Expression, purification, characterization and in silico analysis of newly isolated hydrocarbon degrading bleomycin resistance dioxygenase

Article abstract of DOI:10.1007/s11033-019-05159-x

In the present investigation, we report cloning, expression, purification and characterization of a novel Bleomycin Resistance Dioxygenase (BRPD). His-tagged fusion protein was purified to homogeneity using Ni-NTA affinity chromatography, yielding 1.2 mg of BRPD with specific activity of 6.25 U mg^?1 from 600 ml of E. coli culture. Purified enzyme was a dimer with molecular weight ~ 26 kDa in SDS-PAGE and ~ 73 kDa in native PAGE analysis. The protein catalyzed breakdown of hydrocarbon substrates, including catechol and hydroquinone, in the presence of metal ions, as characterized via spectrophotometric analysis of the enzymatic reactions. Bleomycin binding was proven using the EMSA gel retardation assay, and the putative bleomycin binding site was further determined by in silico analysis. Molecular dynamic simulations revealed that BRPD attains octahedral configuration in the presence of Fe²⁺ ion, forming six co-ordinate complexes to degrade hydroquinone-like molecules. In contrary, in the presence of Zn²⁺ ion BRPD adopts tetrahedral configuration, which enables degradation of catechol-like molecules.

Full text of DOI:10.1007/s11033-019-05159-x

Molecular Biology Reports
https://doi.org/10.1007/s11033-019-05159-x
ORIGINAL ARTICLE
Expression, purifcation, characterization and in silico analysis
of newly isolated hydrocarbon degrading bleomycin resistance
dioxygenase
Vinay Sharma1 · Rajender Kumar2 · Vishal Kumar Sharma3 · Ashok kumar Yadav3 · Marja Tiirola4
Pushpender Kumar Sharma1
·
Received: 9 July 2019 / Accepted: 23 October 2019
© Springer Nature B.V. 2019
Abstract
In the present investigation, we report cloning, expression, purifcation and characterization of a novel Bleomycin Resist-
ance Dioxygenase (BRPD). His-tagged fusion protein was purifed to homogeneity using Ni-NTA afnity chromatography,
yielding 1.2 mg of BRPD with specifc activity of 6.25 U mg−1 from 600 ml of E. coli culture. Purifed enzyme was a dimer
with molecular weight ~ 26 kDa in SDS-PAGE and ~ 73 kDa in native PAGE analysis. The protein catalyzed breakdown of
hydrocarbon substrates, including catechol and hydroquinone, in the presence of metal ions, as characterized via spectro-
photometric analysis of the enzymatic reactions. Bleomycin binding was proven using the EMSA gel retardation assay, and
the putative bleomycin binding site was further determined by in silico analysis. Molecular dynamic simulations revealed
that BRPD attains octahedral confguration in the presence of Fe2+ ion, forming six co-ordinate complexes to degrade
hydroquinone-like molecules. In contrary, in the presence of Zn2+ ion BRPD adopts tetrahedral confguration, which enables
degradation of catechol-like molecules.
Keywords Metagenomics · Dioxygenase · Aromatic hydrocarbons · Pesticides · Pollutant · Molecular modelling
Introduction
physico-chemical techniques are often inefcient in remov-
ing them. Bioremediation, a cost-efective technique that
involve microbes can remove these persistent environmen-
tal pollutants via enzymatic catabolism [4]. Soil bacteria
especially uncultured subset are potential catalytic sources
for biodegradation of aromatic hydrocarbons [5]. Ring-
cleaving dioxygenases play an important role in aromatic
metabolism, both in eukaryotes and prokaryotes. These
dioxygenases are classifed as intradiol dioxygenases which
catalyzes ortho cleavage and extradiol dioxygenases which
catalyze meta cleavage. Both classes of enzymes have dis-
tinct features and specifcities [4]. The extradiol dioxyge-
nases are generally more versatile, cleaves broader number
of substrates including antibiotics [3, 5]. In this category,
hydroquinone dioxygenases (HQDO) are key enzymes
as they act on hydroquinone (HQ), a key intermediate of
several aromatic hydrocarbons such as 4-nitrophenol [6,
7], γ-hexachlorocyclohexane [8], 4-hydroxyacetophenone
[9] and 4-aminophenol [10]. Hydroquinone dioxygenases
(HQDOs) are divided into 2 subtypes sharing few similari-
ties [11]. Members of type I dioxygenases are phylogeneti-
cally correlated with the well-described extradiol catechol
Aromatic hydrocarbons (AHs) including pesticides are
highly toxic compounds and are major chemical hazards dis-
charged into surroundings from diferent sources, including
agricultural and industrial wastes [1–3]. These pollutants
are very stable within the surroundings, and the existing
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11033-019-05159-x) contains
supplementary material, which is available to authorized users.
