In silico approach to support that p-nitrophenol monooxygenase from Arthrobacter sp. strain JS443...

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Interdisciplinary Sciences:Computational Life SciencesComputational Life Sciences ISSN 1913-2751Volume 7Number 2 Interdiscip Sci Comput Life Sci (2015)7:157-167DOI 10.1007/s12539-015-0018-x

In Silico Approach to Support that p-Nitrophenol Monooxygenase fromArthrobacter sp. Strain JS443 Catalyzes theInitial Two Sequential Monooxygenations

Monika Kallubai, UmamaheswariAmineni, Megharaj Mallavarapu &Venkateswarlu Kadiyala

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ORIGINAL RESEARCH ARTICLE

In Silico Approach to Support that p-Nitrophenol Monooxygenasefrom Arthrobacter sp. Strain JS443 Catalyzes the Initial TwoSequential Monooxygenations

Monika Kallubai1 • Umamaheswari Amineni2 • Megharaj Mallavarapu3 •

Venkateswarlu Kadiyala1

Received: 19 November 2013 / Revised: 25 June 2014 / Accepted: 29 October 2014 / Published online: 14 August 2015

� International Association of Scientists in the Interdisciplinary Areas and Springer-Verlag Berlin Heidelberg 2015

Abstract p-Nitrophenol (PNP), used primarily for man-

ufacturing pesticides and dyes, has been recognized as a

priority environmental pollutant. It is therefore important

to reduce the input of this toxicant into the environment

and to establish approaches for its removal from the con-

taminated sites. PNP monooxygenase, a novel enzyme

from Gram-positive bacteria like Arthrobacter sp. and

Bacillus sp., that comprises two components, a flavoprotein

reductase and an oxygenase, catalyzes the initial two

sequential monooxygenations to convert PNP to trihy-

droxybenzene. Accurate and reliable prediction of this

enzyme–substrate interactions and binding affinity are of

vital importance in understanding these catalytic mecha-

nisms of the two sequential reactions. As crystal structure

of the enzyme has not yet been published, we built a

homology model for PNP monooxygenase using crystal-

lized chlorophenol 4-monooxygenase from Burkholderia

cepacia AC1100 (3HWC) as the template. The model was

assessed for its reliability using PROCHECK, ERRAT and

ProSA. Molecular docking of the physiological substrates,

PNP and 4-nitrocatechol (4-NC), was carried out using

Glide v5.7 implemented in Maestro v9.2, and the binding

energies were calculated to substantiate the prediction.

Docking complexes formed by molecular level interactions

of PNP monooxygenase-PNP/4-NC without or with the

cofactors, FAD and NADH, showed good correlation with

the established experimental evidence that the two-com-

ponent PNP monooxygenase catalyzes both the hydroxy-

lation of PNP and the oxidative release of nitrite from

4-NC in B. sphaericus JS905. Furthermore, molecular

dynamics simulations performed for docking complexes

using Desmond v3.0 showed stable nature of the interac-

tions as well.

Keywords PNP monooxygenase � Arthrobacter sp. strainJS443 � Bacillus sphaericus JS905 � Homology modeling �Molecular docking � Molecular dynamics simulations

1 Introduction

With the rapid thrust in industrial and agricultural activity,

the recent times have witnessed vast quantities of soil and

groundwater resources becoming contaminated with haz-

ardous chemicals. p-Nitrophenol (PNP) is probably the

most important contaminant among the mononitrophenols

since 27 % of its use in industry is for pesticide manu-

facture and 13 % is for synthesis of dye components [1].

PNP is also found as a major metabolite of microbial

degradation of organophosphorous insecticides, parathion

and methyl parathion [2] and is thus an important envi-

ronmental pollutant [3, 4] posing even odor problems to

water bodies [5, 6]. PNP is, therefore, one of the eleven

phenolic compounds listed as priority pollutants by the US

Environmental Protection Agency [7].

Electronic supplementary material The online version of thisarticle (doi:10.1007/s12539-015-0018-x) contains supplementarymaterial, which is available to authorized users.

