BMC Research Notes BioMed Central

Data Note Open Access

OxDBase: a database of oxygenases involved in biodegradation
Pankaj K Arora1, Manish Kumar2, Archana Chauhan1, Gajendra PS Raghava2
and Rakesh K Jain*1

Address: 1Environmental Biotechnology, Institute of Microbial Technology, Sector 39-A, Chandigarh-160036, India and 2Bioinformatics Centre,
Institute of Microbial Technology, Sector 39-A, Chandigarh-160036, India
Email: Pankaj K Arora - parora@imtech.res.in; Manish Kumar - manish@imtech.res.in; Archana Chauhan - archana@imtech.res.in;
Gajendra PS Raghava - raghava@imtech.res.in; Rakesh K Jain* - rkj@imtech.res.in
* Corresponding author

Published: 30 April 2009 Received: 21 November 2008

Accepted: 30 April 2009
BMC Research Notes 2009, 2:67 doi:10.1186/1756-0500-2-67
This article is available from: http://www.biomedcentral.com/1756-0500/2/67
© 2009 Jain et al; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract
Background: Oxygenases belong to the oxidoreductive group of enzymes (E.C. Class 1), which
oxidize the substrates by transferring oxygen from molecular oxygen (O2) and utilize FAD/NADH/
NADPH as the co-substrate. Oxygenases can further be grouped into two categories i.e.
monooxygenases and dioxygenases on the basis of number of oxygen atoms used for oxidation.
They play a key role in the metabolism of organic compounds by increasing their reactivity or water
solubility or bringing about cleavage of the aromatic ring.
Findings: We compiled a database of biodegradative oxygenases (OxDBase) which provides a
compilation of the oxygenase data as sourced from primary literature in the form of web accessible
database. There are two separate search engines for searching into the database i.e. mono and
dioxygenases database respectively. Each enzyme entry contains its common name and synonym,
reaction in which enzyme is involved, family and subfamily, structure and gene link and literature
citation. The entries are also linked to several external database including BRENDA, KEGG,
ENZYME and UM-BBD providing wide background information. At present the database contains
information of over 235 oxygenases including both dioxygenases and monooxygenases. This
database is freely available online at http://www.imtech.res.in/raghava/oxdbase/.
Conclusion: OxDBase is the first database that is dedicated only to oxygenases and provides
comprehensive information about them. Due to the importance of the oxygenases in chemical
synthesis of drug intermediates and oxidation of xenobiotic compounds, OxDBase database would
be very useful tool in the field of synthetic chemistry as well as bioremediation.

Background pounds constitute a major group of environmental pollut-

In the last few decades, extensive urbanization and rapid
industrialization has resulted in the addition of a large
number of xenobiotic compounds into the environment.
The chemical properties and quantities of the xenobiotic
compounds determine their toxicity and persistence in
the environment. Organic (aromatic/non-aromatic) com-
ants [1]. These compounds are highly persistent in the
environment due to their thermodynamic stability [2].
Many of these compounds have been reported to be toxic
to the living organisms [3]. Increased public awareness
about the hazards and toxicity of these compounds has
encouraged the development of technologies for their

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BMC Research Notes 2009, 2:67
remediation. Bioremediation, which utilizes the micro-
bial metabolic potential of the degrading microorgan-
isms, has come up as an efficient and cost-effective means
of large scale removal of these compounds in comparison
to the physico-chemical means of bioremediation. A
number of bacteria that can degrade a variety of aromatic
compounds have been identified and the pathways
involved in the degradation have been extensively charac-
terized [3,4]. Based on the complexity of the degradation
pathways, the phenomenon of biodegradation is catego-
rized into two types: convergent and divergent modes of
degradation (Fig. 1). In the convergent mode, structurally
diverse aromatic compounds are converted to one of a few
aromatic ring cleavage substrates such as catechol, gent
sate, protocatechuate and their derivatives [5]. Peripheral
enzymes, particularly oxygenases and dehydrogenases,
were found to transform structurally diverse substrates
into one of these central intermediates by bringing about
the hydroxylation of the aromatic nucleus (Fig. 2A), and
hence it is thought that bacteria have developed these
enzymes to extend their substrate range [5]. There are a
number of benefits of channeling diverse compounds
into a few central aromatic ring cleavage substrates; the
foremost among these being reduction of genetic load and
simplification of regulatory circuits. Further, the central-
ized degradation pathways mean synthesis of fewer degra-
dative enzymes requiring less metabolic energy. This is
clearly a major advantage to soil microbes which often
find themselves in unfavorable environments containing
low concentrations of carbon sources suitable for growth
[6]. However, further conversion of these intermediates
into tricarboxylic acid (TCA) cycle intermediates was
found to be highly diverged (divergent mode) (Fig. 1). In
this divergent mode, a metal-dependent dioxygenase
channels these dihydroxylated intermediates into one of
the two possible pathways: the meta-cleavage pathway or
the ortho-cleavage pathway [7-9] (Fig. 1). The substrate
specificity of these metal-dependent dioxygenases has
been found to play a key role in the overall determination
of pathway selection [5] and the dioxygenases have been
grouped into two classes namely extradiol and intradiol
dioxygenases [7]. Extradiol dioxygenases have nonheme
iron (II) at their active site and catalyze ring cleavage at the
carbon-carbon (C-C) bond adjacent to the vicinal
hydroxyl groups (meta-cleavage) (Fig. 2B) whereas intra-
diol dioxygenases have non-heme iron (III) in their active
site and catalyze ring cleavage at the C-C bond between
the vicinal hydroxyl groups (ortho-cleavage) (Fig. 2C).
Extradiol dioxygenases channel substrates into a meta-
pathway whereas intradiol dioxygenases channel these
substrates into an ortho-pathway. Similarly, monoxygen-
ases catalyze the transfer of one atom of molecular oxygen
to the organic compound with other being reduced by
electrons from cofactors to yield water thereby increasing
their reactivity and water solubility.
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Oxygenases are one of the key enzymes that play a central
role in the degradation/detoxification of compounds.
Without the activity of these oxygenases the mineraliza-
tion of these xenobiotic compounds is not possible.
Despite the fact that the oxygenases play such a crucial
role, limited information is available with respect to these
enzymes. None of the existing databases provide com-
plete and/or comparative information on all the oxygen-
ases known till date. Recent genomic, kinetics and
crystallographic studies on oxygenases have increased our
understanding of the distribution, evolution and mecha-
nism of these enzymes [10]. Studies on oxygenases have
also shown that extradiol dioxygenases are also involved
in the biosynthesis of a variety of biologically active com-
pounds e.g. lincomycin [11]. Keeping above in mind we
have developed a database of oxygenases mainly involved
in biodegradation of organic molecules. The oxygenases
having anabolic properties have also been included in this
database.
Construction
Database design and development
The PostgreSQL relational database management system
(RDBMS) is the main work-horse of OxDBase. It has been
used for storing, retrieval and managing the data. The
scripts, which provide interface between user and data-
base, were written in PERL and CGIPerl. For accessing
information from PostgreSQL Pgperl has been used. The
server OxDBase has been developed and launched on
SUN solaris 10.0 environment on T1000 machine using
Apache sever. Database entries were collected from differ-
ent sources such as the published literature like PubMed
http://www.ncbi.nlm.nih.gov/pubmed/, different exist-
ing databases such as UM-BBD http://
umbbd.msi.umn.edu/, KEGG http://www.genome.ad.jp/
kegg/, ENZYME http://www.expasy.ch/enzyme/,
BRENDA http://www.brenda-enzymes.info/index.php4.
The overall architecture of OxDBase is shown in Fig. 3.
The database contains two tables housing information of
118 monooxygenases and 119 dioxygenases respectively.
Data content and scope
OxDBase is a comprehensive database to provide infor-
mation about oxygenases (both mono- and di-oxygenase)
compiled from published literature and databases. The
information about each entry includes: i) name and
chemical structure of substrate and product; ii) link to the
gene or protein sequence using NCBI database; iii) link to
related PDB structures in the Protein Data Bank; iv) link
to the key external databases such as SWISS-PROT
ENZYME, BRENDA, KEGG and UM-BBD databases
(wherever possible, the International Union of Biochem-
istry and Molecular Biology (IUBMB) name along with
different synonyms by which that enzyme is known); and
v) link to the related published literature at PubMed jour-
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BMC Research Notes 2009, 2:67
Schematic
from Khajamohiddin
Figure 1 diagram showing
et al., 2008)
the role of aromatic dioxygenases in the bacterial degradation of aromatic compounds (Adapted
Schematic diagram showing the role of aromatic dioxygenases in the bacterial degradation of aromatic com-
pounds (Adapted from Khajamohiddin et al., 2008).
nals database has also been provided (Table 1). All entries
of the database are assigned a unique accession number to
identify them unambiguously.
Categorization and classification of data
All entries of OxDBase are divided into two broad classes
i.e. monooxygenases and dioxygenases depending on the
number of atomic oxygen used during oxidation. On the
basis of their mode of action dioxygenases are further cat-
egorized into aromatic ring cleavage dioxygenase (ARCD)
and aromatic ring hydroxylating dioxygenase (ARHD)
[12]. Depending upon the position of ring cleavage with
respect to the hydroxyl groups, ARCDs are again divided
into intradiol aromatic ring cleaving dioxygenase
(IARCD) and extradiol aromatic ring cleavage dioxygen-
ase (EARCD).
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Searching the database
OxDBase provides a number of methods to search the
database. The following are the major ways: (i) general-
ized search using keywords to search in all fields of data-
base; (ii) The Enzyme Commission number (EC number)
based searching that allows extraction of a unique OxD-
Base entry; and (iii) class based searching which restricts
the search within a specified class (described in categori-
zation and classification of enzymes).
In short, keyword search allows users to data mine on all
fields of the database ('EC number', 'IUBMB as well as
other popular names', 'Publication Reference', 'Reactant
and Substrate'). The keyword search could also be
restricted to a particular field and it also allows users to
select the fields to be displayed. An example of keyword
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Figureillustrating
Figure 2 the mechanism of action of aromatic dioxygenases
Figure illustrating the mechanism of action of aromatic dioxygenases. A) aromatic ring hydroxylating dioxygenase;
B) extadiol ring cleavage dioxygenase; and C) intradiol ring cleavage dioxygenase.

