Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 1980 / Vita. / Includes bibliographical references. / by Victor Manuel Rios-Mercadillo. / Ph. D. / Ph. D. Massachusetts Institute of Technology, Department of Chemistry

Propionyl-CoA carboxylase has been purified to a state of near nomogeniety, and some of its enzymatic properties relating to substrate binding and mechanism of action have been studied. The enzyme was not found to catalyze the incorporation of solvent tritium at the c-carbon of propionylâ CoA in the absence of ATP. Absolute stereospecificity was observed with regard to which a-hydrogen is replaced during the addition. / Ph. D.

Includes bibliography / 100 leaves, [8] leaves of plates : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1987

No description available.

This thesis describes an investigation of the mechanism of the bifunctional, a-ketoglutarate dependent dioxygenase, deacetoxy/deacetylcephalosporin C synthase (DAOC/DACS), which catalyses the ring-expansion of penicillin N to deacetoxycephalosporin C (DAOC) and the hydroxylation of this to deacetylcephalosporin C (DAC). The conversion of the unnatural substrate 3-exomethylene cephalosporin C by DAOC/DACS has been investigated in detail. A new metabolite was isolated from incubations of the deuterated [4-²H]-3-exomethylene cephalosporin C, and was identified as the 3β-spiroepoxide cepham, (2Ṟ,3Ṟ,6Ṟ,7Ṟ)-l-aza-[2-²H]-3-spiroepoxy-7-[(5Ṟ)-5-amino- 5-carboxypentanamido]-8-oxo-5-thiabicyclo[4.2.0]octane-2-carboxylic acid. The results obtained indicate that this metabolite is a shunt product whose formation is enhanced by the operation of a deuterium kinetic isotope effect on an enzyme-bound intermediate. It has also been found that this 3β-spiroepoxide cepham is further converted by DAOC/DACS to 3-formyl cephalosporoate products. The mechanism of oxygenation of DAOC/DACS was investigated through ¹⁸O-labelling studies. Incubations of [2-¹³C,3-²H]penicillin N and [4-²H]-3-exomethylene cephalosporin C with DAOC/DACS were carried out under ¹⁸O₂ or in H₂¹⁸O. Incorporation of ¹⁸O-label into the products [3-¹³C]DAC, [3-¹³C,4-²H]-3β-hydroxycepham and 3β-spiroepoxide cepham was observed from both sources. The results suggest that intermediates capable of oxygen-exchange are formed during the enzymatic reactions. Two substrate analogues, the 5-epipenicillin N and the 2β-difluoromethyl penicillin N, have been synthesised in order to probe the substrate specificity of DAOC/DACS with respect to the ring-expansion activity. The 5-epipenicillin N was not accepted as a substrate by DAOC/DACS, and the observations made indicate that it was unstable under the incubation conditions. No product was either observed from incubations of the 2β-difluoromethyl penicillin N with DAOC/DACS, although bioassay tests suggested a cephem product had been formed in very small amounts. Finally, the results of a substrate specificity comparison between the soluble recombinant enzymes deacetoxy/deacetylcephalosporin C synthase (DAOC/DACS) from Cephalosporium acremonium and deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus are described.

C. elegans worms with mutations in the gene CLK-1 develop slowly and have an extended lifespan. CLK-1 encodes a mitochondrial protein that is responsible for the hydroxylation of 5-demethoxyubiquinone (DMQ), the penultimate step of ubiquinone (Coenzyme-Q or UQ) biosynthesis. Structural homologues of CLK-1 are found in mammals, fruit flies, yeast and some types of bacteria. Interestingly, however, there is no structural homologue of CLK-1 in the Arabidopsis genome and no plant homologue can be found in other sequence databases. Yeast with the CLK-1 homologue COQ7 deleted fail to grow on non-fermentable carbon sources. To identify a plant functional homologue of COQ7/CLK-1, an Arabidopsis cDNA expression library was screened for complementation of a yeast coq7 deletion mutant. A clone was identified that rescued the coq7 respiratory deficiency. Although the sequence of the encoded protein has no structural similarity to proteins in the COQ7/CLK-1 family, it contains a monooxygenase/hydroxylase domain that has sequence similarity with the E. coli DMQ hydroxylase encoded by the UBIF gene. Like the structural homologues of COQ7/CLK-1 found in other eukaryotes, the gene (AQX for 'Alternate Quinone monooXygenase') contains a likely mitochondrial targeting presequence at its N-terminus. HPLC analysis of quinone extracts from rescued cog7 strains does not detect ubiquinone, but instead shows another peak that may be DMQ. It is likely that AQX does not hydroxylate yeast DMQ effectively enough to generate detectable levels of UQ. A unique pathway for UQ biosynthesis in plants is proposed that is defined by AQX and Arabidopsis genes identified on the basis of homology to known E. coli and yeast UQ biosynthesis genes.

