Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 111-137).

Thesis (Ph.D.) - University of Queensland, 2003. / Includes bibliography.

The work described in this thesis attempts to analyse the accumulation and degradation of cytochrome P-450 in Saccharomyces cerevisiae. The effect of environmental parameters such as oxygen and constituents of the growth medium have been examined here in an attempt to understand the mechanism underlying the accumulation and degradation of this enzyme. The highest level of S. cerevisiae cytochrome P-450 accumulation was recorded with a new strain NCYC 754 obtained from NCYC 240 and first investigated here. Cytochrome P-450 was found to accumulate during growth of S. cerevisiae only at high glucose concentrations under conditions of mitochondrial repression. It was found that in non-growing yeast a 100 ml 8% glucose (w/v) solution would enhance cytochrome P-450 accumulation. Scale-up of this effect in a 5 l bioreactor was attempted. In experiments on the removal of oxygen during the exponential growth of S. cerevisiae there was found to be a decline in cytochrome P-450 accumulation in which case it is suggested that oxygen may be acting as a substrate inducer of yeast cytochrome P-450. Culture shake speed was also used to control oxygen availability. An optimum shake speed was found which allowed the greatest rate of cytochrome P-450 accumulation, it was also found that the same shake speed caused the greatest rate of degradation of the enzyme during stationary phase. It was also discovered that semi-anaerobic conditions caused less degradation than aerobic conditions. The agents chloramphenicol, dinitrophenol and cycloheximide offered less protection against degradation than semi-anaerobic conditions. Ethanol was found to induce cytochrome P-450 in S. cerevisiae under conditions where cytochrome P-450 is not normally detectable. Added alkanols, other than ethanol, cause rapid degradation of cytochrome P-450 in non-growing yeast.

This thesis is largely concerned with the study of a cytochrome P-450 enzyme mimic consisting of an Iron porphyrin and iodosylbenzene. Chapter 1 consists of a review of the work that has been published on the above and other related metalloporphyrin biomimetic systems. The contents bear witness to the relatively recent interest in these particular enzyme mimics. A kinetic investigation forms the substance of the second chapter. The effect of structural and electronic changes on the iron porphyrin catalyst and the iodosylbenzene oxidant are measured in terms of their effect on the rate of cyclohexene oxidation. Other variables, such as the structure of the substrate and the nature of the solvent system, lead to a proposed mechanism. In Chapter 3 the iron porphyrin system is applied to 15,16-dihydrocyclopenta[a] phenanthren-17-one and its carcinogenic 11-methyl homologue. An attempt is made to selectively oxidise the terminal rings of the cyclopenta[a] phenanthrenes by Incorporating sterically bulky groups around the periphery of the iron porphyrin. The chemically active K-region remained the target for the majority of the iron porphyrins used, however some porphyrin substituents played an active role in encouraging the approach of the cyclopenta[a] phenanthrene substrate. Chemical modelling studies of the active site of the iron porphyrins and the question of steric hindrance as it relates to the approach of the cyclopenta[a] phenanthrenes are reported in Chapter 4.

Spectrophotometric studies have been carried out to determine the pH dependence of binding of SKF 525 A, Brietal sodium and carbon monoxide to cytochrome P-450. The optimal pH for metabolic conversion of SKF 525 A has been investigated and this agent and its major metabolite, SKF 8742 A, have been metabolised in vitro by swine and rat hepatic microsomes. A suitable gas liquid chromatography assay has been developed and used to analyse metabolic production. The effects of carbon monoxide, dithiothreitol, n-octylamine and of induction of cytochrome P-450 by phenobarbital on metabolism of SKF 525 A and SKF 8742 A have been investigated. Attempts have been made to synthesise SKF 525 AN-oxide. / KMBT_363 / Adobe Acrobat 9.54 Paper Capture Plug-in

