Mn2+ and Mg2+ synergistically e

ORIGINAL ARTICLE

Mn2+ and Mg2+ synergistically enhanced lactic acidproduction by Lactobacillus rhamnosus FTDC 8313 viaaffecting different stages of the hexose monophosphatepathwayL.-C. Lew, S.-B. Choi, P.-L. Tan and M.-T. Liong

School of Industrial Technology, Universiti Sains Malaysia, Penang, Malaysia

Keywords

biotechnology, Lactobacillus, modelling,

probiotics.

Correspondence

Min-Tze Liong, School of Industrial Technol-

ogy, Universiti Sains Malaysia, 11800 USM,

Penang, Malaysia.

E-mail: [email protected]

2013/1207: received 17 June 2013, revised

10 October 2013 and accepted 15 November

2013

doi:10.1111/jam.12399

Abstract

Aims: The study aimed to evaluate the effects of Mn2+ and Mg2+ on lactic

acid production using response surface methodology and to further study their

effects on interactions between the enzymes and substrates along the hexose

monophosphate pathway using a molecular modelling approach.

Methods and Results: A rotatable central composite design matrix for lactic

acid production was generated with two independent factors namely,

manganese sulfate and magnesium sulfate. The second-order regression model

indicated that the quadratic model was significant (P < 005), suggesting thatthe model accurately represented the data in the experimental region. Three-

dimensional response surface showed that lactic acid production was high

along the region where the ratio of MnSO4 to MgSO4 was almost 1 : 1,

justifying the need for both Mg2+ and Mn2+ to be present simultaneously in

stimulating the production of lactic acid. Molecular docking simulation was

performed on a total of 13 essential enzymes involved in the hexose

monophosphate pathway for the production of lactic acid with four different

conditions namely in the presence of Mg2+, Mn2+, both Mg2+ and Mn2+ and

in the absence of metal ions. Results showed that the presence of both Mg2+

and Mn2+ within the binding site improved the binding affinity for substrates

in five enzymes namely, glucose-6-phosphate dehydrogenase, phosphogluconate

dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, phosphopyruvate

hydratase and pyruvate kinase.

Conclusions: Using response surface methodology and molecular modelling

approach, we illustrated that Mg2+ and Mn2+ synergistically enhanced lactic

acid production by Lactobacillus rhamnosus FTDC 8313 via affecting different

stages of the hexose monophosphate pathway.

Significance and Impacts of the Study: Mg2+ and Mn2+ synergistically

improved lactic acid production of Lact. rhamnosus via improved binding

affinity of the enzymesubstrate along the hexose monophosphate pathway,instead of purely affecting growth as previously understood.

Introduction

Lactic acid bacteria (LAB) are characterized as Gram-posi-

tive, nonspore forming, facultative anaerobic bacteria that

produce lactic acid as the major fermentation product either

homo- or heterofermentatively. The homofermentative

pathway results in the transformation of glucose to pyruvate

through the EmbdenMeyerhofParnas pathway, yielding2 mol of lactic acid from 1 mol of glucose. Meanwhile, in

heterofermentation, 1 mol of glucose produces 1 mol each

of lactate, carbon dioxide and either acetic or ethanol, via

the hexose monophosphate pathway. The ability of LAB to

Journal of Applied Microbiology 116, 644--653 2013 The Society for Applied Microbiology644

Journal of Applied Microbiology ISSN 1364-5072

produce lactic acid as the main product from a range of car-

bon sources has been a key contribution to industries typi-

cally as preservative, acidulant and flavouring for food

products. Lactic acid has also been widely used for many

years in textile and pharmaceutical industries, as well in cos-

metic and skin care products. In addition, the demand for

lactic acid has also increased considerably over the years

attributed to its role as monomer in the production of bio-

degradable plastics (Wee et al. 2006). According to a recent

forecast by Global Industry Analysts Inc. (GIA), the world

market for lactic acid will reach 3673 thousand metric tonsby 2017. Although lactic acid can be produced via chemical

synthesis, microbial fermentation is commonly preferred

attributed to a higher optical purity, a characteristic impor-

tant for biodegradable polymers. Chemical synthesis from

petrochemical resources often produced racemic of DL-lac-

tic acid, while an optically pure L(+) or D()-lactic acidcan be produced from fermentation of renewable sources

with the appropriate microbial strains (Hofvendahl and

Hahn-Hagerdal 2000). In addition, growing environmental

concerns arising from the pollution caused by the petro-

chemical industry and soaring international oil prices fur-

ther strengthen the preference of microbial fermentation

over chemical synthesis.

