No description available.

No description available.

Abstract
 Acetyl-CoA
carboxylase
(ACC)
is
a
key
enzyme
of
fatty
acid
metabolism
with
multiple
 isozymes
often
expressed
in
different
eukaryotic
cellular
compartments.

 In
agriculture,
inhibitors
of
plastid
ACC
are
used
as
efficient
herbicides
against
grass
 weed.
However,
grass
weed
populations
resistant
to
aryloxyphenoxypropionate
(APP)
and
 cyclohexanedione
(CHD)
herbicides
represent
a
major
problem
for
sustainable
agriculture.
 Using
PCR
and
sequencing
it
was
found
out
that
five
amino
acid
substitutions
in
plastid
ACC
 were
 correlated
 with
 herbicide
 resistance
 of
 Avena
 sterilis
 ssp.
 ludoviciana
 Durieu
 populations
from
the
northern
grain-growing
region
of
Australia:
Trp-1,999-Cys,
Trp-2,027- Cys,
 Ile-2,041-Asn,
 Asp-2,078-Gly
 and
 Gly-2,096-Ala.
 We
 showed,
 using
 a
 yeast
 gene- replacement
 system,
 that
 these
 single-site
 mutations
 also
 confer
 herbicide
 resistance
 to
 wheat
plastid
ACCase:
Asp-2,078-Gly
confers
resistance
to
APPs
and
CHDs,
Trp-2,027-Cys
 and
Ile-2,041-Asn
confer
resistance
to
APPs,
and
Trp-1,999-Cys
confers
resistance
only
to
 fenoxaprop.
These
mutations
are
very
likely
to
confer
resistance
to
any
grass
weed
species
 under
selection
imposed
by
the
extensive
agricultural
use
of
the
herbicides.

...

Reductive acetogenesis via the acetyl-CoA pathway is a hydrogenotrophic pathway that has the potential to reduce methanogenesis from ruminant livestock. However our understanding of the organisms capable of this transformation (acetogens) is hindered by a lack of specific molecular tools for this group. In the present thesis, a PCR primer set specific for a wide range of acetogens was developed, targeting the acetyl-CoA synthase (ACS) gene which is unique to the acetyl-CoA pathway. ACS was found to be useful marker for potential acetogens and ACS sequences could be used to infer family-level phylogeny for many acetogens. ACS gene specific primers were used in combination with existing molecular tools targeting the gene encoding formyltetrahydrofolate synthetase (FTHFS, present in the acetyl-CoA pathway but not unique to it) and 16S rRNA genes, as well as cultivation techniques, to investigate acetogen diversity in the rumen and two analogous gut systems where microbial hydrogenotrophy differs: the forestomach of a native Australian marsupial, the tammar wallaby Macropus eugenii; and the developing rumen of young lambs. Novel potential acetogens present naturally in the rumen of pasture fed and grain fed cattle affiliated with the Ruminococcaceae/Blautia group and distantly with the Lachnospiraceae. A large diversity of potential acetogens with functional genes affiliating broadly between the Lachnospiraceae and Clostridiaceae though without a close sequence from a cultured relative were also detected. Rumen acetogen enrichment cultures revealed the presence of a known acetogen, Eubacterium limosum, in grain fed cattle, as well as novel acetogens affiliating with the Lachnospiraceae and Ruminococcaceae/Blautia group. The novel potential acetogen population detected in this study may represent an important hydrogenotrophic group in the rumen that we understand very little about and that requires further investigation. The tammar wallaby, which exhibits foregut fermentation analogous to that of the rumen but resulting in lower methane emissions, housed a different acetogen population to that of the bovine rumen (LIBSHUFF, p <0.0001) though novel potential acetogens in the tammar wallaby forestomach affiliated broadly in the same family groups (Blautia group, Lachnospiraceae and between Lachnospiraceae and Clostridiaceae without a close cultured isolate). Acetogen enrichment cultures from the tammar wallaby forestomach facilitated isolation of a novel acetogen, which was closely related to potent reductive acetogens from kangaroos. The differences between the acetogen population of the tammar wallaby forestomach and the bovine rumen may be a factor in explaining lower methane emissions and methanogen numbers in tammar wallabies relative to ruminants. Using a gnotobiotically reared lamb model, the unique acetogen population present in the developing rumen was identified and it’s response to methanogen colonisation examined. The acetogen E. limosum and potential acetogen Ruminococcus obeum were identified as well as a small diversity of novel potential acetogens affiliating with the Blautia group and the Lachnospiraceae. A small but diverse population of naturally resident methanogens were also identified in gnotobiotically reared lambs that had been isolated at 17 hours of age. After inoculation with Methanobrevibacter sp. 87.7, methanogen numbers in gnotobiotically reared lambs significantly increased but acetogen diversity was not altered, indicating that this population is resilient to methanogen colonisation to some degree. The potential acetogen population in gnotobiotically reared lambs was significantly different (LIBSHUFF, p < 0.0001) to that in conventionally reared sheep, which indicates that factors other than methanogen establishment alone, probably relating to other microbes and associated hydrogen concentrations in the rumen, affect acetogens during rumen development.

