The role of individual amino acid residues of the enzyme dienelactone hydrolase was investigated. Using the polymerase chain reaction (PCR), a 1.9 kbp clcD fragment was amplified and subcloned yielding a 821 bp BamHI to EcoRI clcD subclone in the plasmid pUC19. Site-specific mutants of dienelactone hydrolase were created using mismatched oligonucleotides to prime DNA synthesis. Specifically modified proteins from mutated clcD genes (Arg 81 to alanine, Tyr 85 to phenylalanine and Arg 206 to alanine), were encoded by the mutant clones. Enzyme assays showed that dienelactone hydrolase activity of the mutants Arg 81 and Arg 206 was totally abolished. The DLHase enzyme activity of mutant Tyr 85 is greatly decreased by approximately two thirds.

No description available.

No description available.

The production of enantiomerically pure amino acids is of industrial importance as they are used in the synthesis of a number of pharmaceuticals, insecticides and herbicides and biologically active peptides and hormones. A number of microorganisms have been identified which possess hydantoin hydrolysing enzymes that stereoselectively convert racemic hydantoins into anantiomerically pure amino acids. Consequently these microorganisms and their enzymes are sought after as biocatalysts for the production of amino acids. The isolation of novel hydantoin hydrolising enzymes with unique or improved biocatalytic characteristics is of importance for the development of potential biocatalysts to be used in the production of enantiomerically pure amino acids. The genes encoding an N-carbamoyl-amino acid amidohydrolase, an enzyme involved in the hydrolysis of hydantoin, was isolated by screening a genomic DNA library of Agrobacterium tumefacience RU-AE01. Nucleotide sequence analysis of the region upstream of this gene revealed a fragment of a gene encoding the hydantoinase enzyme. I this study, a DNA probe consisting of the gene encoding the N-carbamoyl amino acid amidohydrolase, on a large enough fragment of the genomic DNA library which would allow for the simultaneous isolation the hydantoinase gene located upstream. Recombinant expression of the genes encoding hydantoin hydrolysing enzymes has been used to facilitate the production and purification of these enzymes for their use as biocatalysts. Two genes (ncaR1 and ncaR2) encoding different N-carbamoyl-amino acid amidohydrolases with distinct nucleotide and deduced amino acid sequences were isolated from the genome of A, tumefaciens RU-OR. In this study, the heterologous expression of ncaR1 and ncaR2 was explored. Investigation into the optimisation of the heterologous expression of ncaR1 showed that reducing the growth temperature of the recombinant E. coli producing NcaR1 resulted in a two-fold increase in N-carbamoyl-amino acid amidohydrolase activity and solubility. Furthermore, NcaR1 was produced with a C-terminal 6xHis tag, but NcaR1-6xHis did not possess N-carbamoyl amino acid amidohydrolase activity. Furthermore, purification of NcaR-6xHis under native conditions using affinity chromatography performed, and used for the production of antibodies.