Le cytochrome p450 CYP102A1, mieux connu sous le nom de BM3, provient de la bactérie Bacillus megaterium. Cette enzyme possède un groupement prosthétique hémique lui permettant de catalyser l’insertion d’oxygène dans un lien carbone-hydrogène menant généralement à une hydroxylation du substrat, ce qui en fait une monooxygénase. Ce genre de réaction demeure jusqu’à aujourd’hui difficile à effectuer par chimie traditionnelle ce qui confère un intérêt particulier à cette enzyme. Au contraire des autres cytochromes p450, BM3 est soluble (et non membranaire) et est naturellement fusionnée à son partenaire réductase formant ainsi une seule chaîne polypeptidique. Ainsi, au cours des dernières années, BM3 a attiré beaucoup d’attention de la part de l’industrie de la chimie fine et pharmaceutique due à son potentiel biocatalytique important. Cependant, son usage en industrie est restreint par son instabilité ainsi que par le coût prohibitif du cofacteur qui lui est nécessaire pour catalyser ses réactions, le NADPH. Cette thèse décrit le développement de différentes stratégies visant à libérer les réactions effectuées avec BM3 de leur dépendance au NADPH, tout en maximisant le rendement spécifique de la monooxygénase. En place du NADPH, deux autres cofacteurs de moindre coût furent utilisés comme alternative, soit le NADH et le N-benzyle-1,4- dihydronicotinamide (NBAH) en utilisant le mutant R966D/W1046S de BM3. Afin de maximiser le rendement spécifique de BM3, l’une des stratégies de cette thèse, l’optimisation du milieu réactionnel, repose sur deux éléments clés, soit favoriser la stabilité du cofacteur, car celui-ci est plus instable que l’enzyme elle-même, ainsi que d’abaisser au minimum la température de la réaction, car nous avons constaté que ceci avait pour effet d’augmenter le couplage entre les réactions réductase et monooxygénase et donc la stabilité de l’enzyme. L’effet net de la réaction ainsi optimisée fut d’augmenter le rendement spécifique du mutant R966D/W1046S par un facteur situé entre 2 et 2.6 en fonction du cofacteur utilisé. D’autre part, deux stratégies d’ingénierie enzymatique furent explorées afin de générer des mutations pouvant augmenter la performance de BM3. L’une d’entre elles, la mutagenèse par consensus guidé, généra une librairie de mutants de laquelle les mutants NTD5 et NTD6 furent identifiés, augmentant le rendement spécifique de l’enzyme comparativement à leur parent, R966D/W1046S, par un facteur de 5.2 et 2.3 pour le NBAH et le NADH, respectivement. L’autre stratégie explorée fut d’appliquer une pression sélective sur la bactérie Bacillus megaterium pour forcer, par évolution expérimentale, la performance de l’enzyme. De cette stratégie, un nouveau mutant de BM3 nommé DE, possédant 34 acides aminés substitués sur sa séquence, fut généré. Ce dernier a démontré une plus forte résistance aux solvants organiques ainsi qu’une augmentation de son rendement spécifique vis-à-vis le NADPH et le NADH d’un facteur de 1.23 et 1.76, comparativement à BM3 sauvage, respectivement. Les stratégies décrites dans cette thèse présentent une amélioration significative du rendement spécifique de BM3 ainsi que deux iii nouvelles méthodologies avec lesquelles une enzyme peut être optimisée et de nouvelles mutations bénéfiques identifiées. / The p450 cytochrome CYP102A1, better known as BM3, comes from the bacteria Bacillus megaterium. This enzyme possesses a prosthetic heme group enabling it to catalyze the insertion of oxygen into a carbon-hydrogen bond generally resulting in the hydroxylation of the substrate, the enzyme is therefore a monooxygenase. This type of reaction remains difficult to achieve by traditional chemistry. Unlike other p450 cytochromes, BM3 is soluble (is not membrane bound) and is naturally fused to its reductase partner forming a single polypeptide chain. As such, in recent years, BM3 has garnered much attention from the pharmaceutical and fine chemical industries, due to its high biocatalytic potential. However, its use in industry remains constrained by its instability as well as by the prohibitive cost of its cofactor, NADPH. This thesis describes the development of different strategies aiming at liberating reactions driven with BM3 from their dependence to NADPH whilst maximizing the specific yield of the monooxygenase. Instead of NADPH, two other inexpensive cofactors were used, namely NADH and N-benzyl-1,4-dihydronicotinamide (NBAH) by using the BM3 mutant R966D/W1046S. To maximize BM3 specific yield, one of the strategies used in this thesis work, the optimization of the reaction medium, rested on two key elements. Firstly, favouring the stabilization of the cofactor, as it was found to be more unstable than the enzyme itself and secondly lowering the reaction temperature as this effectively augmented oxidase/reductase reactions coupling and as such the stability of the enzyme. The net effect of the optimized reaction was to enhance the specific yield of the BM3 mutant R966D/W1046S by a factor of 2 and 2,6 depending on which cofactor was used. Two other enzymatic engineering strategies were explored to generate mutations which could enhance the performance of BM3. One of these, consensus guided mutagenesis, generated a library of mutants from which mutants NTD5 and NTD6 were identified enhancing the specific yield of the enzyme comparatively to their parent, R966D/W1046S, by a factor of 5,24 and 2,3 for NBAH and NADH respectively. The other strategy explored was to apply a selective pressure on Bacillus megaterium to force, by experimental evolution, the performance of the enzyme. From this strategy, a new mutant of BM3 called DE, possessing 34 new amino acid substitutions, was generated. This new mutant displayed a greater resistance to organic solvents as well as an augmentation of specific yields when used alongside NADPH and NADH comparatively to wild type BM3 by a factor of 1,23 and 1,76 respectively. The strategies described in this thesis allowed a significative enhancement of BM3 specific yield as well as represent two new methodologies by which new beneficial mutations can be identified.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 99-107).