<p>Acetyl-CoA holds a prominent position as the common metabolic intermediate of glucose, amino acid and fatty acid oxidation. Because acetyl-CoA fuels the tricarboxylic acid (TCA) cycle, the primary source of reducing equivalents that drives mitochondrial oxidative phosphorylation, understanding acetyl-CoA pool regulation becomes imperative to understanding mitochondrial energetics. Carnitine acetyltransferase (CrAT), a muscle-enriched mitochondrial enzyme, catalyzes the freely reversible conversion of acetyl-CoA to its membrane permeant carnitine ester, acetylcarnitine. Because CrAT has long been thought to regulate the acetyl-CoA metabolite pool, we investigated the role of CrAT in acetyl-CoA regulation. Although the biochemistry and enzymology of the CrAT reaction has been well studied, its physiological role remains unknown. Investigations herein suggest that CrAT-mediated maintenance of the mitochondrial acetyl-CoA pool is imperative for preservation of energy homeostasis. We provide compelling evidence that CrAT is critical for fine-tuning acetyl-CoA balance during the fasted to fed transition and during exercise. These studies suggest that compromised CrAT activity results in derangements in mitochondrial homeostasis.</p><p>In chapter 3, we examined the effects of obesity and lipid exposure on CrAT activity. Recent studies have shown that acetyl-CoA-mediated inhibition of pyruvate dehydrogenase (PDH), the committed step in glucose oxidation, is modulated by the CrAT enzyme. Because PDH and glucose oxidation are negatively regulated by high fat feeding and obesity, we reasoned that nutritional conditions that promote lipid availability and fat oxidation might likewise compromise CrAT activity. We report an accumulation of long chain acylcarnitines and acyl-CoAs but a decline in the acetylcarnitine/acetyl-CoA ratio in obese and diabetic rodents. This reduction in the skeletal muscle acetylcarnitine/acetyl-CoA ratio was accompanied by a decrease in CrAT specific activity, despite increased protein abundance. Exposure to long chain acyl-CoAs in vitro demonstrated that palmitoyl-CoA acts as a mixed model inhibitor of CrAT. Furthermore, primary human skeletal muscle myocytes exposed to fatty acid and or CPT1b overexpression had elevated long chain acylcarnitines but decreased production and efflux of CrAT-derived short chain acylcarnitines. These data suggest that exposure to fatty acids in obesity and diabetes can counter-regulate the CrAT enzyme leading to decreased activity. </p><p>Alternatively, chapter 4 addresses the importance of acetyl-CoA buffering during exercise and suggests that a deficit in CrAT activity leads to fatigue. Because CrAT is highly expressed in tissues specifically designed for work and because acetylcarnitine, the primary product of the CrAT reaction, is increased during contraction, we reasoned that CrAT could play an important role in exercise. To investigate this possibility, we employed exercise intervention and ex-vivo analysis on a genetically novel mouse model of skeletal muscle CrAT deficiency (CrATSM-/-). Though resting acetyl-CoA levels were elevated in CrATSM-/- mice, these levels dropped significantly after intense exercise while acetylcarnitine content followed the opposite pattern. This contraction-induced acetyl-CoA deficit in CrATSM-/- mice was coupled with compromised performance and diminished whole body glucose oxidation during high intensity exercise. These results imply that working muscles clear and consume acetylcarnitine in order to maintain acetyl-CoA buffering during exercise. Importantly, provision of acetylcarnitine enhanced force generation, delayed fatigue and improved mitochondrial energetics in muscles from CrATfl/fl controls but not CrATSM-/- littermates, emphasizing the importance of acetyl-CoA maintenance. In aggregate, these data demonstrate a critical role for CrAT-mediated acetyl-CoA buffering in exercise tolerance and suggest its involvement in energy metabolism during skeletal muscle contraction and fatigue. These findings could have important clinical implications for individuals with muscle weakness and fatigue due to multiple conditions, such as peripheral vascular or cardiometabolic disease. </p><p>In summary, data herein emphasize the role of CrAT in regulation of mitochondrial acetyl-CoA pool. We demonstrate that CrAT is critical for fine-tuning acetyl-CoA balance both during the fasted to fed transition and during exercise. These data suggest that a deficit in CrAT activity leads to glucose intolerance and exercise fatigue. We examine these studies and suggest future areas of study.</p> / Dissertation