* Pushpender Kumar Sharma
pushp_78@rdifmail.com
1
Department of Biotechnology, Sri Guru Granth Sahib World
University, Fatehgarh Sahib, Pb, India
2
Department of Clinical Microbiology, Umeå University,
90185 Umeå, Sweden
3
4
PGI, Chandigarh, India
Department of Biological and Environmental Science,
Nanoscience Center, University of Jyväskylä,
40014 Jyvaskyla, Finland
Vol.:(0123456789)
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Molecular Biology Reports
dioxygenases [12] and share similarity with VOC (vicinal
oxygen chelate) superfamily [13] that harbour highly con-
served structural motif (βαβββ) [14]. Some of the extradiol
dioxygenases like 2,3-dihydroxybiphenyl 1,2- dioxygenase
and cathecol 2,3-dioxygenase are part of the same super-
family [5, 15]. All these proteins belong to VOC super fam-
ily and contain a 2-His-1-carboxylate metal binding motif.
These proteins uses variety of mechanisms to hamper the
efects of antibiotics including bleomycin [16]. Bleomycin
resistance protein (BRP) share homology with this super
family and confer bleomycin activity [17]. Type II hydroqui-
none dioxygenases accommodates two diferent subunits of
α2β2, forming a hetero-tetramer. These enzymes cleave HQs
formed during degradation pathway of hydroxyacetophe-
none [9] and p-nitrophenol [18, 19]. Type II hydroquinone
dioxygenase from Pseudomonas fuorescens ACB, Pseu-
domonas sp. WBC3 (PnpCD) Sphingomonas sp. TTNP3,
Burkholderia sp. SJ98, Pseudomonas sp. 1–7, have been
purifed and partially characterized so far [9, 11, 19, 20].
Members of this family are structurally well characterized,
and are known to be part of the cupin superfamily, one of
the most functionally diverse protein classes [21]. However,
both the cupin β-barrel fold and the paired βαβββ modules
of the VOC superfamily proteins forms a scafold for a metal
coordination environment [21, 22].
length gene sequence was provided a gene accession num-
ber MK766458.1 for nucleotide sequence of BRPD while
QED41507 for full length protein sequence. Further, the cul-
ture was induced for 4 h by addition of 1 mM isopropyl-β-d-
thiogalactopyranoside (IPTG) at 37 °C. Cells were harvested
by centrifugation, lysed and suspended in the binding bufer.
The soluble fraction was then used for purifcation of recom-
binant protein using nickel-nitrilotriacetic acid (Ni2+–NTA)
agarose (Merck Biosciences). Binding bufer consists of
500 mM NaCl, 10 mM imidazole, and sodium phosphate
bufer (250 mM, pH 8.0). The elution bufer contained all
above bufer components except that the fnal concentra-
tion of imidazole was 50 mM, 100 mM and 150 mM. Pro-
tein concentration was determined according to the Lowry
method with bovine serum albumin as the standard. The
eluted fractions were aliquot and stored in 50% glycerol at
−70 °C. All purifcation steps were performed at 4 °C unless
and otherwise stated.
Enzyme activity assay
Enzyme activity was routinely measured at 25 °C in the
UV–VIS spectrophotometer (Perkin Elmer, Hitachi) by
monitoring the formation of 4-hydroxymuconic semi-
aldehyde at 320 nm (ε320 =11.0 mM−1 cm−1) [6, 23]. One
unit of enzyme activity was defned as the production of
1 μmol 4-hydroxymuconic semialdehyde per minute. The
assay mixture (1.0 ml) typically contained 10 mM hydro-
quinone, 1 mM ZnSO4, 1 mM FeSO4 0.8–10 μg of protein
and 50 mM phosphate bufer (pH 7.4). The reference cuvette
contained all of these compounds except the substrate, and
as the enzyme was subject to suicide deactivation upon incu-
bation with HQ, only initial rates were recorded within 20 s.
Enzyme activity for other aromatic substrate like catechol
was also measured within 1 h incubation at 30 °C in the
presence of Zn2+(1 mM), phosphate bufer (50 mM, pH 7.4)
at diferent concentrations of 10 mM and 50 mM by moni-
toring the formation of 2-hydroxymuconic semi-aldehyde at
375 nm (ε=33.400 mM−1 cm−1).
