Prediction of the antibacterial silver nanoparticles interaction on L ...

Int. J. Biosci.

2014 International Journal of Biosciences | IJB | ISSN: 2220-6655 (Print) 2222-5234 (Online) http://www.innspub.net Vol. 5, No. 7, p. 241-247, 2014

RESEARCH PAPER

OPEN ACCESS

Prediction of the antibacterial silver nanoparticles interaction on L- lactate dehydrogenase (LDH1) in Staphylococcus aureus Javad Sharifi-Rad1, 2, Seyedeh Mahsan Hoseini-Alfatemi3, Fatemeh Taktaz4* 1

Zabol Medicinal Plants Research Center, Zabol University of Medical Sciences, Zabol, Iran

2

Department of Pharmacognosy, Faculty of Pharmacy, Zabol University of Medical Sciences, Zabol,

Iran 3

Department of Bacteriology and Virology, Shiraz Medical School, Shiraz University of Medical

Sciences, Shiraz, Iran 4

Department of Biology, Faculty of Basic Sciences, Hakim Sabzevari University, Sabzevar, Iran

Key words: Antibacterial Silver Nanoparticles, Lactate Dehydrogenase, Biocompatibility, Molecular mechanism.

http://dx.doi.org/10.12692/ijb/5.7.241-247

Article published on October 15, 2014

Abstract The aim of present study was to study the Bioinformatics interaction of silver nanoparticle and L- Lactate Dehydrogenase enzyme in Staphylococcus aureus in order to determine the harmful effects of silver nanoparticle on the this enzyme. The possible interaction between silver nanoparticles and L- Lactate Dehydrogenase enzyme was assessed. Metal Detector Predicts v2.0 software, DiANNA 1.1 web server, Molecular docking web server, 3DLigand Site server, were used to analyse enzyme structure and silver effect on its structure. The results obtained from docking showed that the free energy of binding from docking score for LLactate Dehydrogenase was -2.76 kcal/mol and Inhibition Constant (Ki) was equal to 9.52 mM. The silver nanoparticles and L-Lactate Dehydrogenase demonstrated interaction with an Interact Surface score of about 237.3. Silver nanoparticles may have made interactions with Cysteine 28, 86, 70,115,141 and 160 positions in the enzyme. It can be safely suggested that antibacterial nanosilver can get attached to L- Lactate Dehydrogenase in Staphylococcus aureus cells thus leading to deactivation of the enzyme. * Corresponding

Author: Fatemeh Taktaz  [email protected]

241 Sharifi-Rad et al.

Int. J. Biosci.

2014

Introduction

particles exhibit antimicrobial properties (Petica et

In the field of medicine, nanoparticles are extensively

al., 2008). Silver nanoparticles have been studied as a

explored because of their size dependant chemical

medium for antibiotic delivery, and to synthesize

and physical properties (Rad et al., 2013a). The size of

composites for use as disinfecting filters and coating

nanoparticles is similar to that of most biological

materials. Silver is generally used in the nitrate form

molecules and structures (Rad et al., 2013b). Another

to induce an antimicrobial effect, but when silver

interesting viewpoint of their exploration in medicine

nanoparticles are used, there is a huge increase in the

is the use of nanoparticles as antimicrobials to target

surface area available for the microbe to be exposed

highly pathogenic and drug resistant microbes. But,

to. Though silver nanoparticles find use in many

as far as the application of nanoparticles in biology, is

antibacterial applications, the action of this metal on

concerned, biocompatibility is a highly desired trait

microbes is not fully known. It has been hypothesized

(Miri et al., 2013; Rad et al., 2013). Biocompatibility

that silver nanoparticles can cause cell lysis or inhibit

is the material’s ability to perform medically without

cell transduction. There are various mechanisms

exertion of undesired local or systemic effects (Samia

involved in cell lysis and growth inhibition (Prabhu et

et al., 2006 ). The emerging infectious diseases and

al., 2012).

the development of drug resistance in the pathogenic bacteria and fungi at an alarming rate is a matter of