Due to the extensive industrial use and enormous eco-

nomical value, production of lactic acid from LAB has

been intensively studied throughout the years and opti-

mization of lactic acid production has become the ulti-

mate objective in most of the studies conducted. The

effects of different fermentation medium such as beet

molasses (Kotzamanidis et al. 2002), vine-trimming waste

(Bustos et al. 2004), soybean hydrolysate (Kwon et al.

2000), lactose and concentrated cheese whey (Schepers

et al. 2002) on lactic acid production has been investi-

gated. Other means of increasing lactic acid production

includes, optimization of fermentation systems by utiliz-

ing the cell-recycle system, together with repeated batch

and continuous processes (Oh et al. 2003); utilization of

genome shuffling approach (Yu et al. 2008); development

mutant strain with high lactic acid production (Bhatt and

Srivastava 2012); utilization metabolic engineering

approach (Kyla-Nikkila et al. 2000); cell immobilization

(Senthuran et al. 1999); and development of continuous

electrodialysis fermentation system for higher production

of lactic acid (Min-tian et al. 2005). The effects of culture

temperature, nitrogen sources, pH, oxygen and growth-

stimulating elements such as B-vitamins and amino acids

on lactic acid production by LAB have also been reported

(Hujanen and Linko 1996; Fu and Mathews 1999). Lactic

acid production by Lactobacillus rhamnosus have been

demonstrated to be predominantly growth-associated

(Berry et al. 1999), while our previous study demon-

strated that growth enhancement via the addition of

divalent metal ions led to enhanced production of lactic

acid (Lew et al. 2012). Despite various data supporting

the growth promoting effects of divalent metal ions,

information on the exact targets of divalent metal ions

during synthesis of lactic acid by LAB remains scarce.

Thus, to further understand the influence of divalent

metal ions on the production of lactic acid, a molecular

modelling approach was adopted with the aim of observ-

ing and studying the interactions between the enzymes

and substrates involved along the hexose monophosphate

pathway in the presence of divalent metal ions.

Materials and methods

Bacteria and media preparation

Lactobacillus rhamnosus FTDC 8313 was obtained from

the Culture Collection Centre of Bioprocess Technology

Division, School of Industrial Technology, Universiti

Sains Malaysia (Penang, Malaysia). The strain was acti-

vated in sterile de Man, Rogosa and Sharpe (MRS) broth

(Hi-Media, Mumbai, India) containing 02 mmol l1MnSO4 and 08 mmol l1 MgSO4 for three consecutivetimes using 10% (v/v) inoculum and incubated at 37Cfor 24 h ahead of use. The stock cultures were stored at

20C in 40% (v/v) sterile glycerol.

Determination of lactic acid

Determination of lactic acid was performed according to

Lew et al. (2012). Sterile reconstituted skimmed milk (8%;

w/v) was supplemented with manganese sulfate, MnSO4(Sigma-Aldrich, Steinheim, Germany) and magnesium sul-

fate, MgSO4 (Sigma-Aldrich) as according to Table 1. The

medium was then inoculated with 10% (v/v) inoculum and

incubated at 37C for 12 h. A 100 ll of 158 mol l1 HNO3was added to 15 ml of sample to digest the protein. The fer-mentation broth was then centrifuged at 10 000 g for

15 min at 25C, filtered through a 02 lm cellulose acetatesyringe filter and stored at 20C prior to analyses. A high-performance liquid chromatography (HPLC) equipped with

UV/Vis detector (Jasco 875-UV, Tokyo, Japan) set at

220 nm was used to determine the concentration of lactic

acids. An Aminex HPX-87H column (300 9 78 mm; Bio-Rad Laboratories, Richmond, CA, USA) maintained at 65Cwas used with a degassed mobile phase of 0004 mol l1H2SO4 at a flow rate of 06 ml min1.