search is shown in Fig. 4A, where keyword 'catechol' is
searched in any field of database. The output/result of this
keyword search is shown in Fig. 4B.
Potential utility and limitations
OxDBase is a knowledge based database that provides
comprehensive information about oxygenases including
both monooxygenases and dioxygenases. The mechanism
of action of the oxygenases is based on hydroxylation of
the target molecule. During the recent years, selective
hydroxylation of aromatic ring has gained attention in the
synthetic biology because of the use of hydroxylated aro-
matics as drug intermediates. For example, the large scale
industrial production of carticosterone, cis-cis muconic
acid, pravastatin, indigo and 4-hydroxyproline have been
achieved by hydroxylation mechanism of oxygenases
[13]. Therefore, the information provided by OxDBase,
particularly reaction catalyzed by oxygenases would be a
very useful tool for synthesis of various biologically active
compounds. OxDBase also provides information of the
genes and three dimensional structure of the oxygenases
which can help in site directed mutagenesis of the
enzymes to improve their catalytic properties. The entries
of the oxygenases in OxDBase are linked to various exist-
ing database to provide detailed information of oxygen-
ases. Since oxygenases-catalyzed biotransformations of
the toxic xenobiotic compounds help in reducing the tox-
icity of the xenobiotics, therefore, detailed information of
these oxygenases would increase our understanding of
biodegradation process. The potential uses of these oxyge-
nases have been shown in fig. 5. We hope the OxDBase
would be very useful tool for development of better biore-
mediation strategies as well as synthesis of biologically
active compounds.
At present, OxDBase has 237 entries of distinct oxygen-
ases. Among them, 118 belong to monooxygenases and
119 related to dioxygenases. The primary aim of OxDBase
is to provide detailed information of all known oxygen-
ases because of their wide use in synthetic chemistry and
bioremediation. Hence, inspite of the limited informa-
tion available about oxygenases, OxDBase is largely com-

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Figure architecture
Overall 3 of OxDBase
Overall architecture of OxDBase.

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Table 1: Description and content of fields associated with each entry of OxDBase database.

Entry Field Description

Name Wherever possible IUBMB Name or common name of the enzyme

Category Chemical nature of substrate of dioxygenase
Subcategory Specificity and nature of oxidation of dioxygenase
Enzyme database The Enzyme Commissions identification (EC) number, link to the SWISS PROT ENZYME database
BRENDA database The EC number and link to the BRENDA Enzyme information system
KEGG database Link to molecular interaction network of Kyoto Encyclopedia of Genes and Genomes (KEGG) along with EC number
UMBBD database The accession number of University of Minnesota Biocatalysis/biodegradation database
Reaction The name and chemical structure of substrate and product
Synonyms Popular names other than the IUBMB
Related genes Link to the NCBI Entrez-gene database
Related structures PDB four letter identification code plus a link to the corresponding information
PubMed reference Link to the relevant literature to the PubMed database

Figure 4 of the OxDBase search A) for keyword search; and B) output of keyword search
Overview
Overview of the OxDBase search A) for keyword search; and B) output of keyword search.