A calcium alginate bead-immobilised biocatalyst was developed utilising the D-hydantoinase and D-N-carbamoylase from a novel, mutant Agrobacterium tumefaciens strain RU-ORPN1. The growth conditions for the inducer-independent strain were optimised for production of hydantoinase and N-carbamoylase activities. Methods for the preparation of crude enzyme extracts were evaluated in terms of hydantoinase and N-carbamoylase activities produced. After comparison of the enzyme activities and stabilities in various extracts from fresh and frozen cells, sonication of frozen cells for 5 minutes was found to be the best method for the production of the enzyme extract. The optimal pH and temperature for the hydantoinase activity were pH 10 and 30°C, respectively, while pH 9 and 40°C were optimal for Ncarbamoylase activity. The hydantoinase activity was enhanced by the addition of Mg^(2+) ions to the enzyme extract and the N-carbamoylase was enhanced by the addition of Mg^(2+), Mn^(2+) or Zn^(2+) ions to the enzyme extract. The enzyme activities increased in the presence of ATP suggesting that the enzymes may be ATP-dependent. The addition of DTT and PMSF to the enzyme extract enhanced the hydantoinase activity but had no effect on the N-carbamoylase activity. The N-carbamoylase was unstable at 40°C and was almost completely inactivated after 24 hours incubation at this temperature. The hydantoinase and N-carbamoylase appeared to be insoluble. Various techniques were investigated for the solubilisation of the enzymes including various cell lysis methods, cell lysis at extremes of pH and ionic strength, addition of a reducing agent and protease inhibitors, and treatment with hydrolysing enzymes and detergents. Treatment with Triton X-100 was most effective for the solubilisation of the enzymes indicating that the enzymes were membrane-bound. Hydropathy and transmembrane prediction plots of the predicted amino acid sequences for two identified N-carbamoylase genes from A. tumefaciens RU-ORPN1 revealed possible transmembrane regions in the amino acid sequences, and thus supported the hypothesis that the enzymes were membrane-bound. Various methods were evaluated for the immobilisation of the enzymes in whole cells and enzyme extracts. Immobilisation of the enzyme extract in calcium alginate beads was found to be the best method in terms of enzyme activity retention and stability. The hydantoinase retained 55% activity while the N-carbamoylase exhibited a remarkable sevenfold increase in activity after immobilisation by this method. Furthermore, the hydantoinase activity increased after storage at 4°C for 21 days, while the N-carbamoylase retained 30% activity after this storage period. The calcium alginate bead-immobilised enzymes were further biochemically characterised and then applied in a bioreactor system for the production of D-hydroxyphenylglycine (D-HPG) from D,L-5-hydroxyphenylhydantoin (D,L-5-HPH). The pH and temperature optima for the immobilised hydantoinase were pH 7 and 50°C, respectively, while pH 8 and 40°C were optimal for the immobilised N-carbamoylase enzyme. The immobilised enzymes showed improved thermostability at 40°C in comparison to the free enzymes and retained high levels of activity after five repeated batch reactions. Low levels of conversion were obtained in a packed-bed bioreactor containing the A. tumefaciens RU-ORPN1 biocatalyst due to the low hydantoinase activity present in the strain, relative to N-carbamoylase. A novel, packed-bed bioreactor system was therefore developed for the production of D-HPG from D,L-5-HPH using the A. tumefaciens biocatalyst in combination with a Pseudomonas sp. biocatalyst having high hydantoinase activity. A conversion yield of 22 to 30% was achieved for the production of D-HPG from D,L-5-HPH over 5 days operation demonstrating that the hydantoin-hydrolysing enzymes from A. tumefaciens RU-ORPN1 could be stabilised by immobilisation and, in combination with a biocatalyst with high hydantoinase activity, could be applied to the fully enzymatic conversion of D,L-5-HPH to D-HPG.

This work presents the development of a highly sensitive and selective chemical assay for mono(ADP-ribose) residues covalently bound to proteins in vivo. An extensive review of the literature is presented in the introduction of this work. The physiological.functions of mono(ADP-ribosyl)transferase activities associated with certain bacterial toxins (e.g., diphtheria, cholera and pertussis toxins) are well established. However, the roles of endogenous vertebrate transferases are unknown. The elucidation of the roles of these cellular transferases will likely require identification of the physiologically relevant target proteins. Toward this end, it will also be important to identify the types of (ADP-ribose)-protein linkages present in vivo. ADP-ribosylation reactions catalyzed by the different bacterial and vertebrate transferases are specific for different amino acid acceptors in vitro. However, the vertebrate transferases that have been characterized thus far are NAD:arginine mono(ADP-ribosyl)transferases. The work presented here describes the development of a chemical assay for the detection of in vivo modified, ADP-ribosylated proteins containing N-glycosylic linkages to arginine. The assay was applied to the analysis of ADP-ribose residues in adult rat liver. The strategy employed for detection of protein-bound ADP-ribose residues eliminated potential artifacts arising from trapped nucleotides (or their degradation products), since the acid-insoluble material was completely dissolved in a strongly denaturing solution and freed of non-covalently bound nucleotides prior to chemical release from proteins. Thus, the studies presented here demonstrate the unambiguous detection and quantification of protein-bound ADP-ribose residues in adult rat liver. "Arginine-linked" mono(ADP-ribose) residues (31.8 pmol/mg protein) were present in vivo at a level almost 400-fold higher than poly(ADP-ribose). A minor fraction (23%) of the ADP—ribose residues detected were bound via a second more labile linkage with chemical properties very similar to those described previously for carboxlylate esterlinked ADP-ribose. After fractionation of rat liver proteins by gel filtration HPLC, the major peak of "arginine-linked" ADP-ribose residues eluted in the 40-60 kDa region. The later result is consistent with previous suggestions that G-proteins (40-50 kDa) of the adenylate cyclase complex, which are targets for toxin-catalyzed ADP-ribosylation, may also represent target proteins for endogenous transferases.