Herein, we report expression, purifcation and characteri-
zation of a novel type I hydroquinone dioxygenase which
showed>95% similarity with bleomycin (antimitotic-antibi-
otic) resistance proteins, the gene was cloned from metagen-
omic DNA extracted from contaminated agricultural soil.
Materials and methods
Expression and purifcation of BRPD
BRPD full length gene sequence was amplifed using spe-
cifc primers containing Bam H1 (F-5′-CGGGATCCAAC
CAATTAAAAGG-3′ and Xho1 site R-5′-GTACTCGAG
CTCTTTAATAAATT). These primers were designed
based on the sequence information of a partial fragment
(Accession no. MH643810) amplifed previously in our
lab from metagenomic DNA using degenerate set of prim-
ers. Sequencing and bioinformatic analysis of the par-
tial DNA fragment revealed that it had shared more than
95% sequence similarity with uncharacterized bleomycin
resistant dioxygenase of B. Cereus. Double digested full-
length gene was ligated in frame with the BamH1 and Xho1
digested pET23a containing C-terminal six-His-tag. The
recombinant plasmid transformed in E. coli BL21 (DE3)
carrying BRPD was grown in Luria Broth (LB) at 37 °C
till OD600 reaches 0.6. After that, the plasmid DNA was
extracted from Bl21(DE3) manually and sequenced. Full
Enzyme inhibition assay
Time-dependent inhibition of BRPD was determined by
incubating purifed enzyme at 25 °C in the presence of
20 mM 4-hydroxy benzoate (4-HBA), para-nitrophenol
(PNP) and phenol for 1 h followed by addition of hydroqui-
none (10 mM) to measure its degradation within 20 s.
BRPD‑bleomycin molecule binding assay
The binding afnity of BRPD to bleomycin (Sigma-Aldrich)
was confirmed by electrophoretic mobility shift assay
(EMSA) where purifed BRPD (10 μg) was incubated with
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increased concentrations of bleomycin (10, 20, 30 μg) in
30 μl of Tris–HCl (10 mM, pH 7.5) at 16 °C. After 3 h
incubation, 30 μl of reaction mixture was loaded onto a 12%
non-denaturing polyacrylamide gel, as described previously
[17, 24].
performing MD simulation in AMBER 16.0 software pack-
age using f14SB force felds [32]. Using the t leap module,
all required parameters were applied, considering ionisable
residues set at their default protonation states at pH 7 value.
Each system was neutralized by adding Na+/Cl− ions and
solvated in a truncated octahedron box of TIP3P [33] water
model with a margin distance of 10 Å. For long-range Cou-
lombic interactions calculation, the particle mesh Ewald
method was used [34]. The SHAKE algorithm [35] was
employed to restrain all atoms covalently bonded to hydro-
gen atoms. The protocol was adopted that previous describe
by author [36] for system minimization, equilibrium, etc.
Finally, a MD simulation production was run for 100 ns
using NPT ensemble at room temperature with 1.0 atm pres-
sure. Coordinate trajectories were saved every 20 ps and
further analyzed using VMD programs.
Molecular diversity analysis
Nearly 1000 sequences were retrieved from Uniprot data-
base after performing Basic Local Alignment Search Tool
(BLAST) of quarried sequence (BRPD). Sequence redun-
dancy was removed at 75% sequence identity for set of
representative sequences, these sequences also includes
protein sequences for which crystal structure is available.
Multiple sequence alignment (MSA) was performed using
the MUSCLE program. The molecular evolutionary history
was inferred using Neighbor-Joining method [25]. The evo-
lutionary distances were computed using the Poisson correc-
tion method [26] and included units of the number of amino
acid substitutions per site, analysis involved 25 amino acid
sequences. All ambiguous positions were removed from the
comparison. There was a total of 350 positions in the fnal
dataset. Evolutionary analysis was performed using MEGA
7 [27], and the secondary structure prediction was done
using I-TEASSER [28].
Molecular docking studies
Molecular docking studies of experimental tested substrates
were performed using the AUTODOCK 4.2 program. The
substrates and refned modelled protein molecule were ana-
lyzed using standard docking ligand and protein fles prepa-
ration protocols. The docking protocol was set as per previ-
ous reported studies [37]. The grid size was set as follows, it
includes the metal ion and complete active site. The docking
conformations were set to 30 and all other docking param-
eters were set to default values. All 30 docked conformations
were analyzed.
