Browsing by Subject "vitamin B1"
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Item Mechanistic Studies on the Radical S-Adenosylmethionine Enzymes Involved in Molybdopterin, Thiamin and Vitamin B12 Biosynthesis(2015-02-09) Mehta, Angad PThis dissertation focuses on radical S-adenosylmethionine enzymes involved in cofactor biosynthesis. Mechanistic studies discussed here include: (i) molybdenum cofactor biosynthetic enzyme - MoaA, (ii) thiamin pyrimidine synthase ? ThiC (iii) hydroxybenzimidazole synthase, HBI synthase, involved in anaerobic vitamin B12 biosynthesis. MoaA catalyzes the first step in molydopterin biosynthesis where GTP is converted to pterin. This catalysis involves a remarkable rearrangement reaction where the C8 of guanosine-5?-triphosphate (GTP) is inserted between the C2? and C3? carbon atoms of GTP to give the final pterin. Mechanistic studies involved characterization of the products of the reaction, identification of the position of hydrogen atom abstraction by 5?-deoxyadenosyl radical and trapping of intermediates by using 2?,3?-dideoxyGTP, 2?-deoxyGTP and 2?-chloroGTP as substrate analogs. Thiamin pyrimidine synthase, ThiC, catalyzes a complex rearrangement reaction involving the conversion of aminoimidazole ribotide (AIR) to thiamin pyrimidine (HMP-P). A hydrogen atom transfer from S-adenosylmethionine (AdoMet) to HMP-P was demonstrated. Also, the stereochemistry of this transfer was elucidated. Bioinformatics studies on ThiC revealed that a paralog of ThiC was clustered with vitamin B12 biosynthetic genes in several anaerobic microorganisms. The gene responsible for the anaerobic vitamin B12 ? benzimidazole biosynthesis was previously unknown. We demonstrate that the gene product of this ThiC paralog is a radical S- adenosylmethionine enzyme. Remarkably it catalyzes the conversion of aminoimidazole ribotide (AIR) to 5-hydroxybenzimidazole (5-HBI) and formate, and S-adenosylmethionine to 5?-deoxyadenosine. We determine the hydrogen atom abstracted by 5?-deoxyadenosyl radical. We also performed carbon, nitrogen and hydrogen labeling studies and characterized the labeling pattern on 5-HBI. Based on these studies we propose a reaction mechanism of this remarkable conversion of AIR to 5-HBI.Item Studies on Uptake of Thiamin Analogs by a Thiamin Deficient E. coli Mutant Strain(2012-11-28) Olivard, SarahThiamin transport in Escherichia coli is a model system to establish the tolerance of derivatives for transport into the cell. Since little is known about what types of thiamin derivatives may be successfully taken into the cell through the transport system, a series of thiamin derivatives are synthesized. A thiamin amino analog is synthesized and tested to determine the use of the analog as an alternate source of thiamin for growth of an E. coli thiamin mutant. Formate, acetate, and benzoate thiamin esters are synthesized and tested as alternate sources for growth of an E. coli thiamin mutant. Thiamin esters or amides may provide a scaffold for attaching other small molecules of interest to be imported into the cell by thiamin transport system. Thiamin containing formate, acetate, and benzoate esters were synthesized and tested as alternative growth source for thiamin using an E. coli mutant strain incapable of synthesizing thiamin. All three synthesized ester thiamin forms gave a zone of growth determined by disk-assay study. Also, an amino thiamin is synthesized to determine uptake through thiamin transport system by growth study using an E. coli mutant incapable of synthesizing thiamin. The growth curves resulting show concentration-dependent growth in the absence of natural thiamin, indicating amino thiamin is taken up by thiamin transport system as an alternate source of thiamin for growth. More characterization of the thiamin transport system is desired in order to develop thiamin conjugates of interest such as a photoaffinity probe for isolating thiamin-utilizing enzymes.