Response surface methodology

Response surface methodology was applied with two

independent factors namely, manganese sulfate (X1) and

magnesium sulfate (X2), to generate a central composite

Journal of Applied Microbiology 116, 644--653 2013 The Society for Applied Microbiology 645

L.-C. Lew et al. Mn2+ and Mg2+ enhanced lactic acid production

design (CCD) matrix with an alpha value of 1414. Thetreatment combinations were allocated into two blocks,

with the first and second block representing the first day

and second day of experiment, respectively. The first

block contained the full-factorial runs accompanied by

three centre runs while the second block contained the

axial runs accompanied by three centre runs. All experi-

mental points are presented as the mean values of a trip-

licate determination. Screening and selection of factors

and their varying concentrations were determined as pre-

viously described (Lew et al. 2012).

The modelling and statistical analyses were carried out

using DESIGN EXPERT 5 (software version 5.07; Stat-Ease

Corp., Minneapolis, MN, USA). An experiment for vali-

dation purposes was carried out to confirm the legitimacy

and reproducibility of the model. The concentration of

lactic acid was assessed using a random point, and the

actual result was compared with the predicted value by

the model.

Molecular docking

A total of 13 essential enzymes involved in the hexose

monophosphate pathway for the production of lactic acid

were selected from Protein Data Bank (PDB) for the

computational study (Table 4). Molecular docking simu-

lation was performed on these enzymes with four differ-

ent conditions namely in the presence of magnesium

(Mg2+), manganese (Mn2+), both Mg2+ and Mn2+ and

without any metal ions. Different divalent metal ions

were docked using AUTODOCK 4.2 (Morris et al. 2009). A

total of 100 runs with 250 population size, with Lamarki-

an genetic searching algorithm and root mean square

tolerance of 10 A were set as the docking input parame-ter. The lowest free energy of binding (FEB) of each con-

formation in the most populated cluster was selected.

Analysis and visualization of the docking results was per-

formed using VMD (Humphrey et al. 1996) and LIGPLOT

(Wallace et al. 1995).

Statistical analysis

Data analysis was performed using SPSS Inc. software

(version 20.0; Chicago, IL, USA). Two-way analysis of

variance (ANOVA) was used to evaluate the significant dif-

ferences between sample means, with significance level at

a = 005. Mean comparisons were assessed by Tukeystest, and all data presented were mean values obtained

from three separate runs. P-values were stipulated to

indicate the general inclination of the factors studied

on the response variables with the respective statistical

significance.

Results

Lactic acid response surface

A rotatable CCD with an alpha value of 1414, with afixed middle point of X1 (4142 mmol l1 MnSO4) andX2 (3205 mmol l1 MgSO4), was used to generate thedesign matrix and response (production of lactic acid;

Table 1). By fitting the experimental data with least

squares method, a simulated second-order expression was

obtained as follows:

Y 035 000069X1 00032X2 00062X21 0002X22 00085X1X2

1

where Y is the predicted response of lactic acid, while X1and X2 are the coded values of MnSO4 and MgSO4,

respectively.

The adequacy and fitness of the model were evaluated

using analysis of variance (ANOVA), and the data obtained

is presented in Table 2. The regression analyses indicated

that the quadratic model was significant (P < 005) sug-gesting that the model accurately represented the data in

the experimental region. The insignificant P-value

(01370) of lack-of-fit indicated a reasonable fit of themodel as an approximation to the true response. The

Table 1 Matrix of the central composite design in coded levels for

the factors and response, for the production of lactic acid by Lactoba-

cillus rhamnosus FTDC 8313 in the presence of MgSO4 and MnSO4

Standard

run Block*

MnSO4(X1)

MgSO4(X2)

Response

(Y),

1 1 0 0 0355 00012 1 0 0 0358 00013 1 1 1 0365 00024 1 1 1 0336 00015 1 1 1 0346 00016 1 0 0 0352 00037 1 1 1 0341 00028 2 0 0 0349 00029 2 0 0 0356 0001

10 2 0 a 0349 000311 2 0 a 0350 000212 2 a 0 0338 000113 2 0 0 0356 000114 2 a 0 0344 0002

*1 = batch 1; 2 = batch 2.

