Mechanistic Analysis of the Organophosphonate-Degrading Enzymes PhnY and PhnZ
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Authors
Séguin, Jacqueline
Date
2015-09-14
Type
thesis
Language
eng
Keyword
Organophosphonates , Oxygenase , PhnZ
Alternative Title
Abstract
PhnY and PhnZ comprise a new microbial pathway for the degradation of the naturally occurring phosphonate 2-aminoethylphosphonic acid into glycine and inorganic phosphate. PhnY is an α-ketoglutarate / Fe(II)-dependent dioxygenase that hydroxylates the α-carbon of 2-aminoethylphosphonic acid. PhnZ is a di-iron-dependent oxygenase that cleaves the C-P bond of the PhnY product, 2-amino-1R-hydroxyethylphosphonic acid, using a mechanism unlike that used by other C-P bond cleaving enzymes. Kinetic parameters for PhnY were determined with 2-aminoethylphosphonic acid. A series of substrate analogues were also tested for reactivity, but PhnY was strictly specific for its native substrate. Attempts to crystallize PhnY to provide insight into its mechanism were unsuccessful in spite of the development of improved procedures to obtain concentrated samples of PhnY suitable for crystallography. PhnZ and active site variants were assayed with the native substrate 2-amino-1R-hydroxyethylphosphonic acid and a series of analogues. A primary deuterium kinetic isotope effect of 1.36 ± 0.06 was measured for the PhnZ reaction, indicating that cleavage of the αC-H bond of the substrate is only partly rate limiting. PhnZ was observed to convert 2R-amino-1R-hydroxypropylphosphonic acid, 3-amino-1R-hydroxypropylphosphonic acid, and N-methyl-2-amino-1-hydroxyethylphosphonic acid to inorganic phosphate. New X-ray crystal structures of PhnZ complexed with 2R-amino-1R-hydroxypropylphosphonic acid, 3-amino-1R-hydroxypropylphosphonic acid, and 1R-hydroxyethylphosphonic acid provided further insight into its substrate specificity revealing that it can accommodate some additional bulk in the active site, however the amino group and its location on the substrate are crucial for activity. The structures of PhnZ also provided new insights into the importance of the flexible loop that closes the active site upon substrate binding, and the roles of two residues along this loop, Y24 and E27. Based on these results, a more complete mechanism for C-P bond cleavage by PhnZ is proposed.
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Thesis (Master, Chemistry) -- Queen's University, 2015-09-07 11:44:43.6
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ProQuest PhD and Master's Theses International Dissemination Agreement
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Copying and Preserving Your Thesis
This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.