& Venkateswarlu Kadiyala

[email protected]

1 Department of Microbiology, Sri Krishnadevaraya

University, Anantapur 515055, India

2 Department of Bioinformatics, Sri Venkateswara Institute for

Medical Sciences, Tirupati 517507, India

3 Centre for Environmental Risk Assessment and Remediation,

and CRC for Contamination Assessment and Remediation of

the Environment, University of South Australia, Adelaide,

SA 5095, Australia

123

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Two alternative pathways for aerobic bacterial degra-

dation that convert PNP to maleylacetate have been elu-

cidated [8]. The first pathway that results in the formation

of hydroquinone from PNP, probably via 1,4-benzo-

quinone, with concomitant release of nitrite [9], is more

common in Gram-negative isolates. In the second catabolic

pathway, Bacillus sphaericus JS905 [10] hydroxylates PNP

to produce 4-nitrocatechol (4-NC), and subsequent oxida-

tive removal of the nitro group from 4-NC as nitrite yields

1,2,4-trihydroxybenzene (THB) as shown in Fig. 1. PNP

monooxygenase, a novel enzyme from B. sphaericus JS905

that belongs to a two-component flavin-diffusible

monooxygenase family, consisting of two components—a

flavoprotein reductase and an oxygenase, catalyzes the first

two sequential reactions in the degradation of PNP to THB

via 4-NC. The enzyme uses loosely bound FAD as the

redox chromophore and is NADH dependent. Genetic

evidence that the oxygenase component of the enzyme

hydroxylates PNP as well as 4-NC following two sequen-

tial monooxygenations has been provided for Arthrobacter

sp. strain JS443 [11] and Pseudomonas sp. 1-7 [12]. The

nitro substituent from 4-NC is directly removed from the

aromatic ring in the form of nitrite.

Understanding the microbial metabolism of nitroaro-

matic compounds will assist in management options to

minimize their persistence in the environment, and one

needs to have specific information on various components

involved in catalysis that explains the overall function of

the system. Our present study concerning analysis of

functional sites in PNP monooxygenase relies on the bio-

chemical information provided for B. sphaericus JS905 and

the genetic information reported for Arthrobacter sp. strain

JS443, since the proposed pathways for PNP degradation in

both the bacterial strains are the same involving two

sequential monooxygenation reactions. Experimental evi-

dence also implicates four conserved residues (Arg100,

Gln158, Arg161 and Thr193) for the substrate binding with

oxygenase component of the enzyme [13]. The objective of

the present study was primarily to verify whether the same

amino acid residues at the catalytic site of the enzyme are

involved in the two sequential monooxygenations. Since

molecular docking study using 3D structure of an enzyme

is one way to predict interaction between the substrate and

the active site, we also report the 3D model of PNP

monooxygenase and prediction of interaction sites for the

substrates, PNP and 4-NC, through molecular modeling,

docking and molecular dynamics (MD) simulations.

2 Methodology

2.1 Homology Modeling of PNP Monooxygenase

The protein sequence of PNP monooxygenase from

Arthrobacter sp. strain JS443 (accession number ABL7

5143.1)was retrieved from theNCBI protein database (http://

www.ncbi.nlm.nih.gov/protein). BLASTp [14, 15] search for

PNP monooxygenase was performed against the protein

databank [16] to find a structural template for homology

modeling. Chlorophenol 4-monooxygenase of Burkholderia

cepacia AC1100 (3HWC) was selected as the structural

template and aligned with PNP monooxygenase using

Fig. 1 Proposed pathway for

degradation of PNP by Bacillus

sphaericus JS905

158 Interdiscip Sci Comput Life Sci (2015) 7:157–167

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http://www.ncbi.nlm.nih.gov/protein

http://www.ncbi.nlm.nih.gov/protein

ClustalX 1.83 [17]. Modeller9v7 [18, 19] that performs

comparative modeling based on satisfaction of spatial

restraints was used to build 3D structure of PNP monooxy-

genase. Twenty 3D structures of PNP monooxygenase were

generated, and the structure with the lowest discrete opti-

mized protein energy (DOPE) score was chosen for further

analysis [20]. The stereochemical excellence of the selected

homology model was confirmed by PROCHECK analysis

[21]. The structure of each residue in the model was ensured

for their authenticity by WHATCHECK [22], ERRAT [23]

and ProSA [24].

2.2 Molecular Docking

As PNP monooxygenase catalyzes the two sequential

monooxygenations in both B. sphaericus JS905 [10] and

Arthrobacter sp. strain JS443 [11], we performed docking

studies for PNP monooxygenase from strain JS443 with the

established physiological substrates (PNP, 4-NC) and the

cofactors (FAD and NADH) for the enzyme in order to

gain insights into the most probable binding interactions.