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BMC Research Notes 2009, 2:67
OxDBase
A knowledge based database
Oxygenases
(monooxygenases and dioxygenases)
Bioremediation
Hydroxylation of xenobiotic
Aromatic ring cleavage compounds
Figure 5uses of oxygenases
Potential
Potential uses of oxygenases.
plete and of considerable importance. As new data
becomes available, the database will also increase in size.
Submission and updation of OxDBase
The web server allows user to submit new entry of oxyge-
nase on-line by filling a HTML form. However, before
including in OxDBase we will confirm the validity of new
entry in order to maintain the quality. Our team is also
searching and adding new entries of oxygenases in data-
base from published literature. The mechanism followed
for the curation and updation of the database has been
shown in fig. 6. In order to maintain the consistency we
will revive the OxDBase database quarterly.
Conclusion
OxDBase is a unique database which provides compre-
hensive information about oxygenases. It is a platform
from which users can easily retrieve information on all
available oxygenases. The present database would
increase our understanding of the biological, biochemi-
cal, genomic, evolutionary and structural properties of
oxygenases that could be exploited for industrial and
bioremediation applications.
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Industrial applications
In synthetic biology In protein engineering
Future work
As regards to the future work the database needs to be
maintained and developed further, ensuring the links to
all external databases remain correct and newly published
data is added. We hope, over the time, database size will
increase with accumulation of more experimental infor-
mation. In addition we also hope that data compilation
and distribution through a publicly available medium
will help in biodegradation research.
Availability and requirements
OxDBase is freely available at http://www.imtech.res.in/
raghava/oxdbase/.
List of abbreviations used
FAD: Flavin Adenine Dinucleotide; NADH: Nicotinamide
Adenine Dinucleotide Reduced; NADP: Nicotinamide
Adenine Dinucleotide Phosphate Reduced; PERL: Practi-
cal Extraction and Report Language; NCBI: National
Center for Biotechnology Information; PDB: Protein Data
Bank.; BRENDA: The Comprehensive Enzyme Informa-
tion System; UM-BBD: University of Minnesota Biocataly-
sis/Biodegradation Database; IUBMB: International
Union of Biochemistry and Molecular Biology; KEGG:
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Existing Databases Published literature

Search Entries submitted by Search

users in OxDBase

Validation of
data

Extract the new entries

Updation of OxDBase with new entries

Figure
A flow chart
6 showing the mechanism for the curation and updation of the database
A flow chart showing the mechanism for the curation and updation of the database.

Kyoto Encyclopedia of Genes and Genomes; ENZYME:
Enzyme Nomenclature Database.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
PKA and AC collected the data from literature and existing
databases. MK and PKA created tables in PostgreSQL and
developed web server. RKJ and GPSR conceived the
project, coordinated it and refined the manuscript drafted
by PKA and AC. All authors have read and approved the
final manuscript.
Acknowledgements
We are thankful to our Bioinformatics Centre for excellent technical help.
Authors are thankful to Council of Scientific and Industrial Research (CSIR)
and Department of Biotechnology (DBT), Govt. of India for financial sup-
port. This is IMTECH communication number 050/2008.
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Dihydroxyphenyl alanine-extradiol cleavage is followed by
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Rev Environ Sci Biotechnol
DOI 10.1007/s11157-010-9211-x
WEB ALERT
Tools of bioinformatics in biodegradation
Pankaj Kumar Arora • Wenxin Shi
Ó Springer Science+Business Media B.V. 2010
Rapid industrialization and urbanization cause
environmental pollution and release several toxic
compounds into the environment. Many of them have
been listed as priority pollutants by the United States
Environment Protection Agency (http://www.epa.gov)
due to their toxic nature. Further, the presence of
these compounds in the environment for a long time
can create severe problems to human health and
animals. This is a subject of serious concern and the
attention of environmentalists over a world has been
increased towards this problem in recent years. It has
been observed that physio-chemical methods are not
so cost-effective to remove these diffuse polluting
compounds from the environment and microbial
degradation is a potential way to degrade or detoxify
these compounds in the environment. Microbes have
the ability to use these compounds as sole source of
carbon and energy. Many microbial enzymes are
involved in the metabolism of these xenobiotic
compounds. Biochemical pathway(s) of microbial
degradation of several xenobiotic compounds have
P. K. Arora
Environmental Biotechnology, Institute of Microbial
Technology, Chandigarh 160036, India
e-mail: arora484@gmail.com
W. Shi (&)
State Key Laboratory of Urban Water Resources and
Environment, Harbin Institute of Technology,
150090 Harbin, China
e-mail: swx@hit.edu.cn
been studied and the genes and enzymes associated
with degradation pathway(s) were also identified and
characterized. The knowledge of microbes, enzymes,
genes and pathways involved in the biodegradation
of xenobiotic compounds may increase our under-
standing towards biodegradation process in full scale
and help to develop an effective clean up microbial
technology for the bioremediation of contaminated
red soils and sediment. Keeping these things in the
mind, biodegradation knocks the door of bioinfor-
matics to develop some powerful tools including
databases and pathway prediction system(s) that
provide information on enzymes, genes, pathways
and microbes involved in biodegradation process.
Furthermore, the pathway prediction system plays a
critical role to predict the degradation pathway of a
xenobiotic compound of which microbial degradation
was not yet available in the literature. In this article,
we will focus on a few important websites which
provide readers more informative resources related to
biodegradation of xenobiotic compounds.
1 University of Minnesota biocatalysis/
biodegradtion database
The UM-BBD database was developed in 1995 and
freely available on the site at http://umbbd.msi.
umn.edu/. This database provides information on
(a) microbial metabolic pathways of xenobiotic
compounds in both graphical as well as text form,
123