Analysis of evolutionary conserve sequences
For comprehensive analysis of evolutionary conserve
sequences, ConSurf Program [29, 30] was used. The pri-
mary protein sequence of BRPD was quarried to search
homologues sequences in protein database UNIREF-90
(http://www.uniprot.org/help/uniref) using HMMER (hid-
den Markov models) homolog search algorithm with E-value
cutof 0.0001. Best top 500 hits were used for Multiple
Sequence Alignment (MSA). The MSA was performed
using the MAFFT program. All other parameters were kept
at default values for calculation of conservation scores.
Results
Expression and purifcation of BRPD
Recombinant BRPD was over expressed in E. coli BL21
(DE3) as C-terminal His-tagged fusion protein. The
expressed and purifed protein as analyzed in 12% SDS-
PAGE showed induction of distinct protein band with an
apparent molecular mass of ~26 kDa as detected on SDS-
PAGE (Fig. 1a). The purifed protein band as depicted in
Fig. 1b do not ft to molecular mass of BRPD as deduced
from its amino acid sequence (36.8 kDa). Interestingly,
native PAGE revealed size ~ 73 kDa protein band indicat-
ing dimeric nature of protein (Fig. 1c). Furthermore, we
embarked upon investigating the electrophoretic mobility
shift assay (EMSA) using purifed BRPD which showed that
the BRPD protein migration was delayed in the presence
of bleomycin-like molecules in concentration dependent
manner (Fig. 1d). Altogether, Ni–NTA based purifcation
of recombinant protein resulted in large quantities of solu-
ble and active BRPD from a 600 ml culture having specifc
activity of 6.25 U mg−1 as depicted in Table 1.
Structural modelling and molecular dynamics
simulation studies
Since the target protein structure was not reported in the
PDB database, therefore comparative protein modelling
was performed using highly identical template structures
(PDB ID: 1ZSW) via SWISS-MODEL server (https://swiss
model.expasy.org) including metal ion coordinates. Mod-
elled protein structure was energy minimized by 100 steps
steepest descent method following 50 steps of conjugate
gradient method in minimize structure module of USCF
Chimera software [31]. The minimized modelled structures
as well as the close structural homolog’s protein structure
were simulated incusing diferent metal ions (Zn2+, Fe2+) by
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Fig. 1 SDS-PAGE of BRPD. a Lane 1: Molecular mass standards
(molecular masses in kDa) are indicated on the left; lane 2 and 8:
Empty well, lane 3–4 cell extracts of uninduced E. coli BL21 contain-
ing pET23a; lane 5,6,7, and 9: Cell extracts of induced E. coli BL21
containing pET23a with BRPD at diferent time interval (30 min 1 h,
2 h, 3 h, 4 h). b lane1: Molecular weight marker, lane 2,3 and 4 puri-
fed protein sample lane 5 induced protein sample lane 6–7 purifed
protein sample c Native page of BRPD; lane1,BSA; lane 2 Lysozyme;
lane 3 purifed BRPD bands. d EMSA of purifed BRPD and with
varying concentration of bleomycin (lane1-Protein molecular weight
marker, lane 2–5: 10, 20, 30 and 40 µg bleomycin+10 µg BRPD
respectively, lane 6: 10 µg BRPD alone)
Table 1 Purifcation profle of
Purifcation step
Activity U/ml
Protein (mg)
Specifc act.
(U mg−1)
Purifcation fold
Yield
BRPD
Cell Lysate
Ni–NTA
Dialysis
30.5
14.8
7.5
16.2
3.3
1.2
1.88
4.48
6.25
1
2.38
3.32
100
48.52
24.6
towards zinc than iron, which may be attributed to oxida-
tion of ferrous iron to ferric iron, which could be or not
be restored by adding Fe(II) ions, as reported previously
[11, 13]. BRPD also catalyzes conversion of catechol to
2-hdroxymuconic semi aldehyde evidenced from emer-
gence of peak at 375 nm, in presence of Zinc, these results
indicates its substrate versatility as well as more specifcity
towards zinc ion (Fig. 2D). Further, BRPD also showed
hydroquinone degradation in presence of other divalent
metals ions that includes Hg2+, Mg2+, Cu2+ with activ-
ity (%) (i.e., relative absorbance increases and decrease at
320 nm as compared to Zn2+ absorbance) of 96.5, 70.17
and 35.08 respectively (Table 2). Furthermore, activity of
BRPD was strongly inhibited (> 80% inhibition) in pres-
ence of substrate analogue like para nitro phenol but not
by 4-HBA (<2% inhibition) indicating the role of electron
withdrawing (OH functional group) and electron donat-
ing substituent’s (-Nitro functional group) in para to the
hydroxyl group located adjacently to the cleaving site as it
Catalytic activity
BRPD catalyzes ring cleavage of hydroquinone to
4-hydroxymuconic semi aldehyde and exhibited very nar-
row optimal pH values ranging from 7.4 to 8.0 in phos-
phate bufer, no activity was observed in the Tris bufer.
