A structure-function study of the enzymes of the atrazine catabolic pathway

Abstract

The aim of this study was to develop an understanding of the structure-function relationship of the enzymes from atrazine mineralization pathway from Psuedomonas sp. strain ADP. This pathway has evolved recently in response to the selection pressure exerted by the extensive use of herbicide atrazine over the past six decades. The pathway is comprised of six hydrolytic enzymes, some of which have recently evolved to possess novel functionalities making them excellent models for studying enzyme evolution. A literature review is presented in chapter 1 with the focus on how the enzyme evolution works in general and how the atrazine mineralizing enzymes have evolved and assembled into a catabolic pathway that achieves the complete mineralization of atrazine. In chapter 2, the X-ray crystal structure of AtzA, the 'archetypal' atrazine chlorohydrolase is presented. The active site of AtzA is studied by structural and in silico docking studies and the comparisons were drawn with the alternative and superior metal-dependent atrazine chlorohydrolase, TrzN. On the basis of structural and previously obtained biochemical data, a plausible reaction mechanism is also presented. Chapter 3 is about the over-expression, purification and attempts made so far to obtain the X-ray structure of AtzB (hydroxyatrazine hydrolase). This enzyme performs the second step in atrazine mineralisation pathway by removing the alkylamine chain from hydroxyatrazine. In chapter 4, the X-ray structure of AtzC with bound inhibitor is presented. This enzyme perform the third step in this pathway by removing the isopropylamine chain from the N-isopropylammelide. The presence of bound inhibitor in the active site guided the in silico substrate docking experiments. The mutagenesis work was performed in the light of substrate interactions information obtained from the docking experiments which allowed us to propose a plausible reaction mechanism for this enzyme. Chapter 5 describes the structure of AtzD, a cyanuric acid hydrolase. The structure represents a novel fold and is termed as the 'toblerone' fold. The structure of AtzD suggests that this novel fold has evolved by the structural concatenation of monomers of the trimeric YjgF superfamily and acquisition of metal binding site. Based on the crystallographic and biochemical data, a plausible reaction mechanism is also proposed. The residues imparting substrate specificity were also identified by making comparisons with a homology model of related barbiturase and the phylogeny of the 68 AtzD-like enzymes in the database were analysed based on this structure-function relationship. In chapter 6, the X-ray structure of the amidase domain of allophanate hydrolase, AtzF, from Pseudomonas sp ADP was presented. The structure was compared with the structures of allophanate hydrolases from other organisms. We have also provided the preliminary evidence to propose that AtzF forms a large, ca. 660 kDa multi-enzyme complex with other two enzymes of cyanuric acid mineralisation pathway.