Synopsis The thesis entitled “Synthesis of Glyco-amino-acids, Glycosyl-amino-acids, and α-Amino γ-Lactams from Carbohydrate Derived Donor-Acceptor Cyclopropanes” is divided into five chapters. Chapter 1: Introduction and Background: Carbohydrate Derived Cyclopropanes and Glycoconjugates of Amino Acids and Peptides In this chapter, introduction and background on cyclopropanes, carbohydrate derived DA-cyclopropanes, glycopeptides and its mimetics is discussed Chapter 2: Efficient Synthesis of Glycosyl Esters of Amino Acids from Carbohydrate Derived Cyclopropanecarboxylates In this chapter, the N-iodosuccinimide (NIS) mediated ring opening of carbohydrate derived donor-acceptor (DA) cyclopropanes with carboxylic group of various N-protected amino acids is discussed. Under mild conditions, glucosyl esters of amino acids have been synthesized in moderate to good yields. This methodology has also been applied to galactose derived DA-cyclopropanes for the synthesis of galactosyl-amino-acid derivative. Among three N-protected valine derivatives (–Fmoc, –Boc, and –Cbz), the reaction of N-Fmoc protected valine derivative of glycosyl-amino-acid has not been successful due to the steric hindrance of bulky Fmoc group. Chapter 3: Synthesis of O–Linked Glycosyl-amino-acids & C–Linked Glyco-amino-acids. In this chapter, the synthesis of glycosyl-amino-acids and glyco-amino-acids by the NIS mediated ring opening of carbohydrate derived DA-cyclopropanes is reported. To synthesize the precursors of glycopeptides, deprotection of NHBoc has been performed with trifluoroacetic acid (TFA) and trimethylsilyl chloride (TMSCl). Trimethylsilyl chloride is found to be a better reagent than trifluoroacetic acid for this reaction. The synthesis of both O–linked glycosyl-amino acids and C–linked glycopeptides from single starting material using the orthogonal strategy at amine groups has been achieved. In these glycoconjugates of amino acids, the azide group (–N3) has been used as a masked amine (–NH2) which circumvents the protection and deprotection steps. Chapter 4: Synthesis of Carbohydrate Fused α-Amino γ-Lactams. In this chapter a flexible protocol for the synthesis of carbohydrate fused α-Amino γ-Lactams from carbohydrate derived cyclopropanecarboxylates has been disclosed. Also, the synthesis of carbohydrate fused γ-Lactams in a single-step from the iodo-azide by reductive cyclization has been reported. The formation of -lactam is achieved in low yield using both methods (A & B). The utility of the carbohydrate fused α-Amino γ-Lactams in the synthesis of Agl-bridged glycopeptide conjugates in a single-step with high efficiency has been demonstrated. Chapter 5: Studies on the Synthesis of Septanosides from Carbohydrate Derived DA-Cyclopropanes In carbohydrate derived DA 1,2-cyclopropanes, generally, the electron withdrawing group is attached at C-7 (type-I), C-2 (type-II), or C-3 (type-III). In this chapter, studies on the synthesis and use of carbohydrate derived DA-cyclopropanes of type-II & -III to form the the septanoside derivatives have been described. Attempts at the synthesis of 3,4,6-tri-O-methyl-D-glucal derived cyclopropanecarboxylates of type-II have not been successful. The failure of the cyclopropanation reactions might be due to presence of the carbmethoxy group at C-2 causing steric hindrance on the olefinic bond of 3,4,6-tri-O-tri-methyl-D-glucal methyl ester. It was then speculated that replacement of the of the carboxylate group in tri-O-methyl-glucal with hydroxymethyl group can promote the cyclopropanation reaction. Cyclopropanation of benzyl protected corresponding alcohol furnished the desired cyclopropane derivative in moderate yield (48%) as an inseparable mixture of diastereomers (1:1). Hence the synthesis of cyclopropanecarboxylates of type-II has not been achieved using this methodology. Glucose derived 3-oxo-1,2-cyclopropanes is synthesized from 3,4,6-tri-O-acetyl-D-glucal in good yield. This glucose derived cyclopropane of type-III did not furnish the septanoside derivative under different conditions. The synthesis of 3-oxo-1,2-cyclopropanated galactose derivative has been achieved in reasonably good yield from D-galactal with in three steps. When the galactose derived DA-cyclopropane is reacted with NIS and MeOH in the presence of catalytic amount of TMSOTf, it furnished the desired septanoside along with many side-products. The attempts at separation and identification of the septanoside in pure form have not been successful.