X1 = concentration of manganese sulfate added into the fermenta-

tion broth (17756508 mmol l1; a = 07697514 mmol l1);X2 = concentration of magnesium sulfate added into the fermenta-

tion broth (12175274 mmol l1; a = 03656127 mmol l1).Y = production of lactic acid (mg ml1) by Lact. rhamnosus FTDC8313.

All results are means standard deviation from three separate runs;n = 3.


Mn2+ and Mg2+ enhanced lactic acid production L.-C. Lew et al.

statistical analyses with coefficient estimates and the sig-

nificance of the lactic acid response model are presented

in Table 3. Considering that a quadratic model was used,

analyses of coefficient estimates were performed on qua-

dratic effects. Our data indicated that MnSO4 produced a

significant (P = 00121) quadratic effects on lactic acidproduction, while MgSO4 did not. Despite this, the inter-

action of MnSO4 and MgSO4 produced a significant

effect on the production of lactic acid by Lact. rhamnosus

FTDC 8313 (P = 00119), indicating that both factorsexerted a synergistic effect and should be present together

to achieve a high production of lactic acid.

A three-dimensional (3-D) response surface for the pro-

duction of lactic acid (Fig. 1) generated based on the sec-

ond-order equation (Eqn 1) illustrated that an optimum

region could be observed. This region occurred in the

presence of 473 mmol ml1 MnSO4 and 442 mmol ml1MgSO4, in tandem with our previous findings of an opti-

mum growth region (1059 log10 CFU ml1; Lew et al.2012), justifying that the production of lactic acid was

growth-associated. However, from the 3-D response sur-

face of lactic acid, it is also observed that lactic acid pro-

duction was high at along the region where the ratio of

MnSO4 to MgSO4 was almost 1 : 1. When one of the diva-

lent metal ions was at a high concentration and the other

at a low concentration, lactic acid production decreased.

Validation experiment was performed to further ascer-

tain the predictions and the reliability of the regression

model. Lactic acid concentration obtained in the presence

of 473 mmol ml1 MnSO4 and 442 mmol ml1 MgSO4were compared with the predicted value from the model.

Our results showed that the lactic acid concentration

obtained from actual experimentation was 0356 mg ml1,producing an error of 030% as compared to the predictedvalue. The small error indicated that the prediction gene-

rated from the model was reliable and valid.

Molecular docking

The hexose monophosphate pathway for the production of

lactic acid comprises of 13 enzymes (Fig. 2) and all the

enzymes studied had individual binding sites. Molecular

docking of Mn2+ and Mg2+ was performed within the sub-

strate binding site prior to enzymesubstrate docking, forenzymes that were void of divalent metal ions in their co-

crystal structures. The presence of both Mg2+ and Mn2+

synergistically reduced the FEB for substrates, in five

enzymes along the hexose monophosphate pathway

(Table 4; Fig. 3); glucose-6-phosphate dehydrogenase,

phosphogluconate dehydrogenase, glyceraldehyde-3-phos-

phate dehydrogenase, phosphopyruvate hydratase and

pyruvate kinase. In addition to FEB, four of the five

enzymes (glucose-6-phosphate dehydrogenase, phosphog-

luconate dehydrogenase, glyceraldehyde-3-phosphate

dehydrogenase and pyruvate kinase) showed similar

Table 2 Analysis of variance (ANOVA) of the second-order model (Y)*, for the production of lactic acid by Lactobacillus rhamnosus FTDC 8313 in

the presence of MgSO4 and MnSO4

Source Sum of squares df Mean square F-value P-value

Regression

Linear 845 9 105 2 423 9 105 055 05940Quadratic 592 9 104 3 197 9 104 777 00125

Model 677 9 104 5 135 9 104 533 00245Residual 177 9 104 7 254 9 105Lack-of-fit 127 9 104 3 424 9 105 335 01370Pure error 507 9 105 4 127 9 105Correlation total 863 9 104 13

*Y 035 000069X1 00032X2 00062X21 0002X22 00085X1X2 [Y = production of lactic acid (mg ml1) by Lact. rhamnosus FTDC8313; X1 = concentration of manganese sulfate added into the fermentation broth (17756508 mmol l1; a = 07697514 mmol l1);X2 = concentration of magnesium sulfate added into the fermentation broth (12175274 mmol l1; a = 03656127 mmol l1)].R2 = 07920.Significant at an a level of 005.