Each atom of the protein and substrate or cofactor needs

to be fixed before molecular docking for any potential

aberrations, if any, and for accurate prediction of interac-

tions with better binding affinity. The 3D model of PNP

monooxygenase was preprocessed with the protein prepa-

ration workflow in the Maestro v9.2 [25]. Hydrogens were

added to all the atoms of PNP monooxygenase for subse-

quent minimization using OPLS 2005 force field by con-

verging heavy atoms with maximum root-mean-square

deviation (RMSD) of 0.30 A. Minimization was performed

restraining the heavy atoms with the hydrogen torsion

parameters turned off, to allow free rotation of the hydro-

gen atoms. The structures of PNP, 4-NC, FAD and NADH

(Fig. 2) were imported to Maestro v9.2. LigPrep module

along with Epik was used to prepare multiple 3D confor-

mations of the four ligand molecules in a pH range of

7 ± 2.

Molecular docking was carried out using Glide v5.7

applying extra precision (XP) method [26, 27] that gener-

ates 10000 poses for each substrate during docking and

reports the best pose based on the energy term Emodel.

Residues of PNP monooxygenase specific for substrates

and cofactors were selected to generate a grid of

20� 20� 20 A using Glide. The best poses of each ligand

were further ranked based on XP Gscore. Lower XP

Gscore for a ligand indicates better binding affinity toward

the protein. The cutoff XP Gscore parameter for XP

docking was set to 0.0 kcal/mol, a constraint set to discard

ligands with positive XP Gscore from the final docking

output. Binding affinity of 4-NC and PNP with PNP

monooxygenase was estimated using Prime/MM-GBSA

analysis.

2.3 Molecular Dynamics (MD) Simulations

MD simulations were performed for docking complexes

(PNP monooxygenase?PNP?FAD?NADH and PNP

monooxygenase?4-NC?FAD?NADH) to evaluate the

stability and conformational changes and to gain insights

into the natural dynamics on different timescales in solu-

tion. Simulations were carried out using Desmond v3.0 [28,

29], implemented in Maestro v9.2 graphical user interface.

The system was embedded with simple point charge (SPC)

water model and neutralized by replacing solvent mole-

cules with Naþ ions. The final system was simulated

through a multistep protocol devised in Maestro v9.2. In

brief, the full system was minimized with maximum 2000

interactions of a hybrid of the steepest descent and the

limited-memory Broyden–Fletcher–Goldfarb–Shanno

(LBFGS) algorithms, with a convergence threshold of

50.0 kcal/mol/A2 followed by a similar unrestrained min-

imization with a convergence threshold of 5.0 kcal/mol/A2.

The minimized system was relaxed with four subsequent

short-span simulations: (a) 12-ps simulation in NVT

ensemble (temperature 10 K) restraining nonhydrogen

solute atoms, (b) 12-ps simulation in the NPT ensemble

(temperature 10 K) restraining nonhydrogen solute atoms,

(c) 24-ps simulation in the NPT ensemble restrained with

solute nonhydrogen atoms (temperature 300 K) and

(d) 24-ps simulation in the NPT ensemble (temperature

300 K) with no restraints. The temperatures and pressures in

these initial simulations were controlled using Berendsen

thermostats and barostats, respectively. These initial mini-

mization and simulations were performed to relax the model

before implementing a longer simulation time. The relaxed

system was simulated for a simulation time of 5 ns with a

time step of 2 fs, NPT ensemble using a Nose–Hoover

thermostat at 300 K and Martyna–Tobias–Klein barostat at

1.01325 bar pressure. The simulated systems were analyzed

for stability of the docking complex. Energy fluctuations

and RMSD of the complexes in each trajectory were ana-

lyzed with respect to simulation time. The root-mean-square

fluctuations (RMSF) of backbone and side chains atoms of

PNP monooxygenase were analyzed for each residue. The

docking complex was analyzed and monitored for consis-

tency in hydrogen bonding interactions.