β-Aminocarbonyl motifs are a privileged substructures in medicinal chemistry and peptidomimetics. As part of our efforts toward metal free aminations, we developed a method for intermolecular amino-carbonylation of alkenes using hydrazones. This method provides access to cyclic azomethine imines containing a β-aminocarbonyl motif. Conceptually, these dipoles can be derivatized into many bioactive compounds, such as 1,3-diamines, β-amino amides and β-amino acids. The first part of this thesis will present the results on the derivatization of our aminocarbonylation products into various nitrogen-containing molecules, such as β-amino amides, β-amino acids and pyrazolones. More specifically, a short, chromatography-free derivatization of azomethine imines into N-Boc-β-amino amides will be presented. Following these results, the next chapter will focus on attempts at develop novel aminocarbonylation reactivity between 1,2-diacylhydrazines and alkenes followed by results from our reductive N-N bond cleavage experiments on our cyclic hydrazides.

Previous work in many laboratories has established that the addition of L-valine to a culture of Escherichia coli K-12 growing exponentially on minimal medium causes inhibition resulting in persistent linear growth. The inhibition can be removed by addition of isoleucine. Growth of other strains of E. coli e.g. E. coli B and two mutants of K-12 (E. coli AB1020 and AB1005) was not affected by addition of valine. E. coli LL5, another mutant of K-12, showed no inhibition of growth by valine added in concentrations up to 1 x 10ˉ⁴M. However, the addition of 1 x 10ˉ³M valine caused a decrease in the logarithmic growth rate, and 1 x 10ˉ²M valine caused persistent linear growth. The inhibition of growth by valine in LL5 was antagonized by L-isoleucine (as is the case with wild-type E. coli K-12), but not by α-ketobutyrate, threonine or pyruvate. Kinetic studies showed that the maximal inhibition of acetohydroxy acid synthetase (AHAS) by 1.5 x 10ˉ³ M valine was 84% for E. coli K-12, 55% for E. coli B, 75% for E. coli AB1005, 77% for E. coli AB1020 and only 20% for E. coli LL5. It is proposed that the persistence of linear growth of K-12 in valine-containing medium is the result of incomplete (84%) feed-back inhibition of AHAS. Analysis of the data for valine inhibition of AHAS was carried out by the method of Levitsky and Koshland (1969) using Hill Plots. E. coli strains K-12, AB1005 and AB10 20 showed "positive cooperativity" between inhibitor (valine) binding sites at low valine concentrations, and "negative cooperativity" between inhibitor binding sites at high valine concentrations. The AHAS of E. coli strains E and LL5, however, showed only negative cooperativity between binding sites for inhibitor, which could be the mechanism for incomplete inhibition of AHAS by valine. Preliminary kinetic analyses using Michaelis-Menten plots were carried out with strains K-12 and LL5. The levels of threonine deaminase (TD) and AHAS were also examined in four of the E. coli strains, K-12, B, AB1020 and LL5. AHAS was repressed and TD derepressed in E. coli K-12 grown (linearly) with 10ˉ³M valine. In E. coli strains B and AB1020, growth with 10ˉ³M valine had no effect on the levels of AHAS or (derepressed) TD. In E. coli strain LL5, growth with 10ˉ³M valine did not change the AHAS level but caused significant derepression of TD. Sensitivity of growth to valine has been correlated with three properties of the organism: 1) Feed-back sensitivity of acetohydroxy acid synthetase, to L-valine; 2) The level of acetohydroxy acid synthetase in the cell; 3) The level of biosynthetic threonine deaminase - the regulatory enzyme for isoleucine biosynthesis. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

