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