(b) enzymes and reactions catalyzed by them, (c) list
of microbes and their involvement in degradation
pathways, (d) compounds and their involvement in
degradation pathways as either substrates or products
or both, (e) biotransformation rules with descriptions,
(f) details of functional groups which are generally
present in xenobiotic compounds, and (g) multiple
reactions of naphthalene 1,2-dioxygenase and toluene
dioxygenase.
Currently, the UM-BBD database comprises of 194
microbial degradation pathways; 1,324 chemical reac-
tions catalyzed by different enzymes present in
microbes; 885 enzymes including monooxygenases,
dioxygenases, dehalogenases, reductases, hydroxy-
lases and dehydrogenases; 506 microbes; 267 bio-
transformation rules; 50 functional groups; 76
reactions of naphthalene 1,2-dioxygenase and 109
reactions of toluene dioxygenase. The entries of the
database are cross linked to several external databases
to provide a wide range of information. For example,
each enzyme entry is cross linked directly to the Expert
Protein Analysis System (ExPASy, http://www.
expasy.ch/), the Comprehensive Enzyme Information
System BRENDA (http://www.brenda-enzymes.info/),
and the Enzyme database (http://www.enzyme-data
base.org/). The links of genes and protein structures
have also been given. This site updates regularly and
the updated information is provided to the registered
users by e-mail.
The specialized feature of UM-BBD is the pathway
prediction system (PPS) that is available at http://
umbbd.msi.umn.edu/predict/aboutPPS.html. The users
can predict the microbial degradation pathway of a
chemical compound easily using the PPS that works
on biotransformation rules derived from reactions
found in the UM-BBD database or in the scientific
literature. This prediction system can provide an
accurate prediction the biodegradability of com-
pounds which are similar to those compounds whose
microbial degradation is already available in the
literature.
2 OxDBase: a database of biodegradative
oxygenases
Oxygenases belong to the oxidoreductive group of
enzymes, which oxidize the substrates by transferring
oxygen from molecular oxygen (O2) and utilize FAD/
123
Rev Environ Sci Biotechnol
NADH/NADPH as the co-substrate. They play a key
role in the metabolism of organic compounds by
increasing their reactivity or water solubility or
bringing about cleavage of the aromatic ring.
OxDBase (http://www.imtech.res.in/raghava/oxd
base/) is a comprehensive database to provide infor-
mation about oxygenases compiled from published
literature and databases. This database provides the
information of oxygenases that are involved in aero-
bic degradation of xenobiotic compounds. Currently,
OxDBase contained information of about 237 distinct
oxygenases including both monooxygenases (118) and
dioxygenases (119). All enzyme entries contain reac-
tion(s) in which an enzyme is involved, their common
names and synonyms, structure and gene links, family
and subfamily and literature citations. Enzyme entry is
also linked to several external databases including
Kyoto Encyclopedia of Genes and Genomes (KEGG,
http://www.genome.jp/kegg/), UM-BBD, BRENDA
and ENZYME.
The information provided by OxDBase, particularly
reactions catalyzed by oxygenases, is a useful tool for
synthetic biology. OxDBase is also helpful in site
directed mutagenesis of the enzymes to improve their
catalytic properties. Oxygenases-catalyzed biotrans-
formation of the toxic xenobiotic compounds reduce
the toxicity of the xenobiotics, therefore, this database
increases our understanding of biodegradation pro-
cesses. Users can easily retrieve information on all
available oxygenases using OxDBase.
For further details, please visit at http://www.
biomedcentral.com/1756-0500/2/67.
3 PBT profiler
The PBT Profiler (http://www.pbtprofiler.net/) was
designed to be an easy to use, widely available, no-cost
tool to screen chemicals lacking experimental data in
order to help identify pollution prevention (P2)
opportunities. The PBT profiler is a continuation of the
Office of Pollution Prevention and Toxics (OPPT,
http://www.epa.gov/oppt/) Pollution Prevention (P2)
Assessment Framework—a collection of screening
models and methods to help promote the design,
development, and application of safer chemicals and
processes. The P2 Framework uses computerized
methods, such as structure/activity relationships
(SARs) and standard scenarios, to predict risk related
Rev Environ Sci Biotechnol
data (physical/chemical properties, bioconcentration,
environmental fate, carcinogenicity, toxicity to aquatic
organisms, worker and general population exposure,
and other information) on chemicals lacking experi-
mental data. The PBT Profiler arose from experience
gained in the P2 Framework’s outreach program, a
vigorous set of initiatives by collaborators in the
business, government, and academic sectors to pro-
mote the voluntary use of these tools to reduce pollu-
tion and highlight the potential economic benefits of
informed environmental decision making.
Using the PBT profiler, one can identify chemicals
that potentially persist, bioaccumulate and are toxic to
aquatic life. The PBT profiler also provides the
information whether the chemical belongs to a
category that is known its toxic effects on human
health as described in the EPA chemical category
Report (http://www.epa.gov/opptintr/newchems/pubs/
cat02.htm). The PBT profiler is user friendly and users
can obtain data easily by entering a unique identifier
(e.g., a CAS Registry Number, product ID, or acro-
nym) or a chemical structure using a smiles notation.
The basis, on which the PBT profiler works, is avail-
able at http://www.pbtprofiler.net/methodology.asp.
4 Bionemo: biodegradation network-molecular
biology database
The Bionemo Database (http://bionemo.bioinfo.cnio.
es) focuses on molecular characterization of biodeg-
radation metabolism, which combines metabolic,
genetic and regulatory information. The central entities
of the database are the enzymatic complexes. These
enzymatic complexes are linked to the biochemical
reactions they perform defined as the transformation
of substrates into products. Reactions associate into
pathways. Enzymatic complexes are composed of pro-
tein subunits that are encoded by genes. Genes are
associated into transcription units that contain a single
promoter. These transcription units are regulated by
one or more regulatory complexes, comprising a
transcription factor, one or more DNA binding sites
and generally an inducer compound.
Bionemo has been built by manually associating
sequences database entries to biodegradation reac-
tions, using the information extracted from published
articles. Information on transcription units and their
regulation was also extracted from the literature for
biodegradation genes, and linked to the underlying
biochemical network. The bionemo database includes
(a) information on sequence, domains and structures
for proteins sequence and (b) regulatory elements and
transcription units for genes. Currently, this database
comprises of 145 biochemical pathways, 945 reac-
tions in which 342 reaction are with associated
complex, 537 enzymatic complexes, 1,107 proteins,
234 microbial species, 212 transcription units, 90
transcription factors, 90 effectors, 128 TF DNA
binding site and 100 promoters.
The database is also cross linked to several external
databases such as UMBBD for metabolic reactions;
gene bank for DNA sequences; Uniport for protein;
NCBI Taxonomy for microbial species and Pubmed
for references. The key feature of this database is that
the Bionemo covers all molecular aspects of biodeg-
radation. The information provided by Bionemo can be
useful to cloning, primer designing and directed
evolution experiment. The information in the Bionemo
database is available via a web server and the full
database is also downloadable as a PostgresSQL dump.
To facilitate the programmatic use of the information
contained in the database, an object-oriented Perl API
is also provided.
For more details about this database, please visit
http://www.ncbi.nlm.nih.gov/pubmed/18986994.
123
Journal of Bioremediation & Biodegradation - Open Access
Review Article
OPEN ACCESS Freely available online

www.omicsonline.org doi:10.4172/2155-6199.1000112

Application of Monooxygenases in Dehalogenation,

Desulphurization, Denitrification and Hydroxylation of
Aromatic Compounds
Pankaj Kumar Arora1, Alok Srivastava2* and Vijay Pal Singh2
1
Environmental Biotechnology, Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology, Sector-39A, Chandigarh-160036, India
2
Department of Plant Science, Faculty of Applied Sciences, M.J.P. Rohilkhand University, Bareilly -243006, India

Abstract
Monooxygenases act as biocatalysts in bioremediation process and synthetic chemistry due to their highly
regioselectivity and sterioselectivity on wide range of substrates. They are involved in the process of desulfurization,
dehalogenation, denitrification, ammonification, hydroxylation, biotransformation and biodegradation of various aromatic
and aliphatic compounds. In the recent years, the practical applications of monooxygenases have been improved using
the approaches of directed evolution, meta-genomics and bioinformatics. This review is focused on current applications
of monooxygenses especially in biodegradation and biotransformation of aromatic compounds.