3 Results and Discussion

3.1 3D Model of PNP Monooxygenase

The overall sequence identity between PNP monooxyge-

nase and the template chlorophenol 4-monooxygenase of

B. cepacia AC1100 was 45 % with query coverage of

Interdiscip Sci Comput Life Sci (2015) 7:157–167 159

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99 %. The sequence alignment for these two sequences

enabled us to identify sporadically spread conserved resi-

dues all over the sequence (Fig. 3). Conserved regions or

positions indicate residues supposedly under stronger

evolutionary constraints and thus might be more important

for the protein to fulfill its function. Moreover, residues

that are specifically conserved in subfamilies point to

sequence changes that occurred during the divergence of a

common ancestor, and they imply functional changes or the

acquisition of modified specificity [30].

Twenty PNP monooxygenase homology models were

constructed based on the template structure. All models

were assigned a predicted Genetic algorithm 341

(GA341) score and DOPE score. GA341 score of � 0.7

suggests that fold assignments in the homology model

are correct. GA341 scores for the 20 predicted models

were 1.0. Therefore, fold assignments in the homology

models were declared accurate. Selection of the best

model from the twenty was done based on DOPE score.

Measure of conformational energy is represented as

DOPE score in homology modeling. Lower DOPE score

represents relatively more stable 3D conformation [31].

Thus, the 20th model with the lowest DOPE score

(�54799.56 kcal/mol) was selected for further structural

validation.

The overall geometric and stereochemical quality of the

final modeled structure of PNP monooxygenase was ana-

lyzed using PROCHECK, which showed that 92.9 %

residues lie in the most favored region and 6.1 % in

additionally allowed regions (Fig. 4a). Evaluation of PNP

monooxygenase model with ProSA-web revealed the Z-

score value as �8.56, and the overall residue energy of

PNP monooxygenase model was largely negative except

for some peaks at the end region (Fig. 4b, c). The residue

energies of active site and allosteric site regions of the

protein were highly negative. The overall quality factor

checked from the ERRAT graph was 85.09, indicating the

acceptable protein environment (Fig. 4d). The structural

superimposition of the model of PNP monooxygenase with

the template structure showed backbone RMSD of 0.66 A.

The low backbone RMSD value represents that the pre-

dicted structure is reliable. The validated model was sub-

mitted to protein model database [32] with ID number

PM0077668 (Fig. 5).

(a)

(b) (c) (d)

Fig. 2 Structure of ligands PNP, 4-NC, FAD and NADH


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Fig. 3 Alignment of target and template using ClustalX

(a) (b)

(d)

(c)

Fig. 4 Validation of PNP monooxygenase. a PROCHECK evaluation, b energy plot obtained through ProSA-web analysis, c ProSA-web Z-plot

showing the model that corroborates with crystal structures of similar length, d ERRAT server validation


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3.2 Docking of Substrates into the Active Site

of PNP Monooxygenase

Molecular docking helps in determining binding affinity,

binding orientations and various bonded and nonbonded

enzyme–substrates and enzyme–cofactors interactions in

various enzymatic reactions. Docking analysis was imple-

mented for PNP monooxygenase with its two physiological

substrates, PNP and 4-NC, and the cofactors, FAD and

NADH, to corroborate with the experimental findings of

monooxygenation reactions.

Earlier experimental findings showed that active site

residues, Arg100 and Thr193, are important for substrate

(PNP or 4-NC) binding, while allosteric site residues such

as Gln158 and Arg161 are specific for cofactor (FAD or

NADH) interaction [13]. Multiple sequence alignment of

PNP monooxygenase with homologous proteins,

chlorophenol 4-monooxygenase and hydroxyphenylacetate

hydoxylase, revealed that residues of the active site and

allosteric site are conserved.

A 20� 20� 20 A grid was created around centroid of

the active site of PNP monooxygenase for PNP and 4-NC

docking. Both the substrates, PNP (complex 1) and 4-NC

(complex 3), were docked with PNP monooxygenase at the

active site with XP Gscore of �3.434 and �3.828 kcal/-

mol, respectively. The (O) atom of hydroxyl group of PNP

formed hydrogen bond (h-bond) with residue Thr193, and

pi-cation interacted with Arg100 of PNP monooxygenase.

The hydrogen atom of one hydroxyl group of 4-NC showed

one hydrogen bond with Thr193 of PNP monooxygenase

and Arg100 involved in pi-cation interaction with 4-NC

(Table 1; Supplementary Fig. 1a, d). Similar binding ori-

entations were observed in both the substrate–PNP

monooxygenase complexes.