Thesis (M.S.)--West Virginia University, 2004 / Title from document title page. Document formatted into pages; contains ix, 49 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 41-48).

Thesis (Ph. D.)--West Virginia University, 2005. / Title from document title page. Document formatted into pages; contains viii, 141 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Dissertation (PhD)--Stellenbosch University, 2005. / ENGLISH ABSTRACT: This study: 1. Compares various heterologous expression systems for high-level expression of cytochromes P450. Limitations of the existing cytochromes P450 expression systems are discussed and possibilities to improve the expression yields of human steroidogenic enzymes, are suggested. In addition the potential applications of human steroidogenic cytochromes P450 expressed in Pichia pastoris are illustrated. 2. Describes the cloning and extracellular expression of a recombinant full-length human cytochrome P450 17a-hydroxylase (p45017a) enzyme in Saccharomyces cerevisiae. After the optimisation of expression conditions, it was shown that this system is not suitable for the expression of full-length human P45017a. 3. Describes the cloning and extracellular expression of the full-length human cytochromes P45017a, aromatase, bs and truncated human cytochrome P45017a in P. pastoris. The limitations using P. pastoris as an export system for expressed P450 enzymes were pointed out. 4. Describes the cloning and intracellular expression of the full-length human cytochrome P45017a in P. pastoris as well as the functional expression of human P45017a in P. pastoris, showing progesterone conversion to 17ahydroxyprogesterone and 16a-hydroxyprogesterone in vivo, for the first time. 5. Evaluates developed methods for the preparation of mierosomes from P. pastoris expressing human P45017a and the spectral characterisation of detergent solubilised human P45017a. 6. Describes the development of protocols for the purification of human P45017a from P. pastoris microsomes. / AFRIKAANSE OPSOMMING: Hierdie ondersoek: 1. Vergelyk verskillende heterologiese proteïen uitdrukkings-sisteme vir die preperatiewe produksie van sitochrome P450. Die tekortkomings van bestaande sitochroom P450-uitdrukkings-sisteme word bespreek en moontlikhede om die opbrengs van menslike steroïedogeniese ensieme te verbeter word voorgestel. Die potensiële aanwendings van menslike steroïedogeniese sitochrome P450, wat in Pichia pas/oris uitgedruk word, word ook geïllustreer. 2. Beskryf die klonering en ekstrasellulêre uitdrukking van die rekombinante vollengte menslike sitochroom P45017a-hidroksilase (P45017a) ensiem in Saccharomyces cerevisiae. Na optimisering van die kondisies vir die uitdrukking kon aangetoon word dat hierdie sisteem nie geskik is vir die uitdruk en sekresie van vollengte menslike P45017a nie. 3. Beskryf die klonering en ekstrasellulêre uitdrukking van die vollengte menslike sitochrome P45017a, aromatase, b5 en verkorte menslike sitochroom P45017a in P. pas/oris. Die beperkinge van P. pas/oris as 'n uitvoersisteem vir die uitdrukking van P450 ensieme word bespreek. 4. Besryf die klonering en intrasellulêre ekspressie van die vollengte menslike sitochroom P450 17a. Die funksionele ekspressie van menslike sitochroom P450 17a in P. pas/oris is vir die eerste keer gekarakteriseer. 5. Evalueer die ontwikkelde metodes vir die voorbereiding van mikrosome van P. pas/oris wat menslike P45017a uitdruk en karakteriseer die detergent opgelosbare menslike P45017a t.o.v. spektroskopiese eienskappe. 6. Beskryf die ontwikkelling van protokolle vir die suiwering van die uitgedrukte menslike P45017a vanuit P. pas/oris mikrosome.