Báo cáo khoa học: Analyses of co-operative transitions inPlasmodium falciparumb-ketoacyl acyl carrier protein reductase upon co-factor and acyl carrier protein binding pdf

We report here intensive studies on the direct inter-actions of Plasmodium b-ketoacyl-acyl carrier protein reductase with its cofactor, NADPH, acyl carrier protein, acetoacetyl-coenzyme

falciparum b-ketoacyl acyl carrier protein reductase upon co-factor and acyl carrier protein binding

Krishanpal Karmodiya and Namita Surolia

Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore, India

Malaria is one of the leading causes of morbidity and

mortality in the tropics, with 300–500 million clinical

cases and 1.5–2.7 million deaths per year [1,2] Nearly

all the fatal cases are caused by Plasmodium

falcipa-rum The acquisition of resistance by this parasite to

conventional antimalarial drugs, such as chloroquine,

is growing at an alarming rate and the increasing

bur-den of malaria caused by drug-resistant parasites has

led investigators to seek novel antimalarial drug targets [3]

There are two distinct architectures for fatty acid synthesis in living organisms Our recent demonstra-tion of the occurrence of the type II fatty acid synthase (FAS) pathway in the malaria parasite and its inhibi-tion by triclosan, an inhibitor of a key enzyme (enoyl-acyl carrier protein reductase) of the type II FAS

Keywords

b-ketoacyl-ACP reductase; cofactor;

conformational change; fluorescence

quenching; Plasmodium

Correspondence

N Surolia, Molecular Biology and Genetics

Unit, Jawaharlal Nehru Centre for Advanced

Scientific Research, Jakkur,

Bangalore-560064, India

Fax: +91 80 22082766

Tel: +91 80 22082820 ⁄ 21

E-mail: surolia@jncasr.ac.in

(Received 12 April 2006, revised 15 June

2006, accepted 10 July 2006)

doi:10.1111/j.1742-4658.2006.05412.x

The type II fatty acid synthase pathway of Plasmodium falciparum is a validated unique target for developing novel antimalarials because of its intrinsic differences from the type I pathway operating in humans b-Ketoacyl-acyl carrier protein reductase is the only enzyme of this pathway that has no isoforms and thus selective inhibitors can be developed for this player of the pathway We report here intensive studies on the direct inter-actions of Plasmodium b-ketoacyl-acyl carrier protein reductase with its cofactor, NADPH, acyl carrier protein, acetoacetyl-coenzyme A and other ligands in solution, by monitoring the intrinsic fluorescence (kmax 334 nm)

of the protein as a result of its lone tryptophan, as well as the fluorescence

of NADPH (kmax 450 nm) upon binding to the enzyme Binding of the reduced cofactor makes the enzyme catalytically efficient, as it increases the binding affinity of the substrate, acetoacetyl-coenzyme A, by 16-fold The binding affinity of acyl carrier protein to the enzyme also increases by approximately threefold upon NADPH binding Plasmodium b-ketoacyl-acyl carrier protein reductase exhibits negative, homotropic co-operative binding for NADPH, which is enhanced in the presence of acyl carrier pro-tein Acyl carrier protein increases the accessibility of NADPH to b-keto-acyl-acyl carrier protein reductase, as evident from the increase in the accessibility of the tryptophan of b-ketoacyl-acyl carrier protein reductase

to acrylamide, from 81 to 98% In the presence of NADP+, the reaction proceeds in the reverse direction (Ka¼ 23.17 lm)1) These findings provide impetus for exploring the influence of ligands on the structure–activity rela-tionship of Plasmodium b-ketoacyl-acyl carrier protein reductase

Abbreviations

ACP, acyl carrier protein; apo-PfACP, Plasmodium falciparum acyl carrier protein (apo form); holo-PfACP, Plasmodium falciparum acyl carrier protein (holo form); FabG, b-ketoacyl-ACP reductase; FAS, fatty acid synthase, PfFabG, Plasmodium falciparum b-ketoacyl-ACP reductase; SDR, short-chain alcohol dehydrogenase ⁄ reductase.

pathway, pointed to the pivotal role of this pathway

for the survival of malaria parasites [4,5] The type II

FAS pathway of Plasmodium has discrete enzymes for

each step of the pathway, as opposed to the type I

FAS, found in humans, which is a multifunctional

enzyme [6,7] Also, the type II fatty acid biosynthetic

pathway in P falciparum is one of the pathways

speci-fic to its ‘plastid’ and has been validated as a unique

target for developing new antimalarials [8–10]

