It has been well-established that Pseudomonas species possess extremely versatile metabolic systems allowing them to utilise a wide range of nutrient sources and, furthermore, that the regulation of these enzyme systems involves highly evolved and sophisticated regulatory machinery. This study examined the complexity of metabolic regulation in Pseudomonas using the hydantoin-hydrolysing system of the environmental isolate, Pseudomonas putida RU-KM3s. In this system, the genes encoding dihydropyrimidinase and β-ureidopropionase (dhp and bup) are arranged divergently on the chromosome, separated by a 616 bp intergenic region involved in the transcriptional regulation of these genes. The focus was on the transcriptional regulation of dhp expression. DHP activity was found to be sensitive to several environmental signals including growth phase, carbon catabolite repression (CCR), substrate induction and quorum sensing (QS). Bioinformatic analysis of the intergenic region upstream of dhp revealed a number of putative binding sites for transcriptional regulators, including recognition sequences for the alternate sigma factors σ54 and σ38, as well as for the global regulators Anr (for anaerobic regulator) and Vfr (for virulence factor regulator). The targeted disruption of the genes encoding the transcriptional regulators, Vfr and the major CCR protein, Crc, resulted in a partial relief from repression for the vfr- mutant under quorum sensing conditions and a general decrease in activity in the crc- mutant. This data suggested that both Vfr and Crc were involved in regulating DHP activity. Mutational analysis of the dhp promoter revealed that at least two sites were involved in regulating transcriptional activity, one which mediated activation and the other repression. These sites were designated as a putative Anr box, situated 232 bp from the start codon of dhp, and a CRP-like binding site, at a position 213 bp upstream of dhp. Taken together, this data shows the involvement of several global regulatory factors in controlling the expression of dhp. A complex synergistic model was proposed for the transcriptional regulation of dhp, involving alternate sigma factors in addition to both global and specific regulators and responding to a number of environmental signals associated with growth phase, including nutrient availability, cell density and oxygen status.