Zahlreiche strikt anaerob lebende Mikroorganismen, darunter acetogene Bakterien, Sulfatreduzierer und methanogene Archaeen, nutzen den reduktiven Acetyl-CoA-Weg zur autotrophen Kohlenstoff-Fixierung oder Energiegewinnung. Die letzten Schritte der Acetyl-CoA-Bildung beruhen hierbei auf dem Zusammenspiel dreier Proteine, dem Corrinoid-Eisen/Schwefel-Protein (CoFeSP), der Methyltetrahydrofolat:CoFeSP-Methyltransferase (MeTr) und dem Acetyl-CoA-Synthase/CO-Dehydrogenase-Komplex (ACS/CODH). In der vorliegenden Arbeit wurde die Substratbindung an MeTr durch thermodynamische und kinetische Messungen untersucht. MeTHF bindet stärker an das Enzym als das demethylierte Produkt Tetrahydrofolat (THF) und scheint dabei einem einstufigen Bindungsmodell zu folgen. Das Substrat wird bei der Bindung an MeTr protoniert, wobei Asn200 eine protonierte H-N5(+)-CH3-Position des MeTHF durch eine alternative Konformation stabilisieren könnte. Asp44 und Asp76 bilden eine funktionelle Dyade bei der Substratbindung, kommen als Protondonoren zur Substrataktivierung jedoch nicht in Frage. Die Kristallstruktur von CoFeSP wurde erstmals vollständig mit der flexiblen N-terminalen [4Fe4S]-Cluster-Bindedomäne bestimmt. Die für die Cobalamin-Bindedomäne erwarteten Konformationsänderungen wurden anhand der Interaktion mit dem reduktiven Aktivator von CoFeSP (RACo) analysiert. Durch Förster-Resonanzenergietransfer wurde eine Annäherung der ortsspezifisch markierten CoFeSP-Positionen beobachtet und anhand des Fluoreszenzsignals die Kinetik der Komplexbildung mit RACo bestimmt. Durch gepulste Elektronendoppelresonanz konnte ebenfalls eine Abstandsänderung nachgewiesen werden. ACS wurde als apo-Enzym gereinigt und durch NiCl2-Rekonstitution in die aktive Form überführt. Durch die Kristallisation der C-terminalen ACS-Domäne wurden hochaufgelöste Strukturen erzeugt, welche eine Diskussion der strukturellen Details des aktiven Zentrums ermöglichen. / Several anaerobic microorganisms, including acetogenic bacteria, sulfate-reducing bacteria and methanogenic archaea operate the reductive acetyl-CoA pathway for autotrophic carbon fixation or to gain energy. The last steps of acetyl-CoA formation rely on three enzymes, the corrinoid-iron/sulfur-protein (CoFeSP), the methyltetrahydrofolate:CoFeSP methyltransferase (MeTr) and the acetyl-CoA synthase/CO dehydrogenase complex (ACS/CODH). Substrate binding to MeTr was investigated by thermodynamic and kinetic meassurements. MeTHF binds slightly stronger than the demethylated product tetrahydrofolate (THF), likely following a simple one-step-binding mechanism. Substrate binding to MeTr is coupled to proton uptake. A H-N5(+)-CH3-transition state of MeTHF could be stabilized by an alternative conformation of Asn200. Asp44 and Asp76 form a functional dyade in substrate binding but can be excluded as proton donors for substrate activation. The crystal structure of CoFeSP was solved completely, including the previously disordered N-terminal [4Fe4S]-cluster binding domain. The expected conformational change of the corrinoid binding domain was characterized by analyzing the interaction between CoFeSP and its reductive activator (RACo). An approach of the labeled CoFeSP positions in the CoFeSP:RACo complex was observed by Förster resonance energy transfer. Based on the corresponding fluorescence signal, the kinetics of complex formation were meassured in solution. Pulsed electron double resonance also showed that the labeled positions approach upon complex formation. Full-length ACS was purified in the apo state. A reconstitution of the A-cluster with NiCl2 resulted in active enzyme. Different crystal structures of the isolated C-terminal domain of ACS were solved at high resolution. Therefore, structural details of the active site could be discussed.