Lactate dehydrogenase catalyses the inter conversion

serious concern (Alfatemi et al., 2014; Rad et al.,

of pyruvate and lactate with concomitant inter

2014). Despite the increased knowledge of microbial

conversion of NADH and NAD+. It converts pyruvate,

pathogenesis and application of modern therapeutics,

the final product of glycolysis, to lactate when oxygen

the morbidity and mortality associated with the

is absent or in short supply and it performs the

microbial infections still remain high. Therefore,

reverse reaction during the Cori cycle in the liver.

there is a pressing demand to discover novel

Lactate dehydrogenases exist in four distinct enzyme

strategies and identify new antimicrobial agents from

classes. Each one acts on either D-lactate (D-lactate

natural and inorganic substances to develop the next

dehydrogenase (cytochrome)) or L-lactate (L-lactate

generation of drugs or agents to control microbial

dehydrogenase (cytochrome)). Two are cytochrome c-

infections (Sharifi-Rad et al., 2014). Nanoparticles

dependent enzymes. Two are NAD (P)-dependent

usually ranging in dimension from 1-100 nanometres

enzymes. This article is about the NAD (P)-dependent

(nm) have unique properties vis-à-vis their bulk

L-lactate

equivalent. Simultaneously, with a decrease in

Staphylococcus aureus that is an important human

dimensions of the materials to the atomic level, their

pathogen whose ability to acquire resistance to

properties change. The nanoparticles possess unique

various antibiotics and increased expression of

physico-chemical, optical and biological properties

pathogenic determinants has added to its emergence

which can be manipulated suitably for desired

in both acute and community healthcare settings

applications (Feynman et al., 1991). Moreover, as the

(Stockland et al., 1969). L-Lactate Dehydrogenase

biological processes also occur at the nanoscale and

Reaction

due

reaction are shown in the Figures 1 and 2,

to

their

functionalization,

amenability the

to

nanoparticles

biological are

finding

dehydrogenase

and

Lactate

(E.C.1.1.1.27)

dehydrogenase

from

Structure

respectively.

important applications in the field of medicine. The aim of this study was to investigate the Prediction Silver compounds have been used to treat burns,

of the antibacterial silver nanoparticles interaction on

wounds and infections (Dunn et al., 2004). Besides,

L- Lactate dehydrogenase (LDH1) in Staphylococcus

various silver salts and their derivatives have been

aureus.

used as antimicrobial agents (Russell et al., 1994). Recent studies have reported that nano sized silver


Materials and methods

Int. J. Biosci.

2014

In the first step, amino acid sequence of lactate

identified. 3DLigand Site server and Site Hound web

dehydrogenase enzyme with the number of 3D0O was

server was used for prediction of potentially binding

taken from Protein Data Bank (PDB) website. In the

sites of model to metal ligands.

next step, silver with molecular formula Ag (number 22878) was procured via ChemSpider website. The

Results

Molecular docking web server was used for docking

Protein Structure Analysis

study

Also,

The lactate dehydrogenase consisted of two domains

studies on disulfide bonds were carried out using

(http://www.dockingserver.com/web).

and each of them had 317 amino acids, with

Metal Detector Predicts v2.0 software and DiANNA

molecular weight 34,583 KD. Enzyme catalytic

1.1

residues were Arg155 (A) - Asp152 (A) - His179 (A)

web

server

(http://clavius.bc.edu/~clotelab/DiANNA/). All Cys

respectively (Table 1).

and His residues in amino acid sequences were Table 1. Amino acid sequence analysis of L- Lactate Dehydrogenase for one domain (www.uniprot.org). No 1 2 3 4 5 6

Feature key Chain Active site Nucleotide binding Modified residue Binding site Binding site

Position(s) 1 – 317 179 15 – 43 223 92-155-232 124

Length 317 1 29 1 1 1

Description L-lactate dehydrogenase Proton acceptor NAD Phosphotyrosine Substrate NAD or substrate

