activation. AMP binding was shown to produce an alternate conformation, which is reached through a different pathway than the substrate-induced conformation. We solved the structure of the regulatory insert from cpCBS-PPase in complex with AMP and AP4A and proposed that conformational changes in the loops connecting the catalytic and regulatory domains enable activity regulation. We examined the effects of mutations in the CBS domains of mtCBS-PPase on catalytic activity, as well as, nucleotide binding and inhibition.

Advanced biofuel production: Engineering metabolic pathways for butanol and propane biosynthesis.

Greenhouse gases emitted from energy production and transportation are dramatically changing the climate of Planet Earth. As a consequence, global warming is affecting the living conditions of numerous plant and animal species, including ours. Thus the development of sustainable and renewable liquid fuels is an essential global challenge in order to combat the climate change. In the past decades many technologies have been developed as alternatives to currently used petroleum fuels, such as bioethanol and biodiesel. However, even with gradually increasing production, the market penetration of these first generation biofuels is still relatively small compared to fossil fuels. Researchers have long ago realized that there is a need for advanced biofuels with improved physical and chemical properties compared to bioethanol and with biomass raw materials not competing with food production. Several target molecules have been identified as potential fuel candidates, such as alkanes, fatty acids, long carbon‐chain alcohols and isoprenoids. The current study focuses on the biosynthesis of butanol and propane as possible biofuels. The scope of this research was to investigate novel heterologous metabolic pathways and to identify bottlenecks for alcohol and alkane generation using Escherichia coli as a model host microorganism. The first theme of the work studied the pathways generating butyraldehyde, the common denominator for butanol and propane biosynthesis. Two ways of generating butyraldehyde were described, one via the bacterial fatty acid elongation machinery and the other via partial overexpression of the acetone‐butanol‐ethanol fermentation pathway found in Clostridium acetobutylicum. The second theme of the experimental work studied the reduction of butyraldehyde to butanol catalysed by various bacterial aldehyde‐reductase enzymes, whereas the final part of the work investigated the in vivo kinetics of the cyanobacterial aldehyde deformylating oxygenase (ADO) for the generation of hydrocarbons. The results showed that the novel butanol pathway, based on fatty acid biosynthesis consisting of an acyl‐ACP thioesterase and a carboxylic acid reductase, is tolerant to oxygen, thus being an efficient alternative to the previous Clostridial pathways. It was also shown that butanol can be produced from acetyl‐CoA using acetoacetyl CoA synthase (NphT7) or acetyl‐CoA acetyltransferase (AtoB) enzymes. The study also demonstrated, for the first time, that bacterial biosynthesis of propane is possible. The efficiency of the system is clearly limited by the poor kinetic properties of the ADO enzyme, and for proper function in vivo, the catalytic machinery requires a coupled electron relay system.