One of the major physiological responses to insulin secretion is the activation of lipogenesis in target tissues (principally fat and liver). As acetyl-CoA carboxylase (ACC) is the rate limiting enzyme in fatty acid synthesis, the mechanisms involved in the short term regulation of this enzyme represent a pertinent model system for determining elements involved in amplification of the signals produced in response to stimulation of cells with lipogenic and counter regulatory hormones. The regulation of mammalian ACC by hormones is a complex phenomenon involving interplay between allosteric and covalent mechanisms. While the effects of adrenaline and glucagon are well characterised, the mechanism of regulation by insulin has still to be defined and formed the focus for the work presented in this thesis. To study the role of phosphorylation in the response of ACC to insulin, the site-specific phosphorylation of the enzyme observed following exposure of intact cells to insulin has been reproduced in vitro. These studies for the first time describe the conditions required to achieve distribution of [³²P] in vitro among sites of acetyl-CoA carboxylase, very similar to that seen after hormone treatment of intact cells and employing endogenous polyamine-sensitive kinase(s). No corresponding increase in catalytic activity was detected following phosphorylation, in vitro, of insulin directed phosphorylation sites on purified rat liver acetyl-CoA carboxylase in these studies. Subsequently, ACC was phosphorylated by an exogenous protein kinase from maturation activated sea-star oocytes which led to high stoichiometric incorporation of ³²P into the unique site (I-site) phosphorylated in intact cells in response to insulin (0.3 mol phosphate / mol 240,000 kD subunit). Again no change in ACC activity was observed following I-site phosphorylation. The peptide containing the I-site was separated from other tryptic phosphopeptides by reverse phase HPLC and then sequenced. Phosphorylation of serine 1186 was determined to be the major phosphorylation site of ACC in response to insulin. The amino acid sequence corresponding to the peptide containing Ser 1186 is located in the putative "hinge" region of ACQ which is some 300 amino acids towards the C-terminal of the biotin binding site (Lys-784). Subsequent re-evaluation of the kinetic properties of acetyl-CoA carboxylase during purification has led to the identification of a fraction containing low Mr inhibitor(s) and an apparently novel protein activator present in rat liver. Affinity purified rat liver acetyl-CoA carboxylase can be activated 2-3 fold at physiological citrate concentrations (0.1-0.5mM) by the addition of the heat and pro tease-sensitive cytosolic protein. The ACC activator has been extensively purified (though not yet to homogeneity) from a 100,000 g supernatant fractions from rat liver extract, by a combination of ammonium sulphate precipitation, ion-exchange chromatography and gel filtration. From these results we concluded that the activator is a protein and the native molecular weight in solution is estimated to be approximately 75 kDaltons. A popular hypothesis regarding the short term regulation of ACC involves a phosphorylation-dephosphorylation mechanism resulting in inhibition and activation respectively. A number of experiments have been carried out in order to test the hypothesis that the activator preparation may contain protein phosphatase activity directed towards ACC. The results strongly suggest that under the assay conditions described for the expression of activation of catalytic activity of ACC, there is little or no apparent dephosphorylation. Indeed, the most purified preparations of ACC activator do not contain any detectable phosphatase activity towards the model substrates histone III-S and casein. The activation of ACC occurs rapidly, in a time dependent manner (within 20 min at 37°C) and involves protein-protein interaction which is antagonized by avidin. The interactions between ACC, avidin and activator protein suggest that the activator not only induces conformational change at the active site of ACC but may also bind in such a way as to be displaced (perhaps directly) by avidin. From the data presented it is concluded that this acetyl-CoA carboxylase activator protein represents a novel factor which may be involved in the short term regulation of ACC activity. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Initial velocity studies of O-acetylserine sulfhydrylase-B (OASS-B) from Salmonella typhimurium using both natural and alternative substrates suggest a Bi Bi ping pong kinetic mechanism with double substrate competitive inhibition. The ping pong mechanism is corroborated by a qualitative and quantitative analysis of product and dead-end inhibition. Product inhibition by acetate is S-parabolic noncompetitive, indication of a combination of acetate with E followed by OAS. These data suggest some randomness to the OASS-B kinetic mechanism. The pH dependence of kinetic parameters was determined in order to obtain information on the acid-base chemical mechanism for the OASS-B reaction. A mechanism is proposed in which an enzyme general base accepts a proton from α-amine of O-acetylserine, while a second enzyme general base acts by polarizing the acetyl carbonyl assisting in the β-elimination of the acetyl group of O-acetylserine. The ε-amine of the active site lysine acts as a general base to abstract the α-proton in the β-elimination of acetate. At the end of the first half reaction the ε-amine of the active site lysine that formed the internal Schiff base and the general base are protonated. The resulting α-aminoacrylate intermediate undergoes a Michael addition with HS‾ and the active site lysine donates its proton to the α-carbon to give cysteine and regenerate enzyme to start the second half reaction. In addition, substrate specificity, stereochemistry of the internal Schiff base at C4', and sequence around active site lysine of O-acetylserine sulfhydrylase-A have been determined. The [4'-^3H]pyridoxamine generated by reduction of the internal Schiff base with sodium [^3H]borohydride retained most of its tritium after incubation with apoaspartate aminotransferase. These results agree with the hypothesis put forth by Dunathan (Dunathan, 1971; Dunathan and Voet, 1974) that a single surface (Re face) of the active site PLP is accessible to solvent. The sequence around the active site lysine is AsnProSerPheSerValLysCysArg.