Keywords: Monooxygenases; Desulfurization; Dehalogenation; monooxygenase (TcmH) from Streptomyces  glauscens [5]; ActVA-
Aromatic compounds; Biodegradation orf6 monooxygenase from Streptomyces coelicolor A3(2) [6]; quinol
monooxygenase (YgiN) from E.  Coli [7] and Rv0793 a hypothetical
Introduction monooxygenase from Mycobacterium tuberculosis [8].
Aromatic compounds are persistence environmental pollutants Aromatic dioxygenase are also divided into two subclasses
that are widely distributed in the biosphere due to anthropogenic based on mode of action: aromatic ring hydroxylation dioxygenases
activities. These compounds are highly toxic to living beings; therefore, (ARHDs) and aromatic ring cleavage dioxygenases (ARCDs) [1]. ARHD
many of them have been listed as priority pollutants by United catalyzes the incorporation of both atoms of oxygen into aromatic
States Environmental Protection Agency. Microbial degradation has ring (Figure 1b) whereas ARCD catalyzes ring cleavage of the aromatic
emerged as an effective technology to remove these compounds
from environment. Many microbial enzymes such as oxygenases,
dehalogenases, reductases, hydroxylases and dehydrogenases are
involved in the degradation of these environmental pollutants. Among
them, oxygenases are the key enzymes for aerobic biodegradation
of aromatic compounds because they are involved in the initial
reaction of degradation and also catalyze the ring cleavage of the
aromatic compounds which is the essential step for the complete
mineralization of these compounds [1].
Oxygenases catalyze insertion of one or two oxygen atoms into
the substrates. Two classes of oxygeases have been identified based
on number of oxygen atom used in the oxidation: monooxygenases
and dioxygenases [1]. Monooxygenases incorporate one atom of the
oxygen into the substrate (Figure 1a) whereas dioxygenases add both
atoms of the oxygen [1].
Monooxygenases are classified into two subclasses based on
which cofactor is present: Flavin dependent monooxygenases and Figure 1: Reactions catalyzed by oxygenases. (a) Monooxygenation of phenol;
P450 monooxygenases. Flavin dependent monooxygenases contain (b) aromatic ring hydroxylation of benzene; (c) intradiol aromatic ring cleavage
of catechol; (d) extradiol aromatic ring cleavge of catechol.
flavin as prostethic group and require NADP or NADPH as coenzyme
[2]. P450 monooxygenases are hame containing oxygenases and
found in both eukaryotic and prokaryrotic organisms. Best example of
bacterial P450 monooxygenase is CYP102 from Bacillus megaterium *Corresponding author: Dr. Alok srivastava, Department of Plant Science,
BM3 that is able to hydroxylate a variety of alkanes, fatty acids and Faculty of Applied Sciences, Rohilkhand University, Bareilly-243006, India, Tel:
aromatic compounds [3]. Other types of bacterial monooxygenases +919760821347; E-mail: alok.plsc@rediffmail.com
are also known that do not contain flavin or hame as a cofactor Received October 25, 2010; Accepted November 25, 2010; Published November
e.g., pterin-dependent monooxygenases and metal ion-dependent 27, 2010
monooxygenases [4]. Majority of monooxygenases require cofactor Citation: Arora PK, Srivastava A, Singh VP (2010) Application of Monooxygenases
for their activities, however, some monooxygenases have also been in Dehalogenation, Desulphurization, Denitrification and Hydroxylation of Aromatic
Compounds. J Bioremed Biodegrad 1:112. doi:10.4172/2155-6199.1000112
identified and characterized that do not require any cofactor for their
activities. These cofactor independent monooxygenases require only Copyright: © 2010 Arora PK, et al. This is an open-access article distributed under
molecular oxygen for their activities and utilize the substrate as reducing the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and
agent. Examples of these mononoxygenases are tetracenomycin F1 source are credited. 

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Citation: Arora PK, Srivastava A, Singh VP (2010) Application of Monooxygenases in Dehalogenation, Desulphurization, Denitrification and
Hydroxylation of Aromatic Compounds. J Bioremed Biodegrad 1:112. doi:10.4172/2155-6199.1000112
Figure 2: Dehalogenations of polychlorinated nitroaromatic compounds by
monooxygenases. (a) Dehalogenation of pentachlorophenol by pentachlorophenol
4-monooxygenase; (b) dehalogenation of 2,4,5-trichlorophenol by chlorophenol
4-monooxygenase; (c) dehalogenation of 2, 4, 6-trichlorophenol by 2, 4,
6-trichlorophenol monooxygenase (d) sequential dehalogenation of 2, 4,
6-trichlorophenol by 2, 4, 6-trichlorophenol monooxygenase
ring typically carrying two or more hydroxyl groups [1]. ARCDs are
further divided into two groups based on the ring cleavage: intradiol
(IARCD) and extradiol (EARCD) [1]. Intradiol cleaves the aromatic ring
between two hydroxyl groups (ortho-cleavage) (Figure 1c), whereas
extradiol cleaves the aromatic ring between a hydroxylated carbon
and an adjacent non-hydroxylated carbon (meta-cleavage) (Figure 1d).
Several publications have been focused on dioxygenases and
their role in biodegradation [9-12]. But very few reviews are available
that cover the role of the monooxygenases in biodegradation
[13]. In this review, we have discussed the current applications of
the monooxygenases in biodegradation and biotransformation of
aromatic compounds.
Monooxygenases and Dehalogenation of Polychlorinated
Aromatic Compounds
Polychlorinated compounds are widely distributed in the
environment and cause serious health problem to humans beings
and animals. These compounds are considered recalcitrant due
to presence of multiple chlorine atoms at benzene ring. Examples
of these compounds are polychlorinated biphenyls (PCB),
pentachlorophenol (PCP), dichlorophenol (DCP), trichlorophenol
(TCP), dioxin, 2, 4-dichlorophenoxyacetic acid (2,4-D) and
dichloropheyltrichloroethane (DDT).
Microbial degradation of polychlorinated aromatic compounds
initiated with the removal of the chloride atom from a benzene ring.
Chloride atom can be removed from aromatic ring in three different
ways: hydrolytic, reductive and oxygen dependent dehalogenation
[14]. Hydrolytic dehalogenation includes replacement of chlorine
atom with hydroxyl group. This hydroxyl group is derived from water.
Reductive dehalogenation involves replacement of chlorine atom
with hydrogen atom whereas oxygenolytic dehalogenation involves
replacement of chlorine atom by hydroxyl group whose oxygen atom
is derived from O2.
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Oxygenolytic dehalogenation is further divided into two
classes: dioxygenase type dehalogenation and monooxygenase
type dehalogenation. Dioxygenase type dehalogenase adds two
atoms of oxygen into the substrate to remove the chlorine atom.
They involved in dehalogenation of monochlorinated aromatic
compounds such as 4-chlorophenyl acetate and 2-chlorobenzoate
[14]. Monooxygenase type dehalogenase adds one atom of oxygen
to chlorinated compounds to remove the chlorine atom. Examples
of monooxygenase type dehalogenases are pentachlorophenol
4-monooxygenase, chlorophenol 4-monooxygenase and 2, 4,
6-trichlorophenol monooxygenase. These monooxygenases are
involved in dehalogenation of polychlorinated aromatic compounds
and have been characterized and purified from a variety of
microorganisms.
Pentachlorophenol 4-monooxygenase (EC 1.14.13.50)
Pentachlorophenol 4-monooxygenase (PcpB) is a flavin
monooxygenase that catalyzes hydroxylation at para position with
removal of the chloride ion in the initial step of the microbial
degradation of pentachlorophenol (Figure 2a). As a result of
hydroxylation, pentachlorophenol converted to tetrahydroquinone
[15]. PcpB also catalyzes reaction on various substituted aromatic
compounds with release of nitro, amino or cyano group from para
position [16]. PcpB is encoded by the pcpB gene that has been
identified in a variety of PCP degrading bacteria [17,18]. Examples
of PCP degrading bacteria are Sphingonium, Sphingomonads and
Pseudomonas. The pcpB gene was identified in the four strains of
Sphingonium chlorophenolica (ATCC 39723, RP-2, SR-3 and ATCC
33790) in which three strains (ATCC 39723, RP-2, SR-3) had identical
pcpB gene sequence [19, 20] whereas the sequence of the pcpB
gene of strain ATCC 33790 was differed by 10% from rest of three
strains [20]. The pcpB gene sequence of Sphinogomonads strain
UG-30 showed 90% sequence similarity with that of Sphingonium
chlorophenolica ATCC 39723 [21-23]. The homologus of pcpB
gene has been identified in polychlorinated degrading bacterium
Novosphingonium sp. strain MT1 [24] as well as in two non-PCP
degrading β- and γ- proteobacterial strains isolated from soil samples
from a PCP-contaminated wood treatment site [18].
Pentachlorophenol 4-monoxygenase (PcpB) has been purified
and characterized from Sphingomonas chlorophenolica ATCC 39723.
PcpB was comprised of 538 amino acids with molecular weight of
39 kD. PcpB was synthesized in cytoplasm and then translocated to
periplasm via inner memberane [25]. The crystal structure of PcpB
is yet not solved. However, model of 3D structure of PcpB has been
proposed on the basis of homology modelling [26]. The active site
residue has been determined from proposed 3D structure and site
directed mutagenesis was carried out to change the active site residue
[26]. Mutant thus obtained produced mutant proteins that purified
by affinity chromatography. The finding of mutation study supported
the validity of proposed 3D structure based on homology modelling
[26] . On the basis of experiment of site directed mutagenesis of
active site of PcpB, Nakamura et al. (2004) concluded that Phe 85,
Try 216 and Arg 235 are for enzyme activity and Tyr 397 and Phe 87
stablize the structure of the protein [26].
Chlorophenol 4-monooxygenase (EC 1.14.13.-)
This enzyme is involved in the degradation pathway of 2, 4,
5-trichlorophenoxy acetic acid by Burkholderia cepacia AC1100 [27].
This enzyme catalyzes conversion of 2,4,5-trichlorophenol (2,4,5-
TCP) to 2,5-dichloro-p-benzoquinone, which is chemically reduced to
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Hydroxylation of Aromatic Compounds. J Bioremed Biodegrad 1:112. doi:10.4172/2155-6199.1000112