More interestingly, we observed low activity of BRPD
towards hydroquinone in the absence of metal ions. Fig-
ure 2a shows absorption spectrum of Hydroquinone, show-
ing a prominent peak at 289 nm, however, on incubation
with Zn2+(1 mM) and Fe2+ (1 mM), there is emergence
of peak at 320 nm which indicates conversion of Hydro-
quinone to 4-hdroxymuconic semi aldehyde, the enzyme
shows activity in presence of Zn2+(1 mM) and Fe2+
(1 mM) indicating its metal dependent nature as shown
in Fig. 2b, interestingly Zn2+ ion alone was also found
to be equally active as depicted in Fig. 2c and ~ fourfold
increased activity was observed in the presence of Zn2+
alone. This may indicate tight dependency of the BRPD
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Fig. 2 Spectrophotometric measurement of hydroquinone and cat-
echol degradation by BPRD a Standard hydroquinone absorbance
spectra b Spectra recorded during catalytic conversion of hydroqui-
none using BRPD in presence of Zn2+.ions and Fe2+.ions c Spectra
recorded during catalytic conversion of hydroquinone using BRPD in
presence of Zn2+.ions d Spectra recorded during degradation of cat-
echol by BRPD
Table 2 BRPD activity of HQ in the presence of metals and inhibi-
infer the evolutionary relationship. From phylogeny tree it
becomes evident that it has large diversity at primary protein
sequence level, and the homolog protein sequences repre-
senting Glyoxalase/ring-cleaving dioxygenase family can be
divided into two major groups (Fig. 3a). Interestingly, we
observed that metal ion centre (His9, His224 and Glu272,
numbering according to our target sequence) is highly con-
served (Fig. 3b). In addition, the metal ion coordinating
residue His9 toward N- termini holds a highly conserved
motif [G/X–X–H–H–X–T–A/X] while residue His224
toward C- terminal surrounds a highly conserved motif
[G–X–X–H–H–X–A]. The secondary structure prediction
using I-TASSER (Fig. S1) [28] predicted that BRPD con-
tains four similar size βαβββ modules, a highly conserved
structural motif among the vicinal oxygen chelate (VOC)
superfamily. This motif provides a metal co-ordinating
environment connected by long loops in between. These
repeating motifs span residues 7–63, 78–132, 158–207 and
223–275. The protein in this superfamily consists of βαβββ
modules, although the number and relative orientation of
these modules can difer leading to structural diversity at 3D
level [14]. BRPD amino acid sequences showed similarity
tors
Activity (%)
concentration (μM)
Metals
Hg
Mg
96.5 0.8
70.17 0.6
35.08 1.0
1 mM
1 mM
1 mM
Cu
Inhibitors
4-HBA
Phenol
PNP
98 0.4
99 0.7
17.5 0.5
20 mm
20 mM
20 mM
was found to be main discriminator for substrate binding
in hydroquinone dioxygenases (Table 2).
Analysis of evolutionary conserved sequences
To understand molecular diversity and phylogenetic related-
ness of BRPD, comprehensive sequence analysis was carried
out and sequences showing> 30% similarity were used to
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Fig. 3 a. Phylogenetic relatedness of targeted sequence (BRPD) and
other related extradiol dioxygenases. All sequences were aligned
using MUSCLE program with default settings. A distant neighbour
joining tree was then created using the Mega (version 7.0). b Evolu-
tionary conserved sequences of BRPD, the red rectangle box shows
the active site conserved motifs whereas the green rectangle box
depicts the catalytic residues. (Color fgure online)
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with amino acids sequences of structural homologs for
which 3D crystal structures are available (Fig. S2) and were
used for building 3D model of BRPD as discussed below.
using the molecular docking studies. Both catechol and
hydroquinone showed interactions with metal ion during
substrate entry in the active site. The best docked confor-
mation of catechol and hydroquinone into catalytic cen-
tre with metal ions distances is shown in Fig. 4b and c.
