Structure-function study of the enzymes of the cyanuric acid catabolic pathways

Abstract

Up to now the degradation of atrazine by Pseudomonas sp. strain ADP1 bacterium was thought to involve six steps successively catalysed by enzymes: AtzA, AtzB, AtzC, degrading atrazine into cyanuric acid, and AtzD, AtzE and AtzF, successively mineralising cyanuric acid to ammonia and carbon dioxide. The genes atzD-aztE-atzF are arranged in an operon called the cyanuric acid degradation operon. The exploration of cyanuric acid degradation pathways in different bacteria showed that substantial differences exist in the cyanuric acid degradation pathways between microorganisms. In Rhizobium leguminasorum bv. viciae 3841, for example, a biuret hydrolase (BiuH) belonging to the isochorismatase family performs the deamination of biuret to produce allophanate (118); whereas, in the model s-triazine degrading bacterium Pseudomonas sp. strain ADP, it is an amidase, AtzE, that is thought to perform that step. The characterisation of AtzE revealed the existence of two new enzymes in the Pseudomonas sp. strain ADP1 cyanuric acid operon. The first part of this PhD, reports the structure-function study of the BiuH in Rhizobium leguminasorum bv. viciae 3841. The atomic structure of BiuH was solved and site-directed mutagenesis was used to gain a better understanding of the BiuH catalytic mechanism. Additionally, molecular dynamics simulations highlighted the presence of three channels from the active site to the enzyme surface forming a potential substrate channel, a co-product (ammonia) channel and a co-substrate (water) channel. Although the cyanuric acid degradation pathway in Pseudomonas sp. strain ADP1 has been known and studied for more than twenty years, no one had purified and characterised AtzE. The second part of this PhD reports the purification of the native AtzE from Pseudomonas sp. strain ADP, allowing its biochemical and structural characterisation. The structure revealed the presence of a small, essential protein (AtzG), with which AtzE forms a heterotetramer. Biochemical characterisation and molecular dynamics experiments revealed AtzE acts as a 1-carboxybiuret hydrolase, not as a biuret hydrolase as previously thought. Finally, this work suggests that AtzE might have evolved from the GatCAB transamidosome complex. The final part of my PhD presents the discovery and the study of AtzH, a previously unknown small protein encoded by a gene located in the Pseudomonas sp. strain ADP’s cyanuric acid degradation operon. The structural characterisation of AtzH determined it belonged to the versatile NFT2 protein superfamily. A combination of structural modelling and mutagenesis studies was used to provide evidence that AtzH is an allophanate forming, 1,3-dicarboxyurea amidohydrolase. Mutagenesis also indicated that Tyr22 and Arg46 may play an essential role in the catalysis of 1,3-dicarboxyurea. Finally, a comparison of the genomic context suggests AtzH might be involved more broadly in the catabolism of nitrogenous compounds in Proteobacteria. Moreover, this observation also suggests that the atzG-atzE-atzH cluster predates the formation of the cyanuric acid catabolism operon.