The present investigation was conducted to determine the extent to which the effects dietary protein level and amino acid balance have on rate of growth and efficiency of feed conversion can be related to the effects they have upon thyroid activity and tissue respiration. Thyroid activity and body weight were studied in chicks fed diets containing 17.5% and 26% of protein and variously supplemented with amino acids. The basal diets were deficient in methionine and glycine. Growth was stimulated at both protein levels by the addition of methionine or methionine plus glycine to the basal diets. Even when supplemented with amino acids, the low-protein diet was inadequate for maximum growth. Thyroid weight, thyroid weight relative to body weight and I¹³¹ uptake per chick were increased when the protein level was increased from 17.5% to 26% but uptake of I¹³¹ per unit weight of thyroid gland was not affected. Supplementation of the diets with lysine significantly reduced thyroidal uptake of I¹³¹ per chick. Addition of methionine or methionine plus glycine reduced uptake of I¹³¹ per unit weight of thyroid gland. The effect of various levels of dietary lysine, methionine and tryptophan on body weight, efficiency of feed conversion, nitrogen balance and metabolizable energy were studied in relation to concurrent effects of diet on thyroid activity and tissue respiration in chicks. Respiration of liver slices and fiber bundles of skeletal muscle was measured with a Warburg respirometer. Feed and feces were analyzed for nitrogen by the macro Kjeldahl method and for combustible energy with a Parr oxygen bomb calorimeter. Addition of lysine to diets deficient in lysine significantly improved body weight, efficiency of feed utilization, and nitrogen retention in chicks fed the experimental diets. Addition of 0.30% lysine to diets containing 1.09% of lysine improved body weight significantly but had no significant effect on the other parameters mentioned. Addition of methionine to a diet containing 1.39% of lysine improved feed efficiency in birds 17 to 22+ days of age. Amino acid balance had no effect on metabolizable energy. Birds fed lysine deficient diets had lighter thyroids and lower thyroidal uptake of I¹³¹ but had higher uptake of I¹³¹ per mgm. of thyroid gland than those fed diets containing 1.09% or more of lysine. Amino acid balance had no effect on oxygen consumed by muscle tissue, but a deficiency of lysine significantly increased oxygen consumed by liver slices taken from 6 and 8 week old chicks. It is concluded that amino acid composition of dietary protein, as well as the protein level in the diet, affects thyroid activity in the chick. Amino acid imbalance caused by dietary lysine deficiency results in high thyroid activity, low body weight, poor efficiency of feed conversion, and poor nitrogen retention but apparently high respiration of liver slices. Possible shifts in methionine and lysine requirements during the first 25 days after hatch were investigated. It was found that the requirement of the chick for methionine did not change during this time hut that the requirement of the chick for lysine was higher in birds 0 to 20 days of age than in older birds. / Land and Food Systems, Faculty of / Graduate