The role of specific amino acid residues in β-ketoadipate succinyl-coenzyme A transferase II from Acinetobacter calcoaceticus was investigated. A 1412 base pair BamiHI-EcoRI fragment carrying the catIJ genes was amplified by polymerase chain reaction and inserted into pUCl9 to generate the plasmid pCATEl9. Escherichia coli DH5α (pCATEl9) carrying only the catlJ genes expressed 3-fold higher enzyme activity than the parent strain. Two mutants were constructed by site directed mutagenesis so that glutamate was replaced by a glutamine at positions Gln155 and Gln193 in the ß subunit of the primary amino acid sequence of the CoA transferase. Both mutants produced transferase that was catalytically active suggesting that Glu155 and Glu193 do not participate directly in catalysis.

A soluble enzyme system, isolated from livers of biotin-deficient rats, catalyzes the ATP-dependent synthesis of propionyl holocarboxylase from d-biotin and propionyl apocarboxylase. This system has been resolved by alumina C_∂ gel fractionation into two essential components; (a) gel supernatant which contains propionyl apocarboxylase and (b) gel eluate which contains an enzyme which catalyzes the covalent bonding of d-biotin to propionyl apocarboxylase. The propionyl holocarboxylase synthesis catalyzed by these enzyme systems is irreversible and d·biotin specific. The gel supernatant has been further purified by hydroxyapatite chromatography and (NH₄)₂SO₄ fractionation and the gel eluate by (NH₄)₂SO₄ precipitation and cellulose-phosphate chromatography. An enzyme similar to the gel eluate enzyme has been isolated from cell-free extracts of Propionibacterium shermanii. Although cell-free extracts of P. shermanii do not contain propionyl apo- or holocarboxylase, they do catalyze ATP-dependent propionyl holocarboxylase formation from d·biotin and rat liver propionyl apocarboxylase. Biotin-2'-C¹⁴-labelled propionyl holocarboxylaae, synthesized with these enzyme systems, does not transfer C¹⁴O₂ to propionyl-CoA indicating that the ureido carbon of enzyme-bound biotin is not the"active carbon" of biotin. / Master of Science

<p>The 20^th century introduction of antibiotics made once fatal infectious diseases readily treatable. This taken-for-granted therapy is now threatened by rising antibiotic resistance. The ability of pathogens to acquire numerous simultaneous resistance mechanisms has given rise to an alarming number of increasingly difficult to treat multi-drug resistant infections. When coupled with a sharp decline in development of novel antibiotic therapies, health practitioners today are left with limited therapeutic options. Several alternative methodologies have been employed to find novel therapeutics, including new techniques in natural product isolation and the production of semi-synthetic and synthetic antibiotics; however, there has been limited focus on targeting antibiotic resistance mechanisms directly to create synergistic therapies. We demonstrate the potential in using small molecules to target antibiotic kinases, thereby rescuing the antibiotic action of aminoglycosides and macrolides when used in combination. We conducted a thorough examination of these enzymes including: kinetic analysis; an assessment of phosphate donor specificity; and in-depth structural comparison, including a case study on the structure-function relationship of APH(4)-Ia. This analysis culminated in an intensive screening initiative of fourteen antibiotic kinases against a set of well defined protein kinase inhibitors. From this work, we have identified several inhibitors that have the potential for use in future combination therapeutics. This study illustrates the benefit of a structure-activity based approach to drug discovery, an important tool at a time when novel therapeutic strategies are required.</p> / Doctor of Philosophy (PhD)

<p>The current rise in multi-drug resistant Gram-negative bacterial infections is of particularconcern. Gram-negative pathogens are difficult to treat due to their intrinsic resistome.The outer membrane (OM) of Gram-negative bacteria serves as a permeability barrier tomany antibiotics, due in large part to the lipopolysaccharide (LPS) component that isunique to these organisms, and in addition to, the OM is lined with a number of multidrugresistant efflux pumps. As the clinical effectiveness of first line therapies declines inthe face of this resistance, novel strategies to discover new antibiotics are required. Theidentification of new antibiotic targets is one method currently being applied to meet thischallenge. This work examines the permeability barrier of Escherichia coli as a possibletarget for antibiotic adjuvants. A structure-function analysis of GmhA and GmhB, whichcatalyze the first and third conserved steps in LPS ADP-heptose biosynthesis, wasperformed. The active site residues of each of these enzymes were identified viacrystallographic, mutagenic, and kinetic analyses. Potential mechanisms have beenproposed, offering insight into the function of these potential adjuvant targets. In addition,a whole screen of E. coli was performed to identify compounds that potentiatenovobiocin, an antibiotic with limited activity against Gram-negative pathogens due toOM permeability. Four small molecules were found that were able to synergize withnovobiocin. One of these, A22, is known to alter bacterial cell shape, suggesting a newpathway for antibiotic adjuvants to combat Gram-negative infection. Together, thesestudies highlight the varied targets available for novel therapeutic strategies.</p> / Doctor of Philosophy (PhD)

