• Refine Query
  • Source
  • Publication year
  • to
  • Language
  • 1
  • Tagged with
  • 2
  • 2
  • 1
  • 1
  • 1
  • 1
  • 1
  • 1
  • 1
  • 1
  • 1
  • 1
  • 1
  • 1
  • 1
  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

On the reactions of trans-3-chloroacrylic acid dehalogenase and a cis-3-chloroacrylic acid dehalogenase homologue, Cg10062 : mechanistic and evolutionary implications

Huddleston, Jamison Parker 03 September 2015 (has links)
The tautomerase superfamily (TSF) provides an excellent model system to study enzyme specificity, catalysis, and divergent evolution. trans-3-Cholroacrylic acid dehalogenase (CaaD), cis-3-chloroacrylic acid dehalogenase (cis-CaaD), and malonate semialdehyde decarboxylase (MSAD) are three TSF members that catalyze the final reactions in the degradation of the nematocide, 1,3-dichloropropene. All three enzymes have the TSF characteristic beta-alpha-beta fold and catalytic amino terminal proline (Pro-1). Both CaaD and cis-CaaD dehalogenate their respective isomers of 3-chloroacrylic acid yielding malonate semialdehyde. Subsequently, MSAD decarboxylates malonate semialdhyde resulting in acetaldehyde and CO2. Their catalytic and substrate specificities are exquisite considering they share three key and positionally conserved residues. As part of an effort to understand how such specificity evolved, a pre-steady-state kinetic analysis of CaaD was carried out. Alongside a similar study on cis-CaaD, a fluorescent mutant of CaaD was constructed that had minimal kinetic differences from the wild-type. The mutant was validated as an accurate fluorescent reporter of change in enzyme state that allowed for the reaction to be followed using stopped-flow methods. Stopped-flow fluorescence, rapid chemical quench data and ultraviolet spectroscopy were globally fit by computational simulation. The fit resulted in a kinetic mechanism for CaaD affording detailed information about the reaction, including measuring the rate of product release, the rate of chemistry, a previously unknown partially rate-limiting step associated with a conformational change, and the definition of binding constants for both products (MSA and Br-). In addition to the dehalogenation reaction, the reaction of the fluorescent mutant with a mechanism-based inhibitor, 3-bromopropiolate, was characterized. The values for the apparent rate of inhibition and potency were defined and estimates were determined for the values of the rate of chemistry and the release of bromide. The information gathered during these inhibition experiments was used to further refine the CaaD dehalogenation mechanism eliminating ambiguities present in the initial data set. Finally, the reactions of a cis-CaaD homologue, Cg10062 from Corynebacterium glutamicum were characterized. Cg10062 shares high sequence similarity (53%) and the same six critical active site residues as cis-CaaD, but Cg10062 has poor cis-CaaD activity. Moreover, Cg10062 dehalogenates both 3-chloroacrylic acid isomers. The reactions of Cg10062 with propiolate, 2-butynoate, and 2,3 butadienoate were investigated. Cg10062 functions as a hydratase/decarboxylase using propiolate generating malonate semialdehyde and acetaldehyde. Cg10062 catalyzes a hydration-dependent decarboxylation of propiolate as exogenously added malonate semialdehyde is not decarboxylated. With 2,3 butadienoate and 2-butynoate, Cg10062 functions as a hydratase and yields only acetoacetate. Mutations to the activating residues Glu114 and Tyr103 produced a range of results from a reduction in wild-type activity to a switch of activity. Possible intermediates for the hydration and decarboxylation products can be trapped as covalent adducts to Pro-1 when NaCNBH3 is incubated with certain combinations of substrate and mutant enzymes. Three mechanisms are presented to explain these findings along with the strengths and weaknesses of each mechanism in terms of being able to account for experimental observations. / text
2

Homologous Neurons and their Locomotor Functions in Nudibranch Molluscs

Newcomb, James M 04 December 2006 (has links)
These studies compare neurotransmitter localization and the behavioral functions of homologous neurons in nudibranch molluscs to determine the types of changes that might underlie the evolution of species-specific behaviors. Serotonin (5-HT) immunohistochemistry in eleven nudibranch species indicated that certain groups of 5 HT-immunoreactive neurons, such as the Cerebral Serotonergic Posterior (CeSP) cluster, are present in all species. However, the locations and numbers of many other 5 HT-immunoreactive neurons were variable. Thus, particular parts of the serotonergic system have changed during the evolution of nudibranchs. To test whether the functions of homologous neurons are phylogenetically variable, comparisons were made in species with divergent behaviors. In Tritonia diomedea, which crawls and also swims via dorsal-ventral body flexions, the CeSP cluster includes the Dorsal Swim Interneurons (DSIs). It was previously shown that the DSIs are members of the swim central pattern generator (CPG); they are rhythmically active during swimming and, along with their neurotransmitter 5-HT, are necessary and sufficient for swimming. It was also known that the DSIs excite efferent neurons used in crawling. DSI homologues, the CeSP-A neurons, were identified in six species that do not exhibit dorsal-ventral swimming. Many physiological characteristics, including excitation of putative crawling neurons were conserved, but the CeSP A neurons were not rhythmically active in any of the six species. In the lateral flexion swimmer, Melibe leonina, the CeSP-A neurons and 5-HT, were sufficient, but not necessary, for swimming. Thus, homologous neurons, and their neurotransmitter, have functionally diverged in species with different behaviors. Homologous neurons in species with similar behaviors also exhibited functional divergence. Like Melibe, Dendronotus iris is a lateral flexion swimmer. Swim interneuron 1 (Si1) is in the Melibe swim CPG. However, its putative homologue in Dendronotus, the Cerebral Posterior ipsilateral Pedal (CPiP) neuron, was not rhythmically active during swim-like motor patterns, but could initiate such a motor pattern. Together, these studies suggest that neurons have changed their functional relationships to neural circuits during the evolution of species-specific behaviors and have functionally diverged even in species that exhibit similar behaviors.

Page generated in 0.1267 seconds