[N,N??-bis-(mercaptoethyl)-1,5-diazacyclooctanato]NiII, Ni-1, is known to undergo metallation reactions with numerous metals. [N,N??-bis-(mercaptoethyl)-1,5-diazacycloheptanato]NiII, (bme-dach)Ni or Ni-1??, differs from Ni-1 by one less carbon in its diazacycle backbone ring producing subtle differences in N2S2Ni geometry. Metallation of Ni-1?? with PdCl2, Pd(NO3)2, and NiBr2 produced three structural forms: Ni2Pd basket, Ni4Pd2 C4-paddlewheel, and Ni3 slant chair. In attempts to provide a rationale for the heterogeneity in the active site of Acetyl coA Synthase, metal ion capture studies of Ni-1 in methanol found a qualitative ranking of metal ion preference: Zn2+ < Ni2+ < Cu+. Formation constants for metal ion capture of Ni-1?? in water were determined for Pb2+, Ni2+, Zn2+, Cu+, and Ag+. A quantitative estimate places copper some 15 orders of magnitude above nickel or zinc in binding affinity. Sulfur dioxide uptake by Ni-1?? is characterized by significant color change, improved adduct solubility, and reversible binding of two equivalents of SO2. These combined properties establish Ni-1?? as a suitable model for gas uptake at nickel thiolate sites and as a possibly useful chemical sensor for this poisonous gas. Comparisons of molecular structures, ν(SO) stretching frequencies, and thermal gravimetric analyses are made to reported adducts including the diazacyclooctane derivative, Ni-1·2SO2. Visual SO2 detection limits of Ni-1 and Ni-1?? are established at 25 ppm and 100 ppm, respectively. Structural studies of products resulting from reaction at the nucleophilic S-sites of (bme-dach)Ni and [(bme-dach)Zn]2 included acetyl chloride and sodium iodoacetate as electrophiles are shown. The acetyl group is a natural electrophile important to the citric acid cycle. Acetylation of (bme-dach)Ni produces a five coordinate, paramagnetic species. Iodoacetate is a cysteine modification agent known to inhibit enzymatic activity. The reaction of (bme-dach)Ni and sodium iodoacetate yields a blue, six coordinate nickel complex in a N2S2O2 donor environment. The bismercaptodiazacycloheptane ligand binds lead(II) forming an unprecedented structural form of N2S2M dimers, in which Pb2+ is largely bound to sulfur in a highly distorted trigonal geometry. Its unusual structure is described in comparison to other derivatives of the bme-daco ligand. The synthesis and structural characterization of square pyramidal (bme-dach)GaCl are also given and compared to the analogous (bme-daco)GaCl.