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2,5-dichloro-p-hydroquinone (2,5-DiCHQ) (Figure 2b) [28]. The gene
encoding chlorophenol monooxygenase (tftd) has been identified,
sequenced and cloned from Burkholderia cepacia AC1100 and
transformed into E. coli for overexpression [27-30]. The molecular
weight of the purified protein was 58 kD [28]. This protein utilizes
O2, FAD and NADH for catalyzing the reaction but does not utilize
riboflavin and NADPH [28].
2, 4, 6-Trichlorophenol Monooxygenase (EC 1.14.13.-)
This enzyme was first reported in Azotobacter sp. GP1 and catalyzes
conversion of 2,4,6 trichlorophenol to 2,6-dichlorohydroquinone
with release of chloride ion (Figure 2c) [31]. TCP monooxygenase from
Azotobacter was a homotetrameric protein with molecular weight of
240 kD. Another 2,4,6-trichlorophenol (2,4,6-TCP) 4-monooxygenase
was found in Ralstonia eutropha JMP134 that catalyzes sequential
dechlorinations of 2,4,6-TCP to 6-chlorohydroxyquinol [32]. This
monooxygenase oxidize 2, 4, 6-TCP to 2, 6-dichloroquinone that
further hydrolyzed to 2-chlorohydroxyquinone by the same enzyme
(Figure 2d). 2-Chlorohydroxyquinone was then reduced by ascorbate
and NADH to 6-chlorohydroxyquinol [32].
optimum temperature for activity of DszA and DszC was determined
44°C and 65°C respectively.
Recombinant bacteria have also been created to enhance
the activity of biodesulfurization [48,49]. The recombinant strain
desulfurizes DBT more efficiently than the native one, and also provides
new alternative for the development of a commercial desulfurization
process [50]. A genetically modified organism Pseudomonas putida
CECT 5279 carrying dszABC genes from Rhodococcus erythropolis
IGTS8 and a flavin oxidoreductase (hpac) from E.coli was constructed
for biodesulfurization process [51]. This recombinant strain produced
maximum amount of DszB enzyme at the early exponential phase
that rapidly decreased at the middle exponential phase, hampering
an efficient transformation of HBPS into HBP. To overcome this
problem, a model two-step BDS resting cell process using P. putida
cells from the late and early exponential growth phases was designed
to significantly increase biodesulfurization. In the first step, the cells
of late exponential phase (10 h grown cells) of P. putida CECT 5279
showing the maximum activities of DszA and DszC monooxygenases,

Monooxygenases Involved in Biodesulfurization of

Dibenzothiophene
Sulphur containing organic compounds such as dibenzothiophene
(DBT) are present in the fossil fuels. On combustion of these fossil
fuels, sulphur dioxide is released into the environment and causes air
pollution. The complete removal of sulphur from these compounds
is not possible by conventional physical and chemical methods.
Biodesulfurization, therefore, is a process that completely removes
sulphur from these organic compounds. Biodesulfurization of DBT is
well characterized in Rhodococcus erythropolis IGTS8 [33]. Two flavin
dependent monooxygenases, DBT monooxygenase (DszC) and DBT
sulfone monooxygenases (DszA) are involved in the initial steps of
the biodesulfurization. DBT monooxygenase (EC 1.14.13.-) converts
DBT to DBT sulphoxide which is further converted to DBT sulphone
by same enzyme (Figure 3a and 3b). DBT sulfone monooxygenase
Figure 3: Biodesulfurization of DBT. (a) conversion of DBT to DBT sulphoxide
(EC 1.14.13.-) converts DBT sulfone to 2’-hydroxybiphenyl 2-sulfinate by DBT monooxygenase; (b) conversion DBT sulphoxide to DBT sulphone
(HBPS) (Figure 3c). Both of the monooxygenases require another by DBT monooxygenase; (c) conversion of DBT sulphone to HBPS by DBT
enzyme flavin reductase (DszD) for their activities. One more sulfone monooxygenase and (d) conversion of HBPS to HBP by HBPS
desulfinase.
enzyme HBPS desulfinase (DszB) is also involved in the final step of
biodesulfurization and converts HBPS to hydroxybiphenyl (HBP) and
sulphate (Figure 3d). In R. erythropolis, enzymes DszA, DszB and DszC
are encoded by plasmid located dsz operon and another enzyme DszD
is considered as genome encoded [34, 35]. The desulfurization genes
was conserved among Rhodococcus species [36]. Several strains of R.
erythropolis, i.e., SY1, D-1, Ni-36, Ni-43, and QIA-22 exhibited same
DBT-desulfurizing reaction as reported in Rhodococcus erythropolis
IGTS8 [37-40].
Although the biochemistry, genetics and physiology of
desulphurization have been extensively studied in Rhodococcus
sp., the desulfurizing genes (dszABC) have also been identified in
other mesophilc bacteria [41-43] as well as thermophilc bacteria.
The sequences of the dszABC genes of a thermophilic bacterium
Mycobacterium phlei that carry out biodesulfurization at range of 20-
50°C, was found to be identical to that of Rhodococcus erythropolis
IGTS8 [44-46]. The DszA and DszC from the thermophilic bacterium
Paenibacillus sp. strain A11-2 have been purified and characterized
Figure 4: Monooxygenases involved in denitrification. (a) Conversion of
[47]. DszA is dimeric protein with molecular weight of 120 kD and 4-nitrophenol to p-benzaquinone by 4-nitrophenol 4-monooxygenase; (b)
subunit molecular weight of 48 kD whereas DszC was tetrameric conversion of 4-nitrophenol to 4-nitrocatechol by 4-nitrophenol 2-monooxygenase;
protein with molecular weight of 200 kD and subunit mass 43 kD. The and (c) conversion of 2-nitrophenol to catechol by 2-nitrophenol 2-monooxygenase.

