Il meta-cleavage pathway di Pseudomonas sp. OX1: regolazione trascrizionale e studi di attività enzimatiche
Author(s)
Stancarone, Valeria
Date Issued
February 20, 2009
Type
Doctoral Thesis
Abstract
The release of aromatic compounds in the environment, which accumulate due to their
stability, is one of the most important issues in bioremediation. The main pollutants in nature
are components of petroleum and its refined products, which find wide use as solvents or as
precursors for the production of several chemical compounds. Since the distribution of these
pollutants in the environment is ubiquitous and their effect on humans health is extremely
harmful, it is clear the increasing interest in developing processes for their biodegradation.
Among the aromatic hydrocarbons are simple molecules such as toluene and xylenes,
rings replaced alo- and nitro-containing groups, but also much more complex molecules as
polycyclic aromatic hydrocarbons. The biodegradation of toxic and recalcitrant chemicals,
such as the aromatic hydrocarbons, represents an attractive alternative to conventionally used
methods for the disposal of pollutants. Many genera of microorganisms, naturally present in
soil and water, evolved metabolic pathways to survive in the presence of otherwise toxic
chemicals; nevertheless still a large number of aromatic molecules appear to be recalcitrant to
biodegradation.
The thorough knowledge of the structure and function of metabolic pathways involved
in the biodegradation of environmental pollutants, along with genetic engineering, offers the
ability to create enzymes with an increased catalytic activity or to accelerate the evolution of a
metabolic pathway that can degrade only one compound or a whole family of aromatic
recalcitrant compounds.
The bacteria belonging to the genus Pseudomonas represent the largest group of
microorganisms able to use, under aerobic conditions, many aromatic and aliphatic
compounds as the sole carbon and energy source.
Pseudomonas sp. OX1 shows relevant functions to the environmental decontamination;
it has the ability to metabolize o-xylene, toluene, benzene, phenol, 2,3- and 3,4-
dimethylphenol and cresols, but it is not able to use m-and p-xylene as carbon and energy
source.
The genes coding for toluene and o-xylene catabolism are localized in the chromosome
of P. sp. OX1 and they are organized into two operons: the tou operon (toluene o-xylene
utilization) encodes a multienzymatic complex called ToMO (Toluene o-xylene
Monoxygenase); the phe operon includes a gene cluster coding the subunits of a Phenol
Hydroxylase (PH) and the enzymes of the lower-meta pathway.
ToMO and PH catalyze upper pathway reactions: the final products of ToMO and PH
activities (methyl-substituted catechols) undergo a further degradation through the lower-meta
pathway, leading to intermediate of the tricarboxylic acid cycle.
pathway, leading to intermediate of the tricarboxylic acid cycle.
Many informations are available for ToMO and PH. The two recombinant complexes
were reconstituted and expressed in vitro, demonstrating their enzymatic activity, by the Prof.
Di Donato team from Federico II University in Naples.
The characterization of the lower-meta pathway has not yet been completed. It was
shown that the phe operon has an homologous genetic organization to dmp operon (di-methylphenol
operon) of Pseudomonas sp. CF600. The research group previously mentioned also
expressed, purified and characterized the enzymes involved in the initial reactions of the meta
pathway: the catechol 2,3 dioxygenase (C2,3O), encoded by the pheB gene, the 2-
dehydrogenase (HMSD), encoded by the pheC gene, and the 2- hydroxymuconic
semialdehyde hydrolase (HMSH), encoded by the pheD gene. The C2,3O catalyzes the
extradiolic aromatic ring cleavage reaction of upper pathway catechol derivatives, with
production of 2-hydroxymuconic semialdehyde (HMS). The meta cleavage products are
metabolized by HMSD or by HMSH.
The sequencing of the meta pathway genes, for the phe operon complete
characterization, was completed by the group of Prof. Carla Caruso of the University of
Tuscia in Viterbo. The sequence of pheGFHI genes has been elucidated: they encode the 4-
Hydroxy-2-oxovalerate Aldolase (HOA) and the Aldehyde dehydrogenase (acylating) (ADA)
(pheG and pheF genes), the 4-Oxalocrotonate Decarboxylase (4OD) and the 4-Oxalocrotonate
Isomerase (4OI) (pheH and pheI genes).
An high degree of identity is between the aminoacidic sequences of ADA and HOA
enzymes from P. sp. OX1 and the corresponding DmpF and DmpG enzymes characterized in
P. sp. CF600; moreover, the crystallographic structures of DmpF and DmpG are available on
the PDB (Protein Data Bank) database. Therefore, was performed an homology modeling
study to realize the three-dimensional models of the two proteins for their structural
characterization.
The aims of this thesis are biochemical characterization of lower-meta pathway ADA
and HOA enzymes from Pseudomonas sp. OX1 and the elucidation of the transcriptional
mechanism by which the phe genes expression is regulated.