Further, a docking grid was generated centering on

allosteric site of the PNP–PNP monooxygenase complex

(complex 1) and 4-NC–PNP monooxygenase complex

(complex 3). The grids were generated to determine the

probable site of interactions of PNP, 4-NC, FAD and

NADH with PNP monooxygenase. Docking interactions of

PNP (complex 2) (Fig. 6a) and 4-NC (complex 4) (Fig. 6b)

with PNP monooxygenase in the presence of FAD and

NADH remained same as that of complex 1 and complex 2,

respectively (Table 1; Fig. 6a, b; Supplementary Fig. 1a,

d). Binding affinity of NADH and FAD was expressed in

terms of XP Gscore in complex 2 and complex 4 (Table 1).

NADH was observed to have more binding affinity toward

PNP monooxygenase compared to FAD in both the com-

plexes. This result corroborates well with the experimental

evidence that FAD is loosely bound to PNP monooxyge-

nase compared to NADH [10]. The docking interactions of

FAD and NADH in complex 2 revealed that FAD is

interacting with Gln158 and Arg161 through hydrogen

bonds while NADH is also forming hydrogen bond with

Arg161, and good van der Waals interaction with Gln158

of PNP monooxygenase (Fig. 6a; Supplementary Fig. 1a–

d). Similar interactions were also observed in complex 4.

FAD was observed to form intermolecular hydrogen bond

with 4-NC and good van der Waals interaction with

Gln158, while NADH was involved in intermolecular

hydrogen bond formation with Arg161 and van der Waals

interaction with Gln158 (Fig. 6b; Supplementary Fig. 1d–

f). Interaction between FAD and NADH through one

intermolecular hydrogen bond was observed in complex 2

(Fig. 6a), while that of complex 4 did not reveal any

hydrogen bond (Fig. 6b). Prime/MM-GBSA calculations

for PNP showed DG score of �28.79 kcal/mol and that of

4-NC showed DG score of �27.35 kcal/mol.

3.3 MD Simulation Studies of Docked Complexes

The molecular interactions of PNP monooxygenase with

PNP, FAD and NADH, and with 4-NC, FAD and NADH

(Fig. 6a, b) corroborated well with the experimental evi-

dences reported in the literature for the substrate and

cofactor [13]. Therefore, 5-ns molecular dynamics simu-

lations (MD) were performed for complex 2 (Fig. 7a) and

complex 4 (Fig. 7b) to understand the stability of the

interactions during PNP degradation in closer-to-natural

environmental condition. The interactions obtained through

MD simulations are more convincing than docking com-

plexes. Initially, conformational stability of complex 2 and

complex 4 was evaluated. The energy plots of complex 2

and complex 4 showed that the energy of the respective

systems were stable throughout the simulations (Fig. 8a,

b). Analysis of the RMSD plot for protein backbone,

cofactors (FAD, NADH) and the substrates (PNP, 4-NC)

Fig. 5 Three-dimensional structure of PNP monooxygenase


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Table 1 Docking interactions and MD simulations of PNP, 4-NC, FAD, NADH with PNP monooxygenase

S. no. Docked molecules Hydrogen bonds Pi-cation XP

GScore

(kcal/mol)

MD simulation

Hydrogen bonds van der

Waal

interactions

1 PNP monooxygenase ? PNP (docking

in absence of FAD and NADH)

(complex 1)

Thr193, Pro152 Arg100 �3.434 Thr193, Ser151 Arg100

2 NADH (PNP monooxygenase ? PNP ?

FAD ? NADH complex) (complex 2)

Asp156, Val157, Tyr159,

Arg161, Phe448 (2)

– �9.288 Arg161, Val157,

Gln211, Gln215,

Water (19)

Gln158,

Phe148,

Asp156

3 FAD (PNP monooxygenase ? PNP ?


Asp156 (2), Gln158,

Tyr159, Arg161, Thr451

– �8.492 Arg161, Trp207,

Water (19)

Thr451

4 PNP (PNP monooxygenase ? PNP ?


Thr193, Pro152 Arg100 �3.434 Water (2) PNP monooxygenase

(Phe154, Val155, Ile191) through

one water molecule (SPC 4299)

connected to PNP

NO2 group of PNP through the path

SPC12001-SPC9446-SPC4359

connected to FAD. Similarly, NO2

group of PNP through the path

SPC12001-SPC9446-SPC3736 to

NADH

FAD and NADH are connected

through inter molecular hydrogen

bonds

5 PNP monooxygenase ? 4-NC (docking

in absence of FAD and NADH)