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Hydroxylation of Aromatic Compounds. J Bioremed Biodegrad 1:112. doi:10.4172/2155-6199.1000112

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were transformed all DBT into 50% mixture of HBPS and HBP [51]. In
the second step, the cells of late exponential phase have removed
and the early exponential phase cells (5 h grown cells) were added.
After addition remaining HBPS was efficiently converted to HBP [51].
Another recombinant bacteria has been design by transferring
the pVLT31 vector harboring flavin-oxidoreductase gene (dszD) into
the recombinant P. aeruginosa ATCC 9027 which contained dszABC
gene in its chromosome stably [52]. This recombinant bacteria
showed the enhanced activity of biodesulfurization when all four
genes co expressed. The biodesulfurization efficiency has also been
significantly improved using the approach of directed evolution.
Arensdorf et al. (2002) presented an example of directed evolution
of biodesulfurization using chemostat approach [53]. Rhodococcus
erythropolis IGTS8 has not ability to utilize octyl sulphide and
5-methylbenzothiophene (5-MBT) as sole sulphur sources. Arensdorf
et al. (2002) selected gain-of-function mutants of strain IGTS8 using
sulphur limited chemostat [53]. These mutants were grown with octyl
sulphide and 5-MBT as sole sulphur sources. The ability of mutant
to grow on 5-MBT as sole sulphur source was due to a transversion
(guanine to thymine) in dszC codon 261 [53].
Monooxygenases and Denitrification of Nitro Aromatic
Compounds
Nitroaromatic compounds are serious environment pollutants
that are used in the synthesis of pesticides, drugs, dyes and explosives
[54]. Nitro phenols are the most common examples of nitroaromatic
compounds. The toxicity of these compounds is mainly due to the
presence of nitro group in the aromatic ring. The aerobic degradation
of these compounds generally initiate with removal of the nitro group
by oxygenase activity [55]. Examples of monooxygenases involved
in denitrification of nitroaromatic compounds are 4-nitrophenol
4-monooxygenase, 4-nitrophenol 2-monooxygenase and
2-nitrophenol 2-monooxygenase. 4-Nitrophenol 4-monooxygenase
(EC 1.14.13.-) has been purified and characterized from Pseudomonas
sp. strain WBC-3 [56]. This enzyme is a single component and flavin
adenine dinucelotide monooxygenase that catalyzes conversion of
p-nitrophenol to p-benzoquinone in the presence of NADPH (Figure
4a). This enzyme also catalyzes NADPH dependent conversion of
nitrocatechol to 1, 2, 4-benzenetriol [57]. Another denitrifying
4-nitrophenol 2-monooxygenase (EC 1.14.13.29) has been purified
and characterized from Rhodococcus sp. strain PN1 [58]. This enzyme
is a two-component flavin adenine dinucelotide monooxygenase
and converts p-nitrophenol to 4-nitrocatechol in the presence
of NADH (Figure 4b) whereas NADPH dependent 2-nitrophenol-
2-monooxygenase (EC 1.14.13.31) catalyzes the conversion of
2-nitrophenol to catechol (Figure 4c) [55].
Monooxygenases and hydroxylation of aromatic compounds
Several monooxygenases have been identified and characterized
that involved in the biodegradation of various aromatic compounds
and have been listed in the (Table 1). A few monooxygenases acting on
aromatic compounds have been used as biocatalysis for synthesis of
the pharmaceuticals compounds. Examples of these monooxygenases
are styrene monooxygenase, hydroxybiphenyl 3-monooxygenase
and phenylacetone monooxygenase. Styrene monooxygenase (EC
1.14.13.-) from Pseudomonas sp. VLB 120 has ability to convert
styrene to s-styrene oxide with an enantiomeric excess of greater than

Enzyme E.C number Substrate(s) References

4-Methyl 5-nitrocatechol monooxygenase 1.13.12.- 4-Methyl-5-nitrocatechol [58]
Toluene 3-monooxygenase 1.14.-.- 3-Hydroxytoluene, Toluene [59]
Toluene 2-monooxygenase 1.14.13.- 2-Hydroxytoluene, Toluene, 1,1,2-Trichloroethylene [60]
4-Hydroxyacetophenone monooxygenase 1.14.13.- 4-Hydroxyacetophenone [61]
Toluene 4-monooxygenase 1.14.13.- Toluene, N-Nitrosodimethylamine [62]
4-Hydroxybenzoate 3-monooxygenase 1.14.13.2 4-Hydroxybenzoate [63]
http://umbbd.msi.umn.edu/servlets/
2-Mercaptobenzothiazole monooxygenase 2-Mercaptobenzothiazole [64]
pageservlet?ptype=r&reacID=r1178 1.14.-.-
6-Hydroxy-2-mercaptobenzothiazole
1.14.-.- 6-Hydroxy-2-mercaptobenzothiazole [64]
monooxygenase
Benzothiazole monooxygenase 1.14.-.- Benzothiazole [65]
Phenylboronic acid monooxygenase 1.14.-.- Phenylboronic acid [66]
2,6-Dihydroxybenzothiazole monooxygenase 1.14.-.- 2,6-Dihydroxybenzothiazole [67]
2-Hydroxybenzothiazole monooxygenase 1.14.-.- 2-Hydroxybenzothiazole [65]
1-Indanone monooxygenase 1.14.-.- 1-Indanone [68]
2-Indanone monooxygenase 1.14.-.- 2-Indanone [68]
6-Hydroxy-3-methyl-2-oxo-1,2-dihydroquinoline
1.14.-.- 6-Hydroxy-3-methyl-2-oxo-1,2-dihydroquinoline [69]
6-monooxygenase
Toluene-4-sulfonate monooxygenase 1.14.-.- Toluene 4-sulfonate [70]
Dibenzothiophene 5,5-dioxide monooxygenase 1.14.13.- Dibenzothiophene-5,5-dioxide [71]
3-Methyl 2-oxo 1,2-dihydroquinoline
1.14.-.- 3-Methyl-2-oxo-1,2-dihydroquinoline [69]
6-monooxygenase
BCDS monooxygenase 1.14.-.- Branched-chain dodecylbenzene sulfonate [72]
Toluene sulfonate methyl monooxygenase 1.14.13.- Toluene-4-sulfonate [73]
Orcinol 2-monooxygenase 1.14.13.6 Orcinol [74]
3-Hydroxybenzoate 4-hydroxylase 1.14.13.23 3-Hydroxybenzoate [75]
3-Hydroxybenzoate 6-monooxygenase 1.14.13.24 3-Hydroxybenzoate [76]
4-Methoxybenzoate monooxygenase 1.14.99.15 4-methoxybenzoate [77]
Benzoate 4-monooxygenase 1.14.13.12 Benzoate [78]
N-isopropylacetaniline monooxygenase 1.14.15.- N-Isopropylacetanilide [79]
Table 1: Monooxygen6+ases involved in the biodegradation of aromatic compounds.