During the elongation cycle of FAS II, the acyl

chain covalently attached to the acyl carrier protein

(ACP) is elongated successively by two carbon units

by the action of four enzymes acting consecutively

First, b-ketoacyl-ACP synthase (either FabB or FabF)

elongates the acyl-ACP of the Cnacyl chain to a Cn+2

b-ketoacyl form The b-ketoacyl-ACP thus formed

is reduced to b-hydroxyacyl-ACP by an

NADPH-dependent b-ketoacyl-ACP reductase (FabG) The

b-hydroxyacyl group is then dehydrated to an

enoyl-ACP by a b-hydroxyacyl-enoyl-ACP dehydratase (FabZ or

FabA) Reduction of the enoyl group by an

enoyl-ACP reductase (FabI, FabK or FabL) finally produces

Cn+2 acyl-ACP, which is ready to re-enter the cycle,

become hydrolyzed from ACP for the synthesis of

phospholipids or sphingolipids, or become diverted for

other modifications [11,12]

FabG is ubiquitously expressed in bacteria [13], is

highly conserved across species, and is the only known

isoform that functions as a ketoacyl reductase in the

FAS II system The primary structure of FabG from

the parasite reveals that it belongs to the short-chain

alcohol dehydrogenase⁄ reductase (SDR) family of

enzymes, whose members catalyze a broad range of

reduction and dehydrogenation reactions using a

nuc-leotide cofactor [14,15] P falciparum FabG (PfFabG)

has 48% sequence identity with its counterpart from

Brassica napus, with which it shares stronger homology

than with the Escherichia coli enzyme Solution studies

on detailed interactions of PfFabG with its cofactor,

substrate and holo-acyl-carrier protein (ACP, unless

otherwise indicated) to have insight into its

co-opera-tivity and catalytic mechanism, are lacking The

intrin-sic fluorescence of tryptophan in a protein is sensitive

to its surroundings, a characteristic that has made it

an invaluable and popular tool for studying protein–

ligand interactions, and FabG, with a lone tryptophan,

provides an ideal system for studying ligand-induced

conformational changes in it Here, we report subtle

aspects of the interactions between FabG with its

co-factor, acetoacetyl-coenzyme A (acetoacetyl-CoA) and

also with ACP, with emphasis on association

con-stants, number of binding sites and the co-operativities

involved therein

Results Cloning, expression, purification and kinetic analyses of FabG

FabG (acc no.: PFI1125c) potentially resides in the apicoplast of Plasmodium and therefore possesses a bipartite signal and transit peptide at the N terminus for correct targeting to the apicoplast On the basis of the FabG sequence in PlasmoDB, the start site of the mature protein, from nucleotides 132 to 903, was cloned The deduced amino acid sequence correspon-ded to the mature protein, with a predicted molecular mass of 31.0 kDa [16] Mature FabG was expressed in

E coli BL21 (DE3) codon plus cells with a C-terminal His-tag The soluble protein was purified to homogen-eity on a Ni-nitrilotriacetic acid affinity column On SDS⁄ PAGE, the purified protein yielded a monomeric

Mrof 31 000 (Fig 1A) and on Superdextm

200 yielded

an Mr of 110 000 ± 2500 (Fig 1B), demonstrating that it exists as a tetramer in solution (3 mm Hepes,

pH 7.5, 100 mm NaCl, 2 mm b-mercaptoethanol and 10% glycerol) The enzyme has a Kmvalue for the sub-strate acetoacetyl-CoA of 0.43 ± 0.05 mm and a Km

value for NADPH of 42.6 ± 0.05 lm The specific activity of the enzyme with acetoacetyl-CoA is 59.8 UÆmg)1 and the kcat is 259 ± 25 s)1, which are within the range of values reported previously [16]

Co-operative binding of the cofactor to FabG HPLC-purified, fresh NADPH (A258⁄ A340¼ 2.3) was used for studying the conformational changes and co-operativity in FabG The intrinsic fluorescence of FabG, as a result of its lone tryptophan (kmax¼

334 nm), decreased when it was titrated with increasing concentrations of NADPH (Fig 2A,B), with simulta-neous appearance of another peak with a kmax at