(complex 3)

Thr193 Arg100 �4.139 – Arg100

6 NADH (PNP monooxygenase ? 4-NC ?


Asp156 (2), Val157,

Tyr159, Arg161,

Phe448, Leu206

– �10.455 – Gln158,

Phe448,

Leu206

7 FAD (PNP monooxygenase ? 4-NC ?


Asp156, Trp207 – �8.332 Asp156 (2), Arg161,

Trp207, Arg208

Gln158

8 4-NC (PNP monooxygenase ? 4-NC ?


Thr193 Arg100 �4.139 PNP monooxygenase connected to

FAD through hydrogen bond; FAD

and NADH are connected through

hydrogen bonds; NADH is

connected to 4-NC through

hydrogen bond

Fig. 6 Docking interactions. a PNP monooxygenase with PNP, FAD and NADH, b PNP monooxygenase with 4-NC, FAD and NADH


123


for complex 2 (Fig. 9a) and complex 4 (Fig. 9b) showed

that the RMSD was relatively consistent throughout the

MD simulations period. During 5-ns MD simulations of

both the complexes, 4-NC showed lower structural rear-

rangement with PNP monooxygenase compared to PNP.

Similarly, FAD showed less structural rearrangement when

compared to NADH. Lower RMSD of docking complex

during MD simulations represents higher conformational

stability of the docking interactions. The root-mean-square

fluctuations (RMSF) of a given residue in the MD trajec-

tory were calculated for protein backbone and all side chain

atoms of each residue (Fig. 10a, b). RMSF for most of the

residues of both the complexes were within the limit of

2.5 A. The atomic fluctuations of the active site and

allosteric site residue were consistent, and backbone resi-

dues were also consistent with small conformational peak

regions. Analysis of energy plots, RMSD plots and RMSF

plots for both the complexes revealed that the two docking

Fig. 7 MD simulations of docking complexes. a PNP monooxygenase with PNP, FAD and NADH, b PNP monooxygenase with 4-NC, FAD and

NADH

(a)

(b)

Fig. 8 Energy plot for docking

complexes during molecular

dynamics simulations. a PNP

monooxygenase with

PNP ? NADH ? FAD, b PNP

monooxygenase with

4-NC ? NADH ? FAD


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complexes were stable during 5-ns MD simulation. Once

conformational stability of the docking complexes during

5-ns MD simulations was established, interactions of PNP,

4-NC, FAD and NADH with PNP monooxygenase were

analyzed in complex 2 and complex 4.

In molecular docking of complex 2 and complex 4 with

PNP and 4-NC, hydroxyl groups were observed to form

hydrogen bond with Thr193. Both these substrates were

also observed to interact with Arg100 through pi-cation

interaction (Supplementary Fig. 1). Hydrogen bond moni-

toring during MD simulations for substrate–enzyme com-

plexes, PNP and 4-NC showed intermolecular hydrogen

bond between the substrates and Thr193 in some trajecto-

ries (Supplementary Figs. 3a, b, 4a, b). The presence of

hydrogen bond in less number of trajectories for Arg100 is

quite evident for its preference for pi-cation interaction in

the docking complex, but Thr193 is mainly involved with

water-mediated intermolecular hydrogen bond with the

substrates in some of trajectories.

In complex 2, Arg161 of PNP monooxygenase inter-

acted with FAD and NADH through hydrogen bond.

Gln158 interacted with FAD through hydrogen bond and

with NADH through van der Waals interaction. Hydrogen

bond monitoring showed that the interaction of FAD and

NADH with Arg161 is maintained in all trajectories during

MD simulations (Supplementary Fig. 3d, f). MD simula-

tions also revealed that both the cofactors (FAD and

NADH) are forming hydrogen bonds with amino group of

Arg161 but with different atoms. Suitably, Gln158 showed

hydrogen bond with FAD in first � 500 ps and � 4200 to

� 4700 ps (Supplementary Fig. 3e), and no hydrogen bond

was observed between Gln158 and NADH during 5-ns MD

(a) (b)

Fig. 10 RMSF plot of PNP monooxygenase in four docking complexes during molecular dynamics simulations. a PNP monooxygenase with

PNP ? NADH ? FAD, b PNP monooxygenase with 4-NC ? NADH ? FAD

(a) (b)

Fig. 9 RMSD plot of four docking complexes during molecular dynamics simulations. a PNP monooxygenase with PNP ? NADH ? FAD,

b PNP monooxygenase with 4-NC ? NADH ? FAD


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simulations (Supplementary Fig. 3c). The hydrogen bond

between FAD and NADH observed in molecular docking

was also reproduced (Fig. 11a).