Thesis (MSc (Chemistry and Polymer Science))--University of Stellenbosch, 2006. / This work focuses on the biocatalytic production of coenzyme A (CoA) analogues with different tether lengths in its pantetheine moiety, and on analogues where the cysteamine moiety has been replaced with a range of other amines. An attempt was made to develop a simple biocatalytic method for the optimum production of such CoA analogues by chemo-enzymatic means. Pantothenic acid ethyl thioesters with different tether lengths were first synthesized as substrates of the CoA biosynthetic enzymes, CoaA, CoaD and CoaE. The acceptability of these compounds as substrates for the pantothenate kinase (CoaA) from prokaryotic and eukaryotic organisms was investigated through kinetic studies. These substrates were subsequently exposed to CoaA, CoaD and CoaE to produce various general CoA synthons (ethyl pre-CoAs). Finally aminolysis of these ethyl pre-CoAs by cysteamine and homocysteamine gave the various CoA analogues of different tether lengths in their pantetheine moiety. The identical production of a second type of CoA synthon (phenyl pre-CoA) from pantothenic acid phenyl thioesters was also investigated as a means to increase reactivity of the thioester substrates. Aminolysis of the phenyl pre-CoA produced the corresponding CoA derivative, but reactivity was lower than expected. A second strategy was also developed where the pantothenic acid phenyl thioesters were first aminolyzed, resulting in various pantothenamide intermediates. Aminolysis was attempted with thiol-bearing amines such as cysteamine and homocysteamine as well as with amines without sulfhydryl functionalities. These pantothenamide intermediates were then used in the biosynthesis of the corresponding CoA analogues by addition of CoaA, CoaD and CoaE. The ideal method of CoA analogue production will utilize a continuous bioreactor system in which these analogues can be prepared on large scale. However, to construct a bioreactor the enzymes involved need to be immobilized on a matrix in order to transform substrate to product. The enzymes CoaA, CoaD and CoaE can be immobilized on cellulose via a cellulose binding domain (CBD) affinity tag. Various types of CBDs were investigated and used in the construction of suitable expression vectors. Optimum expression conditions to obtain soluble CBD-fused enzymes were developed.

Thèse de doctorat : Sciences du vivant : Strasbourg 1 : 2007. / Titre provenant de l'écran-titre. Bibliogr. p. 227-261.

Title from title page of PDF (University of Missouri--St. Louis, viewed March 3, 2010). Vita. Includes bibliographical references (p. 109-113).

Thesis (Ph. D.)--Ohio State University, 2009. / Title from first page of PDF file. Includes vita. Includes bibliographical references (p. 143-152).

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 180-198).

This thesis is divided into three parts, united by the theme of the development of selective inhibitors of mammalian cell DNA polymerase delta (pol δ). The first part consists of an investigation of the cytotoxic mechanism(s) of certain 2-substituted adenine analogs, selected on the basis of their inhibitory properties towards DNA polymerase alpha (pol α) and mammalian cell DNA synthesis. The second is a direct search for inhibitors of isolated pol δ, and an investigation of inhibitory mechanisms. The third consists of measurement of the effects of a selective pol δ inhibitor on cellular DNA synthesis. Mechanism of Cytotoxicity of 2-substituted adenine analoqs. The mechanism of inhibition by 2-(p-n-butylanilino)-2'-deoxyadenosine (BuAdA), and related compounds, of Chinese hamster ovary (CHO) cell ([3H]thymidine [3H]TdR) incorporation, was investigated. The potency of the compound could largely be explained by its potency (IC50 = 23 μM) as an inhibitor of CHO cell [3H]TdR uptake. BuAdA inhibited incorporation by CHO cells of [32p]phosphate into DNA relatively weakly, displaying an IC50value of 80 μM. Differential inhibition of DNA polymerases alpha and delta. Known DNA polymerase inhibitors of a structurally wide range were screened for their ability to inhibit pol δ derived from calf thymus selectively with respect to pol α derived from the same tissue. Pyrophosphate (PPi) and difluoromethanediphosphonate each inhibited pol δ weakly, but with greater potency than pol α. Based on this lead, an expanded series of PPi analogs was screened. Carbonyldiphosphonate (COMDP) inhibited pol δ with a potency (Ki = 1.8 μM) twenty-two times greater than that displayed for pol α. Kinetic studies indicated that COMDP inhibited pol δ competitively with the dNTP specified by the template, but not competitively with the template:primer. Analogous experiments with pol α showed that the compound inhibited that enzyme uncompetitively with the dNTP, and not competitively with the template:primer. COMDP was a weak inhibitor of the 3' → 5' exonuclease activity of pol δ, displaying an IC50value greater than 1 mM. Inhibition of permeabilized cell DNA synthesis bv a selective pol δ inhibitor. The potency of COMDP as an inhibitor of permeabilized CHO cell DNA synthesis (IC50= 200 μM) did not clearly indicate the participation of pol δ in cellular DNA replication. Prospectus. The thesis concludes with a prospectus for the development of pol δ inhibitors with improved properties compared to COMDP.