We also performed structural modelling and MD simula-
tions studies with two diferent metal ions Fe2+ and Zn2+.
During the entire 100 ns MD simulations the metal ions
were found to be well coordinated with catalytic centre
residues His9, His224 and His272. Furthermore the metal
ion Fe2+ coordinates with three water molecules and cata-
lytic residues His9, His224 and Glu272 and adopts the
octahedral geometry while Zn2+ coordinates with two
water molecules and catalytic residues His9, His224 and
Glu272 and adopts the tetrahedral geometry. In both metal
ions Fe2+ and Zn2+ consistency exhibited the bound aver-
age distance ~ 2.2 Å with NE2 atom of His9 and His224.
Interesting, the side chain fexibly of Glu amino acid was
observed and metal ion coordinates either OE1 or OE2
during the MD simulation in both metal ions as shown in
Fig. 5a–d. Furthermore, we also predicted two putative
binding sites for bleomycin in dioxygenase with similar
3D structural modelling, substrate docking and MD
simulations
In order to investigate 3D structure of target protein, we
performed structural molecular modelling and MD simula-
tions with the modelled protein. Target sequence of BRPD
showed ~ 39% and ~ 31% sequence identify with crystal
structure of Bacillus cereus metallo protein (1ZSW) from
glyoxalase family and crystal structure 2,6-dichloro-p-
hydroquinone 1,2-dioxygenase (PcpA;4HUZ) protein from
Sphingobium chlorophenolicum, respectively. An average
model obtained after 100 ns MD run and superimposed on
template structure showed RMSD value corresponding to
1.15 Å. The overall structural topology retained the same
conformation except some minor loop region fexibility in
overall refne modelled structure after 100 ns MD run as
depicted in Fig. 4a. The active site binding mode of tested
substrates like Catechol and Hydroquinone were predicted
Fig. 4 In silico analysis of BRPD a An average modelled structure after 100 ns MD simulation showing bleomycin binding pocket. b Molecular
docking of Hydroquinone. c Molecular docking of substrate catechol
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Fig. 5 a. The modeled protein simulated with Zn2+ metal ion repre-
senting distance between metal ion and co-ordinating residues, b The
catalytic centre co-ordinated by Zn2+, c the catalytic residues and
water molecules during 100 ns MD simulation The modeled protein
simulated with Fe2+ metal ion showing distance between metal ion
and coordinating residues, d: catalytic centre coordinated by Fe2+, the
catalytic residues and water molecules during 100 ns MD simulation
structural topology and cavity for binding of bleomycin
centre to ferric iron, a process which could be completely
reversed within 32 h by reduction with 100 mM ascorbic acid
(data not shown) and in vivo by redox-dependent reactions
catalyzed by ferredoxins [38]. This characteristic feature is
also reported previously for HQDO from Sphingomonas sp.
strain TTNP3 [11], Lactococcus lactis IL1403 [39] and also
for other extradiol type dioxygenases, such as catechol diox-
ygenases [40, 41] and protocatechuate dioxygenases [42].
However, like PcpA, BRPD also showed its dimeric nature
(Fig. 1c) and further showed uneven faster movement on
SDS-PAGE due to its acidic isoelectric point(pI 5.4;aspartic
acid(D) and glutamic acid(E) content about ~16% of total
protein) and also due to no cysteine amino acid residues in
protein structure. Due to this, non-reduced protein nature
causes BRPD like protein to unfold completely, retaining
somewhat globular shape and bind to SDS less as compared
to its mass and normally causes protein faster run on SDS-
PAGE [43]. BRPD also shown very narrow optimal pH
values ranging from 7.4 to 8.0 in phosphate bufer with no
as shown in Fig. 6.