Table 3 Analysis of the coefficient estimates of the second-order

model (Y)* for the production of lactic acid by Lactobacillus rhamno-

sus FTDC 8313 in the presence of MgSO4 and MnSO4

Variable Coefficient estimate Standard error t-value P-value

Intercept c0 = 035 00021X1 c1 = 000069 00018 039 07103X2 c2 = 00032 00018 178 01178X21 c11 = 00062 00019 336 00121X22 c22 = 00020 00019 107 03213X1X2 c12 = 00085 00025 337 00119

*Y c0 c1X1 c2X2 c11X21 c22X22 c12X1X2 [Y = production oflactic acid (mg ml1) by Lact. rhamnosus FTDC 8313].X1 = concentration of manganese sulfate added into the fermenta-

tion broth (17756508 mmol l1; a = 07697514 mmol l1);X2 = concentration of magnesium sulfate added into the fermenta-

tion broth (12175274 mmol l1; a = 03656127 mmol l1).Significant at an a level of 005.



binding conformation with their respective substrates,

where Mn2+ was located closely to the phosphates moiety

(

-D-Glucose-6P

-D-Glucose-6P

6-P-D-Glucono-1,5-lactone

6-P-D-Gluconate

D-Ribulose-5-P

D-Xylulose-5-P

Glyceraldehyde-3-P

1,3-P-Glycerate

3-P-Glycerate

2-P-Glycerate

PEP

Pyruvate

D-lactatedehydrogenase

D-Lactate L-Lactate

L-lactate dehydrogenase

Pyruvate kinase

Phosphoglycerate kinase

Phosphoglycerate mutase

Phosphopyruvatehydratase

Glyceraldehyde-3-phosphatedehydrogenase

Xylulose-5-phophatephophosketolase

Ribulose phosphate-3-epimirase

Phosphogluconatedehydrogenase

Glucose-6-phosphateisomerase

Glucose-6-phosphatedehydrogenase Divalent metal ions

The presence of Mn2+ and Mg2+significantly affected the bindingof -D-Glucose-6P toGlucose -6-phosphatedehydrogenase

The presence of Mn2+ and Mg2+significantly affected the bindingof 6-P-D-Gluconate toPhosphogluconatedehydrogenase

The presence of Mn2+ and Mg2+significantly affected thebinding of Glyceraldehyde-3-Pto Glyceraldehyde-3-phosphate dehydrogenase

The presence of Mn2+ and Mg2+significantly affected thebinding of 2-P-Glycerateto phosphopyruvatehydratase

The presence of Mn2+ and Mg2+ significantly affected thebinding of PEP to Pyruvate kinase

( Mn2+,

Mn2+

Mg2+)

Mg2+

Mn2+

Mg2+

Mn2+ Mg2+

Mn2+

Mg2+

Mn2+

Mg2+

Divalent metal ions( Mn2+, Mg2+)




6-phosphogluconolactonase

Figure 2 Main enzymes of the hexose phosphate pathway for the production of lactic acid and the specific targets of Mg2+ and Mn2+ (present

synergistically) on enzymesubstrate interactions.



of growth. Our strain of Lact. rhamnosus FTDC 8313 is a

heterolactic fermenter with detectable acetic and lactic

acids via HPLC (Lew et al. 2013); lactic acid is a growth-

associated predominant metabolite of the two acids. We

have previously postulated that Mn2+ and Mg2+ enhanced

growth leading to enhanced production of lactic acid.