Electronic and steric parameters for square planar NiN2S2 complexes as bidentate, S-donor ligands have been established. According to the (CO) stretching frequencies and associated computed Cotton-Kraihanzel force constants of (NiN2S2)W(CO)4 adducts, a ranking of donor abilities and a comparison with classical bidentate ligands are as follows: Ni(ema)= > { [NiN2S2]0 } > bipy phen > Ph2PCH2CH2PPh2 > Ph2PCH2PPh2. In addition, we have demonstrated that the NiN2S2 ligands are hemilabile as evidenced from CO addition to (NiN2S2)W(CO)4, which is in equilibrium with the resulting (NiN2S2)W(CO)5 species (Keq = 2.8 M-1, G = -1.4 kJ/mole at 50C). Complete NiN2S2 ligand displacement by CO-cleavage of the remaining W-S bond to form W(CO)6 was not observed, indicating that the remaining W-S bond is considerably strengthened upon ring-opening. Several new cluster compounds based on the NiN2S2 ligands bound to CuI, RhI, PdII and W0 are reported. Structural analysis of (NiN2S2)MLn complexes show a unique structural feature defined by the dihedral angle formed by the intersection of NiN2S2/WS2C2 planes; placing the NiN2S2 ligand in closer proximity to one side of the reactive metal center. This unique orientational feature of the NiN2S2 ligands in the series of bimetallic compounds contrasts with classical diphosphine or diimine ligands. The "hinge angle" ranges in value from 136 as in the (Ni-1*)W(CO)4 to 101 in the (Ni-1)Pd(CH3)(Cl) complexes. The rigidity of the SR hinge of the nickeldithiolate ligands suggests that they might be suitable for stereochemical and regioselective substrate addition to catalytically active metals such as RhI and PdII.. The structural as well as functional similarities of the acetyl CoA synthase enzyme (ACS) and a palladium-metal based industrial type catalyst led to the preparation of a [(Ni-1)Pd(CH3)]+ bimetallic complex. This complex facilitates CO and ethylene copolymerization to produce polyketone similar to conventional (diphosphine)Pd(X)2 catalysts. However, the diphosphine ligands produce more efficient catalysts as the electron-rich character of the NiN2S2 ligand favors the resting state of the catalyst, [(Ni-1)Pd(C(O)CH3)(CO)]+, over the reactive form (Ni-1)Pd(C(O)CH3)(2-C2H4)]+. An exploratory investigation with the Ni-Pd heterobimetallic showed that this complex also facilitated the C-S coupling reaction to form a thioester similar to the ACS enzyme.