Discussion
Hydroquinone dioxygenases (HQDO) are key enzymes
that act on hydroquinone (HQ), a key intermediate of
several aromatic hydrocarbons such as 4-nitrophenol [7],
γ-hexachlorocyclohexane [8], 4-hydroxyacetophenone [9]
and 4-aminophenol [10]. In the present investigation, we
were able to isolate and characterize a novel hydroquinone
dioxygenase from contaminated agricultural soil that cata-
lyzes Fe2+ and Zn2+ dependent conversion of HQ (Hydroqui-
none) to 4-hydroxymuconic semialdehyde (320 nm). Inter-
estingly, enzyme readily lost activity upon incubation with
its substrate hydroquinone, which distinguishes it from PcpA
and related hydroquinone dioxygenases [13] (Fig. S2). This
may be attribute to oxidation of ferrous iron in the active
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Fig. 6 Predicated active site
using the structural similarity
and binding patterns of bleomy-
cin. Frontal side view and back
side view 180° rotation
activity was observed in the Tris bufer. Previously, MnpC,
a hydroquinone dioxygenase involved in meta-nitophenol
degradation by cupriavidus nectar JMP134 showed amino
acid homology and metals activity as well narrow pH range
as similar to BRPD [23].
His9, His224 and Glu272, adopting octahedral geometry
while Zn2+ ion adopts tetrahedral geometry by coordinat-
ing with two water molecules and catalytic residues. Both
metal ions Fe2+ and Zn2+ consistently exhibited the bound
average distance corresponding to ~ 2.2 Å with NE2 atom
of His9 and His224 (Fig. 4b, c). Interestingly, the side chain
fexibility of Glu amino acid was observed and metal ion
coordinates at either OE1 or OE2 during the MD simulation.
This fexibility allows these metal ions to temporarily dis-
sociate from the active-site, and is required for stereospecifc
reaction in glyoxalase 1 [45]. The arrangement of protein
ligands around diferent metal ions is found to be unique in
relation to their function in dioxygenase family proteins. For
example, protein ligands arrangement around Zn2+ ion and
Fe2+ is the same as for the Zn2+ ion of human GLO and for
the Co2+, Ni2+, or Cd2+ ions bound to E. coli GLO except
that tetrahedral arrangement instead of octahedral and vice
versa. An octahedral or tetrahedral co-ordination geometry
that leaves two or three positions to be flled by water mol-
ecules seems to be essential for proper functioning [46].
In human GLO, a transition state analogue binds with
Zn2+ by occupying two cis positions and displacing the water
[45]. In E. coli GLO, active metal ions (Co2+, Ni2+, and
Cd2+) have two water molecules occupying cis octahedral
sites, however on binding with incorrect metal ion (Zn2+) its
geometry changed to trigonal bipyramidal that results in loss
of enzyme activity [14]. Thus, it can be inferred that these
structural geometries in dioxygenase are important to cata-
lyze degradation of aromatic hydrocarbons [13, 47]. Inter-
estingly, low level of dioxygenase activity observed in the
presence of Cu2+ (Table 2) indicate combined copper/qui-
none stress in the bacterial community of our study sample,
Molecular diversity and analysis of evolutionary con-
served sequences revealed that both its N and C-terminal
domains are conserved at sequence and structural level
(Fig. S2). These domains shared strong similarity with the
conserved domain of structurally related metalloproteins,
including the bleomycin resistance protein, glyoxalase
I, and type I ring-cleaving dioxygenases. It was observed
that metal ion centre His9, His224 and Glu272 are highly
conserved in this protein. In addition, the metal ion coor-
dinating residue His9 toward N- termini hold a highly
conserved motif [G/X–X–H–H–X–T–A/X] while residue
His224 toward C- terminal surrounds a highly conserved
motif [G–X–X–H–H–X–A]. As conserved amino acids in
metal binding comes from topologically equivalent posi-
tions of the two modules i.e. from 1st and 4th module.
Indeed, it was documented that formation of a symmetric
protein with ability to bind a metal ion was a crucial step
in the evolution of this family of proteins [44]. BRPD is
activated in presence of divalent metal ions, with the most
effective being Zn2+ and Fe2+, and the overall order is
Zn2+>Fe2+>Hg2+>Mg2+>Cu2. Previously, it was reported
that this super family of proteins uses a variety of metal ions
for their catalytic activities [16].
MD stimulations and docking studies using modelled
BRPD provided constructive insights about arrangement of
metal ion in the active site. It was observed that Fe2+ ion co-
ordinates with three water molecules and catalytic residues
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Molecular Biology Reports
as also observed in Gram positive bacterium Lactococcus
lactis IL1403 [39]. Further, substrate docking studies with
modelled BRPD (Fig. 4b, c) provided important insights
about the similarities and diferences between BRPD and
the EDOs (extradiol dioxygenases). The model sufciently
highlighted the binding modes of both the catechol and hyd-
roquinone in the active sites by co-ordinating through Zn2+
ions to attain reasonable geometry and other divalent met-
als as also reported previously ([13, 15]. Further, in case of
Fe(II) ions, three water molecule and 2-His-1-carboxylate
facial triad forms six-co-ordinate complex with hydroqui-
none and BRPD to form 4-hydromuconic semi aldehyde as
reported previously [9, 15].