M.Sc. (Chemistry) / Inhibition of mycothiol biosynthesis pathway has attracted attention from chemists and biochemists who aim to develop novel anti-TB drugs. A possible route to inhibit the production of mycothiol in cells may be via the inhibition of enzymes involved in the biosynthetic pathways. Molecular analogues that mimic mycothiol and containing tetrahedral-forming functional groups have been reported to show activity against mycothiol biosynthesis by inhibiting the enzymes in the mycothiol biosynthetic pathway...

Over the past few years, the use of enzymes as catalysts for the preparation of novel organic molecules has received a steadily increasing amount of attention. Lipolytic enzymes are widely distributed in nature and attract great attention because of their biotechnological potential, as they catalyse the enantio- and regioselective hydrolysis and synthesis of a broad range of natural and non-natural esters. Bacteria produce different lipolytic enzymes, such as esterases (EC 3.1.1.1), which hydrolyse ester-containing molecules at least partly soluble in water, and lipases (EC 3.1.1.3), which hydrolyse water-insoluble long-chain triglycerides. In this study, a bacterial isolate, B. coagulans strain 81-11, isolated from popcorn seeds, was characterized with the specific aim of isolating and characterizing genes encoding novel lipolytic enzymes. A genomic library of B. coagulans strain 81-11 was screened in Escherichia coli JM83 for lipolytic activity by using tributyrin agar plates. A 2.4-kb DNA fragment was subcloned from a lipolytic-positive clone and completely sequenced. Nucleotide sequence analysis predicted a 723-bp open reading frame (ORF), designated estCl, encoding a protein of 240 amino acids with an estimated molecular mass of 27 528 Da and a pI of 9.15. The deduced amino acid sequence of the estCl gene exhibited significant amino acid sequence identity with carboxyl esterases and sequence analysis showed that the protein contains the signature G-X-S-X-G included in most esterases and lipases. Enzyme assays using p-nitrophenyl esters with different acyl chain lengths as the substrate confirmed the anticipated esterase activity. EstCl exhibited a marked preference for esters of short-chain fatty acids, yielding the highest activity with p-NP butyrate. Maximum activity was found at pH 8 and 50°C, although the enzyme was active in the pH range 7-9 and displayed activity at temperatures up to 55°C. Since bacterial esterases are potentially important for a variety of biotechnological applications, there is a considerable industrial interest to produce these enzymes at a larger scale. Among the many systems that are available for heterologous protein production, attempts were made to over express the newly identified B. coagulans estCI esterase¬encoding gene in different Gram-positive bacteria, as they are well known for their important contribution to food biotechnology and as production organisms for industrial enzymes. A recombinant expression vector was thus constructed (pMG36-EstCl) and introduced in Lactococcus lactis, Lactobacillus plantarum and Bacillus subtilis strains 154 and lA297. Of these different bacterial hosts, high levels of intracellular esterase activity were detected in B. subtilis lA297 only. In an attempt to increase extracellular expression of the B. coagulans EstCl esterase, a recombinant secretion plasmid (pNW-EstClaps) was constructed that contained an alkaline protease promoter and signal sequence from a Bacillus species. Following introduction of the construct in B. subtilis lA297, the derived recombinant strain displayed 2.3-fold higher extracellular esterase-activity levels than the parent B. coagulans strain, and the extracellular esterase activity represented 82% of the total esterase activity. / Dissertation (MSc (Microbiology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

No description available.

Synchronous cultures of a high temperature strain of Chlorella pyrenoidosa have been used to determine the activity of the TMP kinase during cellular development. It was observed that the apparent enzyme activity was closely correlated with the rate of DNA biosynthesis. During the period of nuclear division, when DNA synthesis is at its maximum apparent TMP kinase activity is highest. The maximum in enzyme activity, however, slightly precedes the peak in DNA synthesis. These results support the hypothesis that TMP kinase activity is a factor controlling the rate of DNA biosynthesis. It appeared that the dramatic shifts in the level of TMP kinase activity were a result of variations in the rate of enzyme synthesis rather than control of enzyme activity by small molecule inhibitors or activators. / Ph. D.