[N,N??-bis-(mercaptoethyl)-1,5-diazacyclooctanato]NiII, Ni-1, is known to undergo metallation reactions with numerous metals. [N,N??-bis-(mercaptoethyl)-1,5-diazacycloheptanato]NiII, (bme-dach)Ni or Ni-1??, differs from Ni-1 by one less carbon in its diazacycle backbone ring producing subtle differences in N2S2Ni geometry. Metallation of Ni-1?? with PdCl2, Pd(NO3)2, and NiBr2 produced three structural forms: Ni2Pd basket, Ni4Pd2 C4-paddlewheel, and Ni3 slant chair. In attempts to provide a rationale for the heterogeneity in the active site of Acetyl coA Synthase, metal ion capture studies of Ni-1 in methanol found a qualitative ranking of metal ion preference: Zn2+ < Ni2+ < Cu+. Formation constants for metal ion capture of Ni-1?? in water were determined for Pb2+, Ni2+, Zn2+, Cu+, and Ag+. A quantitative estimate places copper some 15 orders of magnitude above nickel or zinc in binding affinity. Sulfur dioxide uptake by Ni-1?? is characterized by significant color change, improved adduct solubility, and reversible binding of two equivalents of SO2. These combined properties establish Ni-1?? as a suitable model for gas uptake at nickel thiolate sites and as a possibly useful chemical sensor for this poisonous gas. Comparisons of molecular structures,&#61472; &#957;(SO) stretching frequencies, and thermal gravimetric analyses are made to reported adducts including the diazacyclooctane derivative, Ni-1&#903;2SO2. Visual SO2 detection limits of Ni-1 and Ni-1?? are established at 25 ppm and 100 ppm, respectively. Structural studies of products resulting from reaction at the nucleophilic S-sites of (bme-dach)Ni and [(bme-dach)Zn]2 included acetyl chloride and sodium iodoacetate as electrophiles are shown. The acetyl group is a natural electrophile important to the citric acid cycle. Acetylation of (bme-dach)Ni produces a five coordinate, paramagnetic species. Iodoacetate is a cysteine modification agent known to inhibit enzymatic activity. The reaction of (bme-dach)Ni and sodium iodoacetate yields a blue, six coordinate nickel complex in a N2S2O2 donor environment. The bismercaptodiazacycloheptane ligand binds lead(II) forming an unprecedented structural form of N2S2M dimers, in which Pb2+ is largely bound to sulfur in a highly distorted trigonal geometry. Its unusual structure is described in comparison to other derivatives of the bme-daco ligand. The synthesis and structural characterization of square pyramidal (bme-dach)GaCl are also given and compared to the analogous (bme-daco)GaCl.

The A-cluster of acetyl-CoA synthase (ACS) catalyzes the formation of acetyl- CoA from CO, coenzyme-A, and a methyl group donated by a corrinoid iron-sulfur protein. Recent crystal structures have exhibited three different metals, Zn, Cu, and Ni, in the proximal site, which bridges a square-planar nickel site and a [Fe4S4] cubane. Contradicting reports supported both the nickel and copper containing forms as representing active enzyme. The results presented here indicate that copper is not necessary or sufficient for catalysis and that copper addition to ACS is deleterious. Several proposed mechanisms exist for the synthesis of acetyl-CoA, the two most prominent are the ‘paramagnetic’ and ‘diamagnetic’ mechanisms. The ‘diamagnetic’ mechanism proposes a two electron activation that precedes methylation to produce an EPR silent Ni2+-CH3 species. This then reacts with CO and coenzyme-A to form acetyl- CoA and regenerate the starting species. The ‘paramagnetic’ mechanism assumes a one electron activation prior to the methylation of the paramagnetic Ni1+-CO state to form an unstable Ni3+-acetyl species. This is immediately reduced by an electron shuttle. Results are presented here that no shuttle or external redox mediator is necessary for catalysis. This supports the ‘diamagnetic’ mechanism, specifically that a two-electron reductive activation is necessary and that the Ni1+-CO species is not an intermediate. The two-electron reductive activation required by the ‘diamagnetic’ mechanism results in an unknown electronic state. Two proposals have been made to describe this form of the A-cluster. The first hypothesis from Brunold et al involves a one-electron reduction of the [Fe4S4]2+ cube and a one-electron reduction of the Nip 2+. This should result in a spin-coupled state that is S = integer. The Ni0 hypothesis requires both electrons to localize on the Nip 2+ forming a zero-valent proximal nickel. Mössbauer spectroscopy has been used to probe the oxidation state and spin state of the [Fe4S4] cube in the reduced active form. No integer spin system is found and this is interpreted as supporting the Ni0 hypothesis. Additionally, spectra are presented that indicate the heterogeneous nature of the A-cluster is not caused by the occupancy of the proximal site.