ADA and HOA are associated in a bifunctional enzyme complex by which the HOA
product, acetaldehyde, is channeled directly into the ADA active site, protecting the cell from
its toxicity. ADA requires NAD + and CoA as cofactors to turn acetaldehyde into acetyl-CoA.
Pyruvate and acetyl-CoA are the final products of the pathway and they will be
channeled into tricarboxylic acid cycle.
The functional characterization of ADA and HOA enzymes involved in this
channelling, begins with the construction of a heterologous system for the production of
recombinant proteins in E. coli and it is continued with the purification and the study of their
kinetic parameters. The characterization of recombinant proteins and the analysis of their
catalytic properties, together with the predicted 3D structure, will to achieve a fine structure function
correlation.
Pseudomonas sp. OX1 is able to grow using o-xylene as the sole carbon source but it is
not able to metabolize m-and p-xylene. From these two isomers of xylene, ToMO and PH
catalyze the oxidation of the aromatic ring to give 3,5- and 3,6-dimethylcatechols, that are not
metabolized by C2,3O, resulting toxic to the cell. However, Pseudomonas sp. OX1 retains
genes for the catabolism of m- and p-xylene, omologs to xyl genes of the TOL plasmid of
Pseudomonas putida mt-2, a strain that normally uses these compounds as growth substrates.
In environments contaminated by the two isomers of xylene, spontaneous mutant of
Pseudomonas sp. OX1 able to metabolized these compounds, but not the o-xylene, have been
isolated: the catabolism proceeds through the TOL pathway, through the progressive
oxidation of methyl group that leads to not lethal methylcatechols. It is believed that xyl genes
have been acquired by a catabolic transposons: in wild-type strain that operon is inactivated
by an insertion sequence, preventing the growth on m- and p-xylene; in the mutants, the
insertion sequence transposes in the locus coding ToMO, blocking the use of o-xylene.
During this work, were isolated the genes coding xyl-like enzymes: the Aldehyde
dehydrogenase (acylating) ADA and the Aldolase HOA, corresponding to the xylQK genes
from Pseudomonas putida mt-2. We created an heterologous system for the production of
recombinant proteins in E. coli. After their purification and the study of kinetic parameters,
we characterized Xyl-like ADA-HOA bifunctional enzyme structure by an homology
modeling study to create the 3D structure of the enzyme complex.
In microorganisms, the ability to readily activate or silence the expression of different
metabolic pathways is essential for adapting to a changing environment, ensuring the
performance not only of the single cell, but also of population and even of the microbial
community. Promoters have to process different physicochemical and metabolic signals to
regulate their activities: those signals are mediated by specific regulatory proteins. Many
operons for the catabolism of aromatic compounds are regulated by means of s54-dependent
circuit. The RNA polymerase (RNAP) containing the alternative sigma factor s54 recognizes
and binds a class of promoters, which are characterized by GG and GC motifs at positions -24
and -12, respectively. Enhancer-like elements or UASs (Upstream Activating Sequences),
located about 100 or 200 bp upstream of -12/-24 regions, represent the binding site of regulatory proteins. The s54-RNAP forms stable closed complexes with the promoter and is
unable to catalyze the isomerization to the open complex initiating transcription. The
isomerization takes place only upon interaction with an NtrC-like transcriptional activator.
These activators have three structural and functional domains: the amino-terminal A domain,
which recognizes and binds effector molecules; the carboxy-terminal D domain that binds the
DNA, and the central C domain with ATPase activity. In the absence of effectors, the A
domain acts as an intramolecular repressor, blocking the regulator in an inactive form; the
interaction between the A domain and the specific effector removes this repression, leading to
the active form, able to interact with DNA and s54-RNAP and promoting transcription.
We know the mechanism that regulates the expression of tou genes: a transcriptional
regulator belonging to the family of activators NtrC-like, TouR, positively controls the
expression of ToMO. Pseudomonas sp. OX1 is the first strain that degrades toluene in which
the toluene-monooxygenase encoding operon (tou operon) has been found associated to a
dmp-like operon (phe operon). The presence of a dmp-like operon (phe operon) together with
a phenol responsive regulator (TouR) suggests that, in this strain, the toluene and o-xylene
catabolic pathway evolved by vertical expansion which led to the incorporation of the tou
gene cluster in a preexisting route for phenol catabolism. This observation suggest that the
phe operon expression is under the control of a σ54-dependent promoter and can be regulated
positively by TouR or another activator belonging to the same family.
A further objective of this research is the isolation and characterization of the Pphe
promoter region. The phe operon 5' non-translated region of Pseudomonas sp. OX1 was
isolated and characterized. Were subsequently made a series of experiments that led to
isolation and identification of a putative regulation factor of the phe operon.
The ability shown by Pseudomonas sp. OX1 to grow on different aromatic substrates
with the ability to express otherwise silent genes in response to changes in environmental
conditions, gives an extraordinary metabolic versatility that makes this strain an ideal
candidate to be used in bioremediation.
Additional information
Dottorato di ricerca in Genetica e biologia cellulare
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