Table 2. Disulphide bonds in L- Lactate Dehydrogenase. NO 1 2 3

Cysteine sequence position 28 – 86 70 - 115 141 - 160

Distance 58 45 19

Bond DIALECERYLA-NMVTRCNNVGV VSYKLCTRSGN-GTSSTCGSYFN FNDGKCKTGSG-TQVRDCRLTGL

Score 0.99587 0.79343 0.99897

Disulfide bonds

cell membrane that are involved in trans membrane

The disulfide bonds in L- Lactate Dehydrogenase had

energy production and ion transportation. It is also

number 3 and the Cysteines were predicted with all of

believed that silver can join in catalytic oxidation

their scores (Table 2).

reactions that result in the construction of disulfide bonds (R-S-SR). Silver performs this process by

Furthermore,

small

particles

exhibited

higher

catalyzing the reaction between oxygen molecules in

antimicrobial activity than big particles. This could be

the cell and hydrogen atoms of thiol groups so that

due to high particle penetration when these particles

water is subsequently released as a product and two

have smaller sizes. The antibacterial properties are

thiol groups become covalently bonded to one

related to the total surface area of the nanoparticles.

another through a disulfide bond (Feng et al., 2000).

Smaller particles with larger surface to volume ratios have greater antibacterial activity, although the

Cysteines

antimicrobial properties of silver have been identified

Predictor

for centuries; recently we have only begun to

The Cysteines and Histidines Metal Binding Sites

understand the mechanisms by which silver inhibits

with Site Hound web server and ligand binding sites

bacterial growth. It is believed that silver atoms bind

by computing the interactions with Site Hound web

to thiol groups (-SH) in enzymes and consequently

server are shown in Tables 3 and 4, respectively. Site

cause the deactivation of enzymes. Silver forms stable

Hound-web

S-Ag bonds with thiol-containing compounds in the

computing the interactions between a chemical probe


and

Histidines

identified

Metal

ligand

Binding

binding

Sites

sites

by

Int. J. Biosci.

2014

and a protein structure. By using web server, 7

Molecular docking study

regions with different levels of energy had been

Docking calculations were carried out using Docking

recognized. The regions are divided into A to G (Table

Server. Gasteiger partial charges were added to the

1). The highest energy is related to ligand bound to

ligand atoms. Non-polar hydrogen atoms were

enzyme at a position which is equal to -1905.59.

merged, and rotatable bonds were defined. Essential hydrogen atoms, Kollman united atom type charges,

Table 3. Cysteines and Histidines metal binding sites

and solvation parameters were added with the aid of

with site Hound web server.

Auto

Dock

tools.

Docking

simulations

were

Position

Residue

performed using the Lamarckian Genetic Algorithm

15

H

(LGA) and the Solis & Wets local search method.

28

C

Initial position, orientation, and torsions of the ligand

70

C

86

C

molecules were set randomly. All rotatable torsions

101

H

103

C

115

C

141

C

evaluations. Interaction parameters and structure of

160

C

silver nanoparticles and L- Lactate Dehydrogenase

185

H

enzyme are shown in Table 5 and Figure 3. Also

201

H

analysis of its hydrogen bonding network by hydrogen

215

H

bonding plot shown in Figure 4 was performed

299

H

305

H

because functional and structural characterization of

331

H

were released during docking. Docking experiment was derived from 10 different runs that were set to terminate after a maximum of 250000 energy

a protein is based on analysis of its hydrogen bonding network.

Table 4. Ligand binding sites by computing interactions with Site Hound web server. Rank

Energy

Energy Range

Volume

A

-1905.59

(-16.27, -8.91)

165.00

B

-1236.83

(-16.66, -8.93)

103.00

C

-1206.47

(-20.22, -8.96)

96.00

D

-812.02

(-17.35, -8.92)

72.00

E

-729.72

(-18.57, -8.98)

64.00

F

-707.13

(-16.45, -8.94)

63.00

G

-662.56

(-19.79, -8.91)

59.00

Center (x,y,z) 31.913

56.793

19.506

38.205

63.539

18.808

33.720

60.463

6.019

43.761

84.105

25.447

60.613

65.383

32.051

9.934

53.349

35.634

8.473

35.585

16.365

The silver-catalyzed formation of disulfide bonds

water. An exploration to find the potential toxic

could probably modify the shape of cellular enzymes

mechanisms and long-standing effects by which these

and consequently affect their function. However,

nanomaterials

these effective biocidal properties have the potential

throughout broad production and use is increasing.

to

Extensively

adversely

affect

beneficial

bacteria

in

an

environment especially those existing in the soil and


could

used

cause

environmental

nanoparticles,

such

as

risks silver

nanoparticles will most likely enter the environment

Int. J. Biosci.