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Hydroxylation of Aromatic Compounds. J Bioremed Biodegrad 1:112. doi:10.4172/2155-6199.1000112
99% [81]. Styrene monooxygenase is a two component enzyme: StyA
(oxygenase) and StyB (reductase). A recombinant E. Coli expressing
StyA and StyB has been used as whole cell biocatalyst for a broad
substrate spectrum that gives access to various chiral acryloxides
[82]. Another commercially important enzyme, 2-hydroxybiphenyl
3-monooxygenase (HbpA, EC 1.14.13.44) from Pseudomonas azelaica
HBP1 catalyzes ortho-hydroxylation of 2-hydroxybiphenyl to 2,
3-dihydroxybiphenyl and also catalyzes the conversion of different
2-substituted phenols to 3-substituted catechols that are of interests
of pharmaceuticals industry [83,84] . The substrate reactivity of
2-hydroxybiphenyl 3-monooxygenase (HbpA) have been improved
by directed evolution [84]. A variant of recombinant E. coli JM101
expressing HbpA was used for the production of 3-tert-butylcatechol,
a compound of pharmaceutical interest [86].
Phenylacetone monooxygenase (PAMO, EC 1.14.13.92) is a FAD-
containing Baeyer-Villiger monooxygenase (BVMO). This enzyme was
discovered from Thermobifida fusca that is a moderately thermophilic
soil bacterium and grows at 55°C. PAMO oxidizes phenylacetone,
aromatic and aliphatic ketones, aromatic sulphides and sulphoxides
and organoborn compounds [87]. This enzyme has thermostability
and tolerance towards organic solvents [88], therefore, this enzyme
is suitable for various industrial applications.
Current ways to imporve the efficiency of monooxygenases
Metagenomic approach: Microbial diversity is a rich source
of novel enzymes including monooxygenases. To date, only 1%
microorganisms could be cultured in the laboratory conditions and
monooxygenases associated with these culturable microbes have
been identified and characterized. Most potential monooxygenases
remain untapped because vast majority of bacteria are uncluturable.
In such a case, metagenomics may be powerful tool to explore
novel monooxygenases from entire community of microbes. The
metagenomic approach involves (i) the isolation and purification of
DNA from an environmental sample, (ii) cloning of DNA fragments into
suitable vectors, (iii) the transformation of host cells with construct
and (iv) functional and sequence based screening of constructed
clones. Numerous novel biocatalysts have been isolated from various
metagenomic libraries. A novel styrene monooxygenase (SmoA) was
discovered by the screening of a metagenomic library from loam
soil [89]. SmoA is highly enantioselective, since it is able to convert
aromatic alkenes into the (S)-epoxides with an excellent enantiomeric
excess. Recently, two flavin monooxygenases catalyzing the oxidation
of indole to a mixture of indigo and indirubin pigments have been
identified from an effluent treatment plant sludge metagenomic
library. A new self-sufficient CYP, SYK181was identified during
sequence-based screening of a metagenomic library [91]. SYK181
showed significant hydroxylase activity towards naphthalene and
phenanthrene as well as towards fatty acids [91]. Therefore, it is very
useful for the biodegradation of organic chemicals.
Directed evolution: In the recent years, directed evolution is
playing a major role for engineering of the proteins for bio catalytic
applications. Directed evolution is based on the Darwinian principle
of evolution and involves (i) random mutagenesis to generate the
diversity and (ii) the subsequent selection of the improved variants
by screening. The efficiency of several monooxygenases has been
significantly improved by directed evolution. For example, CYP102
from Bacillus  megaterium BM3 has been turned by directed
evolution into an enzyme that hydrolyze aromatic compounds
like 2,6-dichlorophenol and 2-benzyloxyphenol which cannot be
hydrolyzed by wild type enzyme [92]. Furthermore, a triple mutant
J Bioremed Biodegrad
ISSN:2155-6199 JBRBD, an open access journal
Page 5 of 8
of CYP102A1 has converted β-ionone into flavorant (R)-4-hydroxy-β-
ionone in a regioselective manner and exhibited 80-fold higher activity
compared to the wild type enzyme [93] . Another example of directed
evolution is cyclohexanone monooxygenase (CHMO; EC 1.14.13.22)
from Acinetobacter sp. NCIMB 9871. This enzyme act as a biocatalyst
in the Baeyer–Villiger (BV) reaction of 4-hydroxycyclohexanone and
other 4-substituted cyclohexanone derivatives [94]. It has been
used for commercial production of bicyclic lactone, sulfoxides,
thiosulfinates and cyclic sulphates [95-97]. The range of the
substrates and enantioselectivity of this enzyme have been increased
by directed evolution [98,99]. Reetz et al. (2004) demonstrated that
some CHMO mutants showed improved enantioselectivity, preferring
the (R)-enantiomer (49-54% ee vs 9% ee for the wild type enzyme)
in the oxidation of prochiral 4-hydroxycyclohexanone whereas other
variants favored (S)-enantiomer (79% ee).
Bioinformatic approach: Database providing information about
the biochemistry and genetics of monooxygenases are valuable tools
of the computational biology. Currently, two databases namely UM-
BBD (University of Minnesota Biocatalysis/Biodegradation Database)
[100,101] and OXDBase (A Database of Biodegradative Oxygenases)
[1] are providing the information about all monooxygenases involved
in biodegradation. The characteristics feature of these databases is
to provide information of the genes and three dimensional structures
of the monooxygenases which can help in site directed mutagenesis
of the monooxygenases to improve their catalytic properties [1].
The entries of the monooxygenases in OxDBase as well as UM-BBD
are linked to various existing databases which provide to users the
detailed information of monooxygenases [1]. Since monooxygenases
catalyzed biotransformations of the toxic xenobiotic compounds help
in reducing the toxicity of the xenobiotics, therefore, the detailed
information of these monooxygenases increase our understanding of
biodegradation process [1].
Homology modelling is a tool of bioinformatics that use to
generate reasonable models of protein structures. The determination
of 3D structure of monooxygenase by homology modelling is a helpful
to design the experiment of site directed mutagenesis in active site
of that protein to improve the efficiency of the monooxygenase.
The three dimensional structure of tolune 4–monooxygenase
determined by homology modelling has helped in altering the
activity of this enzyme by active site engineering for the synthesis of
3-methoxycatechol, methoxyhydroquinone and methylhydroquinone
[102].
Conclusion
Monooxygenases are multifunctional enzymes and involved in
biodesulfurization, dehalogenation, denitrification and hydroxylation
of various aromatic compounds. The applications of monooxygenases
have been improved using the recent molecular and bioinformatic
approaches.
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ISSN:2155-6199 JBRBD, an open access journal