bleomycin resistance protein (BRP) [48]. Due to this swap-
ping, there could be criss-cross reversal between two mod-
ules of dioxygeases and BRP domain. As a result, it could be
speculated that in BRPD, metal binding domain is used to bind
and degrade aromatic compounds while other domain binds
and sequester bleomycin. Due to this BRPD, PcpA, human
glyoxalase I [49], Yeast glyoxalase I belong to a family of
protein having four βαβββ modules in one polypeptide [15]. It
also shows how VOC superfamily protein allows very diferent
as well as unique functions on a very similar protein scafold.
Further, at the level of the dimer, the similarity between BRPD
and PcpA persists, and using superposition study BRPD dimer
match the equivalent atoms in the PcpA dimer with minor
loop variation (data not shown). Finally, we concluded that in
these proteins, it is the dimer that provides the common stable
structural unit. Interactions between the two βαβββ modules
that make up the active site “substructure” seems insufcient
to give such close correspondence between BRPD and PcpA;
rather it is the extensive interface between the backs of the
two “substructure” (the BRPD dimer interface) that stabilizes
the overall structure [13, 16].Thus, a very stable BRPD-like
dimer would provide the basis for domain swapping without
disturbing structure and function.
Our results further demonstrate that electron withdrawing
and electron donating substituent’s groups in the para posi-
tion located adjacent to the cleaving site is main discrimina-
tor for substrate binding with BRPD and related HQDO [9,
11]. In our study, 4-nitrophenol containing electron donating
functional (OH) groups in para position exhibited strong
inhibitory efect on enzyme activity, whereas those having
electron withdrawing group substituents in para position
e.g., 4-HBA demonstrated weak or no inhibitory efects.
Interestingly, those lacking substituents in para position such
as phenol inhibited enzyme activity less than 2% (Table 2)
as also reported previously [11]. These results suggest that
the BRPD activity is afected both by electronic and steric
constraints in the active site.
Conclusion
For the frst time, we propose a comprehensive picture of
BRPD with its unique catalytic mechanism that is very
unlikely conserved among the HQDO type I having capa-
bility to degrade hydroquinone, catechol as well resistance
to antibiotic like bleomycin. Knowledge of the structure and
biochemical characterisation reported here will be benef-
cial not only for the basic biochemistry of aromatic hydro-
carbon metabolism, but also increases our knowledge on
antibiotic resistance elements, especially those from natural
environments.
Inspection of the superimposed 3D structures of BRPD,
1ZSW, PcpA (4HUZ) revealed high sequence similar-
ity around the active-site pocket (Fig.S2). Consequently,
the frst and fourth βαβββ motifs generally showed higher
sequence similarity than the second and third motifs (Fig.
S1). Specifcally, the Fe(II) and Zn(II) co-ordinating residues
and critical residues in the second co-ordination sphere were
completely conserved. As PcpA has capability to degrade
hydroquinone and related analogous, while 1ZSW have no
known functions. Interestingly BRPD demonstrate similar
activity and substrate specifcity like PcpA, thus constituting
a unique class of hydroquinone dioxygenases. It also showed
bleomycin resistance property which is unique function of
this family of protein; however, “dual efect” of antibiotic
resistance and aromatic degradation remained elusive in
these types of proteins. For the frst time, Santos et al. [3]
reported this kind of activity from metagenomic clone CRB2
(1), which is extradiol dioxygenase, however mechanism of
antibiotic resistance is not yet clear.
Funding This research Grant was funded by the Science and Engineer-
ing Research Board (SERB) New Delhi, project fle number: SB/YS/
LS-63/2013 under fast track scheme for the young scientists. Dr PKS
would like to thank SERB for fnancial support.
Compliance with ethical standards
Conflict of interest The authors declare that they have no confict of
interest.
Information retrieved after multiple sequence alignment of
BRPD (Fig. S2) and its superimposition study with available
crystal structures, PcpA, 1ZSW (Fig. 4a) as well as from previ-
ously reported literature [5, 15, 44], we propose that domain
must have swapped among the frst and fourth βαβββ modules
between BRPD and PcpA like HQDOs type I as compared to
frst and third in type I EDOs (Extradiol Dioxygenases) and
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