The genes coding for mannitol-1-phosphate dehydrogenases (mtlD) from Aspergillus parasiticus and Eschericia coli were cloned and sequenced. The two coding regions were highly homologous and the identity was 88.7% at the amino acid level and 81.6% at the nucleotide level. The two genes translate into polypeptides of equal numbers (382) of amino acids with M, of 40,880 and 41,221, respectively. The possible NAD binding sites were identified for both enzymes in the N-terminal regions according to the consensus sequence fingerprint. The C-terminal regions of both enzymes were similar in sequence to the kinase domain of human liver or -rat liver fructose-6-phosphate-2- kinase:fructose·2,6-bisphosphatase, suggesting that the C-terminal regions are involved in fructose-6-phosphate binding. This conclusion was further supported by site-specitic mutagenesis experiments near the 3’ end of the A. parasiticus gene. A modified A. parasiticus mtlD gene directed the expression, in E. coli, of an enzyme in which amino acid residues 362-369 were altered and amino acid residues 370-382 were deleted with respect to the wild type enzyme. This enzyme exhibited 15% of wild type activity with a 3-fold increase in K_m for fructose-6-phosphate. In the 5’ upstream region of the A. parasiticus mtlD gene, no sequence was found which is similar to the consensus sequences derived for either procaryotic or higher eucaryotic gene promoters; however, inverted repeats were identified, which may be important for regulation of gene expression. A sequence similar to the Shine-Dalgarno sequence was found preceding the translation start codon of the A. parasiticus mtlD gene, which is important for its expression in E. coli. In the 3’ downstream region of the A. parasiticus mtlD gene, an additional open reading frame was found, which translated into a polypeptide of 153 amino acids with M, of 17,111. This polypeptide was identified using maxicell experiments. / Ph. D.

Graduation date: 1992

Not available

Phenolic compounds are a group of organic chemicals present in the wastewaters of many synthetic industrial processes. Due to their extreme toxicity to man and animals, and deleterious impact on the environment, a range of techniques exist for the effective treatment and disposal of these pollutants. Biological degradation using microbial enzymes presents a valuable alternative to conventional wastewater treatment systems. This research was therefore initiated to investigate the polyphenol oxidase enzyme system and the feasibility of its application for effluent treatment and studies in organic solvents. The enzyme system is widely distributed in nature, with Agaricus bisporus (the common mushroom) being the best known producer. Biochemical investigations of the enzyme system were therefore carried out using this extract. A screening programme was initiated to identify microbial polyphenol oxidase producers which could be cultured in liquid media, thereby enabling the production of large quantities of enzyme in fermentation systems. Extensive growth optimization and enzyme induction and optimization studies were carried out on selected cultures. A number of good producers were isolated, namely a bacterial culture designated AECI culture no. 26, Streptomyces antibioticus, Streptomyces glaucescens and a manipulated strain, Streptomyces lividans (pIJ702). Enzyme production by Agaricus bisporus mycelia was optimized in deep-liquid culture; enzyme extracts showed high phenol removal efficiencies. Streptomyces antibioticus, Streptomyces glaucescens, Streptomyces lividans (pIJ702) and AECI culture no. 26 whole cells were also investigated for phenol-removing ability in simulated phenolic effluents. The use of whole cells reduces enzyme inactivation and instability due to the protection of the enzyme system within the cell. All cultures showed improved removal efficiencies in phenolic growth media. These results strongly suggest their use for phenol removal in continuous systems.

O-Acetylserine Sulfhydrylase (OASS) is a pyridoxal phosphate enzyme that catalyzes the reaction of O-acetyl-Lserine with sulfide to give L-cysteine. OASS is present as two isoforms, designated -A and -B. The kinetic mechanism of OASS-A is well known and there is also much known concerning the acid-base chemistry of the enzyme. However, little is known concerning the location of the rate determining steps, the sequencing of chemical steps that occur at the active site, or the nature of the rate determining transition states. The studies performed to help elucidate these aspects of the OASS-A mechanism included determination of the thermodynamics of both half reactions, along with studies utilizing substrate analogs of OAS halting the reaction at specific points along the reaction pathway allowing the identification of reaction intermediates. The free energy change of the first half reaction was shown to be -5.7 Kcal/mole while the second half reaction was shown to be, for all intents and purposes, irreversible. Intermediates along the reaction pathway that have been previously identified include the internal Schiff base and the a-aminoacrylate. The external Schiff base was identified using the analogs cysteine, alanine, and glycine while the geminal diamine was identified using the analog serine. Formation of the external aldimine was shown to be pH dependent with a pK of 8.1 ± 0.3 most likely representing a general base that accepts a proton from the a-amine of cysteine to facilitate a nucleophilic attack on C4r of the PLP imine. Formation of the geminal diamine was also shown to be pH dependent with two pK values having an average value of 8.1. One of the groups most likely represents the general base which accepts a proton from the a-amine of cysteine while the second group likely interacts with the amino acid side chain to orientate the amino acid into the correct configuration.

Abstract not available

No description available.