2014

and may produce a physiological response in certain

ions, whereas metallic silver is relatively unreactive. It

organisms, possibly altering their fitness and finally

has also been revealed that nanoparticles penetrate

might

community

microbial cells, signifying that lower concentrations

densities. Research concerning the ecotoxicity of NPs

of nanosize silver particles would be adequate for

is still promising and gaps exist in our knowledge of

microbial control. This approach can be more capable

this area. Although there have been studies exploring

than existing treatments, especially for certain

the toxicity of metal NPs and despite their widespread

organisms that are resistant to cell penetration which

application, currently there is inadequate toxicity data

results in less sensitivity to antibiotics (Barani et al.,

necessary to fill the gap in the source pathway

2011). The actual mechanism by which silver

receptor-impact framework necessary to correct risk

nanoparticles interfere with bacteria is not fully

measurement of Ag NPs. These results suggest the

understood. Some researchers suggest that silver

possibility using of silver nanoparticles to eliminate

nanoparticles damage bacterial cells by destroying the

phyto pathogens. Several parameters will require

enzymes that transport the cell nutrient and

estimation

application,

weakening the cell membrane or cell wall. The toxicity

including phyto toxicity and antimicrobial effects in

of nanosilver can be explained through the interaction

situ and development of systems for delivering

of nanoparticles with microbes involving silver ion

particles into host tissues that have been colonized by

release. The nanosilver toxicity is species specific.

phyto pathogens. It is believed that nanometer-sized

Small sized Ag-NPs can inhibit bacterial growth more

silver particles have different physical and chemical

than silver ions at the same silver concentrations.

properties from their macro scale counterparts which

Research has been focused on antibacterial material

alter their interaction with biological structures.

containing various natural and inorganic substances

Undeniably, several pieces of evidence support the

among them, silver or silver ions have long been

hypothesis that silver nanoparticles have enhanced

known to have potential inhibitory and bactericidal

antimicrobial

are

effects as well as a broad spectrum of antimicrobial

Ag+

activities (Gavanji et al., 2013).

affect

their

populations

proceeding

activity.

to

or

practical

Silver

nanoparticles

extremely reactive, which is due to generation of Table 5. Interaction parameters. Rank Silver nanoparticle

Free Energy of Binding -2.76 kcal/mol

Inhibition Constant, Ki 9.52 mM

Electrostatic Energy +0.07 kcal/mol

Interact Surface 237.3

NPs, which are produced by living organisms or biological processes, have synergistic effects with antibiotics such as erythromycin, chloramphenicol, ampicillin, and kanamycin against gram-negative and gram positive bacteria. The combination of biogenic Ag NPs with antibiotics has efficient antibacterial activity. In fact, the ampicillin damages the cell wall

Fig 1. L-Lactate Dehydrogenase Reaction.

and mediates the internalization of Ag NPs into the Discussion

bacteria. These NPs bind to DNA and inhibit DNA

The exact mechanisms of NP toxicity against various

unwinding, which leads to cell death. Moreover, Ag

bacteria are not understood completely. NPs are able

NPs modified with titanium are toxic to S. aureus. Ag

to attach to the membrane of bacteria by electrostatic

NPs naturally interact with the membrane of bacteria

interaction and disrupt the integrity of the bacterial

and disrupt the membrane integrity, and the silver

membrane. The mechanisms of NP toxicity depend

ions bind to sulphur, oxygen, and nitrogen of

on

intrinsic

essential biological molecules and inhibit bacterial

properties, and the bacterial species. Biogenic Ag

growth. The aforementioned studies show that

composition,

surface

modification,


Int. J. Biosci.

2014

suitable NPs can be selected to fight against specific bacteria.

Fig. 3. Interaction of silver nanoparticles and LLactate Dehydrogenase enzyme.

Fig 2. Lactate Dehydrogenase structure.

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