Microorganisms have developed the greatest diversity of enzymatic pathways to allow their successful proliferation in the natural world. Those enzymatic pathways are the result of interactions between microorganisms and their habitat leading to adaptations by the microorganisms. As knowledge about the enzymatic pathways accumulates, increasingly those pathways can be used to degrade recalcitrant pollutants. The biodegradation pathway of s-triazine compounds is an example. Cyanuric acid hydrolase (CAH) is the one of the enzymes in the s-triazine degradation pathway. CAH is of particular interest because it hydrolyses cyanuric acid that is the by-product of tri-chlorocyanuric acid decomposition which has been used in swimming pool disinfection. The first three chapters will deal with the mechanism of CAH mainly using X-ray crystallography. Chapter 2 describes the structural characteristics of the active site of CAH with emphasis on emerging evidence that a serine in domain B is the nucleophilic serine in the catalytic cycle. Evidence from mutational studies, Burgi-Dunitz angle analysis, and sequence alignments all support the hypothesis that the serine in domain B is the nucleophilic serine. Direct evidence is presented in chapter 3 for this serine acting as the nucleophile by our solving an X-ray structure showing a reaction intermediate covalently attached to this serine (the so-called “acyl intermediate”). The X-ray structure also shows 24 monomers in the crystal’s asymmetric unit and different active sites within the asymmetric unit contain different stages of the reaction such as the unreacted substrate, the acyl intermediate, carboxybiuret, biuret, and an empty active that has expelled the product. The thesis concludes with chapter 5 that provides the novel biodegradation pathway of unsubstituted triazine (1,3,5-triazine) by Acinetobacter sp. Trz. Although there have been a lot of studies on substituted triazines biodegradation, no study on unsubstituted triazine (1,3,5-triazine) has been done previously. The chapter 5 describes the isolation of Acinetobacter sp. Trz that utilizes 1,3,5-triazine as its sole nitrogen source and the novel biodegradation pathway of 1,3,5-triazine. The 1H-NMR and HPLC analysis shows that 1,3,5-triazine first transforms to formamidine (FAD), formamide (FD) and ammonia non-enzymatically and then enzymes likely trasnform formamidine to formamide and formamide to formate and ammonia.
University of Minnesota Ph.D. dissertation. October 2015. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: Lawrence Wackett. 1 computer file (PDF); viii, 85 pages.
Structural and mechanistic studies of cyanuric acid hydrolase and biodegradation of 1,3,5-triazine.
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