However, we also hypothesize that Mn2+ and Mg2+ tar-

geted different sites along the hexose monophosphate

pathway. Thus in this study, using the same parameters

obtained from our previous study on optimization of

growth, we would like to justify such a hypothesis. In this

study, it is observed that lactic acid production was high

along the region where the ratio of MnSO4 to MgSO4was almost 1 : 1. When one of the divalent metal ions

was at a high concentration and the other at a low con-

centration, lactic acid production decreased, justifying the

need for both Mg2+ and Mn2+ to be present simulta-

neously in stimulating the production of lactic acid.

A molecular modelling approach was adopted to further

understand the influence of Mn2+ and Mg2+ on the pro-

duction of lactic acid and to determine possible specific

targets of the divalent metal ions on enzymessubstratesalong the hexose monophosphate pathway. The hexose

monophosphate pathway for the production of lactic

acid comprises of 13 enzymes (Fig. 2). The crystallized

structures of all enzymes were selected based on criteria

such as origin, apo or holo structure, existence of divalent

metal ions and the types of structures (wild or mutant)

from the PDB (www.pdb.org). In addition, an assumption

was also made where enzymes with a same Enzyme

Commission (EC) number will adopt the same functional

domain despite originating from different organisms.

The functional domain is also known as binding site, which

will be the core region affecting the binding affinity of

enzymesubstrate. Thus, all the 13 enzymes studied hadindividual binding sites, and molecular docking simulation

was performed utilizing a grid box with the size of

50 9 50 9 50 and grid spacing of 0375 A within thebinding site.

Mn2+ and Mg2+ have the same partial charges and are

known to play significant roles in electrostatic stabiliza-

tion and enzyme activation (Andreini et al. 2008).

However, our current data reflected that both Mn2+ and

Mg2+ functioned differently in affecting the substrate

binding affinity among the five enzymes evaluated. Mn2+

has been reported to mainly affect redox processes due to

its nature as a transition element in enzymatic catalytic

mechanisms while Mg2+ could not (Silva and Williams

2001). Our present data suggested that Mn2+ did not act

as a redox centre, but formed coordination bond with

the electron lone pair from both oxygen atoms of the

substrate phosphorus moiety. This process stabilized the

substrate due to increased nucleophilicity, leading to

improved binding affinity towards the respective

enzymes.

In general, Mg2+ is one of the most abundant diva-

lent metal ions in most organisms (Andreini et al.

2008), greatly needed to balance the polarity of

enzymes involved in catalytic properties (Sissi and Pal-

umbo 2009), and is vital for the activation of various

important biological metabolic pathways (Sanwal 1970;Ozer et al. 2001; Romani and Maguire 2002; Andreini

Table 4 Free energy of binding (FEB) of enzymes involved in the hexose monophosphate pathway upon docking against their natural substrates

in the presence and absence of divalent metal ions

No. Enzyme PDB ID

FEB (kcal mol1)

Without ion Mg2+ Mn2+ Mg2+ and Mn2+

1 Glucose-6-phosphate isomerase 3FF1 695 775 738 6682 Glucose-6-phosphate dehydrogenase 1DPG 479 474 452 859*3 6-phosphogluconolactonase 3OC6 698 666 658 7094 Phosphogluconate dehydrogenase 2IYO 479 1160 893 1205*5 Ribulose phosphate-3-epimerase 2FLI 536 793 770 8216 Xylulose-5-phosphate phosphoketolase 3AHE 528 1184 2040 12397 Glyceraldehyde-3-phosphate dehydrogenase 3LC2 436 407 425 2245*8 Phosphoglycerate kinase 1VPE 977 1779 1757 18069 Phosphoglycerate mutase 1EJJ 1009 1770 1771 1776

10 Phosphopyruvate hydratase 3QN3 359 768 779 1073*11 Pyruvate kinase 3TOT 446 1355 1355 2881*12 D-lactate dehydrogenase 1J49 240 423 1963 63813 L-lactate dehydrogenase 3D4P 510 522 520 639

PDB, Protein Data Bank.