In complex 4, Arg161 was observed to interact through

hydrogen bond with NADH in first � 200 ps and with

FAD in � 4500 to � 5000 ps (Supplementary Fig. 4d, f).

Gln158 was observed to form hydrogen bond in very few

trajectories (Supplementary Fig. 4c). The result coincided

favorably with the docking results observed for FAD and

NADH interactions in complex 4 (Supplementary Fig. 1).

Additionally, hydrogen bond interaction between FAD and

NADH was identified in complex 4 in all trajectories

during MD simulations (Fig. 11b). The final trajectory of

5-ns MD simulations revealed that PNP interacted with

residues Phe154, Val155, Ile191 of PNP monooxygenase

through a water bridge by SPC4299. The NO2 group of

PNP interacted with FAD through intermolecular water

bridge formed by three water molecules, from PNP to

SPC12001-SPC9446-SPC4359 to FAD. Similarly, NO2

group of PNP was connected with NADH through water

bridge with SPC12001-SPC9446-SPC3736 (Fig. 7a; Sup-

plementary Fig. 2). An intermolecular hydrogen bond was

also observed between FAD and NADH in complex 2.

Water-mediated interactions and intermolecular hydrogen

bonds were observed among PNP monooxygenase, PNP,

FAD and NADH in all 1042 trajectories. The two cofactors

FAD and NADH showed more than one intermolecular

hydrogen bonds among themselves (Fig. 11a) in all tra-

jectories. Similar analysis of complex 4 after 5-ns MD

simulations revealed FAD interaction with PNP

monooxygenase through intermolecular hydrogen bond

with residues Asp156, Arg161, Trp207 and Arg208. Two

hydrogen bonds were observed between FAD and NADH.

NADH was connected to 4-NC through hydrogen bond

(Fig. 7b; Supplementary Fig. 2). The intermolecular

hydrogen bond monitoring between FAD and NADH

showed two hydrogen bonds in most of the trajectories

(Fig. 11b).

The analysis of docking complex thus predicted poten-

tial path of PNP monooxygenation. Initially, PNP

monooxygenase induces oxygenation through Thr193 and

Arg100 to form 4-NC. Further, reduction of 4-NC is

induced by NADH through FAD to form THB. The bind-

ing orientations of ligand molecules predicted through MD

simulations showed better correlation to their biologically

active states. Our present results clearly corroborate the

earlier observations that the two-component monooxyge-

nase catalyzes both the hydroxylation of PNP and the

oxidative release of nitrite from 4-NC in B. sphaericus

JS905 [10].

4 Conclusions

In the present study, we attempted to create an in silico

model for PNP monooxygenase by aiming at the physio-

logical substrates, PNP and 4-NC, along with the cofactors,

FAD and NADH. We explored residues Arg100, Gln158

and Thr193 as key catalytic residues which play a major

role in controlling the orientation of the nitro group at the

active site, and Arg161, Phe154, Val155, Ile191, Trp207

and Arg208 as cofactor-binding residues. Binding orien-

tations of the substrates, PNP and 4-NC, and the cofactors,

FAD and NADH, provided a basis for analyzing novel

mechanism of PNP monooxygenation reactions in silico.

Thus, the modeled 3D structure of PNP monooxygenase

showed consistent docking results of enzyme–substrates

interactions which correlated well with the earlier

(a) (b)

Fig. 11 Hydrogen bonds between FAD and NADH during 5-ns MD simulations. a PNP monooxygenase with PNP ? NADH ? FAD, b PNP

monooxygenase with 4-NC ? NADH ? FAD


123


experimental results that the enzyme catalyzes two

sequential monooxygenation reactions to yield THB with

the concomitant release of nitro group as nitrite.

Acknowledgments KM thanks the University Grants Commission,

New Delhi, India, for the financial assistance.

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In silico approach to support that p-nitrophenol monooxygenase from Arthrobacter sp. strain JS443...

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