*Significant reduction of FEB, from the synergism effects of Mg2+ and Mn2+ as compared to the absence of ions and presence of individual ions.



et al. 2008; Sissi and Palumbo 2009). However, our

current data illustrated that Mg2+ was located at dis-

tances surpassing the cut-offs of any important interac-

tions, yet the presence of Mg2+ was significant in five

enzymes along the hexose monophosphate pathway.

This led us to hypothesize that Mg2+ played a different

role, in that of maintaining polarity within the binding

pocket and thus producing a stable binding environ-

ment for catalytic activities and favourable for the

actions of Mn2+.

In conclusion, using response surface methodology,

we have demonstrated that the interaction of Mn2+

and Mg2+ produced a significant effect on the produc-

tion of lactic acid by Lact. rhamnosus FTDC 8313, indi-

cating that both factors exerted a synergistic effect and

should be present together to achieve a high produc-

tion of lactic acid. Meanwhile, the 3-D response surface

indicated that when one of the divalent metal ions was

at a high concentration and the other at a low concen-

tration, lactic acid production decreased, justifying the

need for both Mg2+ and Mn2+ to be present simulta-

neously in stimulating the production of lactic acid.

Using molecular modelling approach, we illustrated that

Mn2+ and Mg2+ targeted different sites along the

hexose monophosphate pathway, leading to improved

binding affinity for substrates in five enzymes namely,

Asp235

His240

Glu216

Gln51 Asp192

Asp183 His191

Lys193

Ans369

179 166

164240

IIe361 Thr353

Thr348Lys341

Phosphoenolpyruvate

Thr239269

211160

188170

His178

Beta-D-glucose-6-phosphate

Glyceraldehyde-3-phosphate

Tyr415

Mn2+

Mn2+

Mn2+

Mg2+

Mg2+

Mg2+

Mn2+

Mn2+

Mg2+

Mg2+

269

Lys148 Lys184

Ans188

Trp267Asn102

His154 2-phospho-glycerate

Glu162

Glu163 Asp239

Asp307

Asp308Lys332

199

144181

160

390

6-phospho-D-gluconate

268

289

317339

269

263

165

165

167

Lys266

Glu270914 302162

(a) (b)

(c) (d)

(e)

Figure 3 Binding interactions of selected enzymes (a) glucose-6-phosphate dehydrogenase, (b) phosphogluconate dehydrogenase (c) glyceralde-

hyde-3-dehydrogenase (d) phosphopyruvate hydratase and (e) pyruvate kinase with their respective substrates in the presence of both Mn2+ and

Mg2+.



glucose-6-phosphate dehydrogenase, phosphogluco-

nate dehydrogenase, glyceraldehyde-3-phosphate dehy-

drogenase, phosphopyruvate hydratase and pyruvate

kinase.

Acknowledgement

This work was financially supported by the Science Fund

Grant (305/PTEKIND/613222) provided by the Malaysian

Ministry of Science, Technology and Innovation

(MOSTI), the FRGS grant (203/PTEKIND/6711239) pro-

vided by the Malaysian Ministry of Higher Education

(MOHE), the Research University grant (1001/PTEKIND/

815056) and USM Fellowship provided by Universiti

Sains Malaysia.

Conflict of interest

The authors declare that there are no conflict of interest.

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Table 5 Distance of Mn2+ and Mg2+ (A) with the phosphorus moiety of substrates

Enzyme Substrate Atom

Distance between

interacting moiety with

divalent metal ion (A)

Mn2+ Mg2+

Glucose-6-phosphate dehydrogenase Beta-D-glucose-6-phosphate O1P 364 914O2P 162 1126O3P 269 1004

Phosphogluconate dehydrogenase 6-Phospho-D-gluconate O1P 165 1744O2P 416 1901O3P 390 1667

Glyceraldehyde-3-phosphate dehydrogenase Glyceraldehyde-3-phosphate O1P 170 1987O2P 188 1948O3P 350 1851

Phosphopyruvate hydratase 2-phospho-glycerate O1P 390 408O2P 403 181O3P 160 409

Pyruvate kinase Phosphoenolpyruvate O1P 338 1629O2P 164 1564O3P 179 1745



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Mn2+ and Mg2+ synergistically e

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