456 nm as a result of NADPH The appearance of fluor-escence with a kmaxat 456 nm is caused by energy trans-fer from the lone tryptophan of FabG to the bound NADPH, as the emission spectrum of tryptophan in the protein has a considerable overlap with the excitation spectrum of the NADPH Binding of NADPH to FabG, as analyzed by quenching of its fluorescence intensity at 334 nm, exhibited a negative, homotropic co-operativity (with a Hill constant of 0.8) (Fig 2C) The NADPH-induced changes in the fluorescence of tryptophan in the protein at 334 nm were analyzed by nonlinear least-squares fit of the data, using the Adair equation with 1–4, equivalent and independent, as well

as equivalent and interdependent, binding sites (n)

As shown in Fig 2D, a model of the binding site with

n¼ 1 does not provide a satisfactory coalescence of the

fit with the data for NADPH binding However, the fit

improves dramatically as the number of sites are

increased from two to four equivalent,

interdependent-binding sites, with n¼ 4 (Ka¼ 40.90 lm)1) being clo-sest to the experimental data (Fig S1 and Table S1) As mentioned above, binding of NADPH to the enzyme leads to the appearance of fluorescence with a maximum

at 450 nm when excited at its excitation maximum (340 nm) From the NADPH concentration dependence

of the increase in fluorescence intensity of NADPH at

450 nm, the Kavalue for its binding to FabG, with n¼

4, was found to be 45.2 lm)1 Binding of other ligands

to the binary complex of NADPH.FabG also alters the cofactor-specific fluorescence intensity (kmax 456 nm) The Kavalues for all the ligands that were determined [i.e acetoacetyl-CoA, Plasmodium falciparum acyl carrier protein (apo form) (apo-PfACP) and Plasmo-dium falciparum acyl carrier protein (holo form) (holo-PfACP)] using the cofactor-specific fluorescence intensity at 456 nm (excited at 280 nm), are identical to those obtained by measurement of the intrinsic trypto-phan fluorescence intensity at 334 nm (Table 1)

Allosteric binding of NADPH to FabG in the presence of ACP

The binding constant of acetoacetyl-CoA to the enzyme

is increased several fold in the presence of NADPH, which motivated us to investigate allostery in its cata-lytic mechanism The affinities (Ka) of FabG for its cofactor, NADPH, determined by quenching of the fluorescence of its tryptophan (kmax 334 nm) in the absence and presence of 20 lm ACP, were found to be 40.9 lm)1 and 48.4 lm)1, respectively (Fig 3A and Table 1) In the presence of ACP, the affinity of FabG for NADPH increased, while the number of cofactor-binding sites decreased, indicating a negative, hetero-tropic co-operative effect of ACP upon binding of NADPH (Table 1) In addition, the degree of negative co-operativity increased in the presence of ACP (Hill constant, nH¼ 0.5) (Fig 2B) In the absence of ACP, as stated earlier, the binding of NADPH exhibited negat-ive, homotropic co-operativity In the absence of ACP, four NADPH-binding sites were present, corresponding

to the four equivalent subunits in FabG, which decreased to two in the presence of ACP Altogether, the negative co-operativity and stoichiometry calcu-lations show that binding of ACP converts the four equivalent negative co-operative homotropic NADPH-binding sites to two high-affinity NADPH sites (Fig 3B)

Interaction of FabG with ACP Fluorescence titration of a fixed concentration of FabG with varying concentrations of ACP gave an

associ-PfFabG (31 kDa)

kDa

118

66

45

35

25

18

14

A

log molecular mass

1

2

3

B

Elution Volume (ml)

0 10 20

Fig 1 Purification and determination of the molecular mass of

b-ketoacyl-acyl carrier protein reductase (FabG) by gel filtration

chro-matography (A) SDS ⁄ PAGE of recombinant FabG Lane 1, protein

molecular mass markers (MBI Fermentas); lane 2, purified FabG.

(B) The standard curve, Ve ⁄ V 0 versus log molecular mass (mol.

mass) was derived from the elution profiles of the standard

molecular weight markers on a Superdex 200 gel filtration column.

Ve, peak elution volume of the protein; V0, void volume of the

col-umn The position of FabG elution (2 mgÆmL)1) is indicated by (.).

The standards used were 1, cytochrome c (12 kDa); 2, carbonic

anhydrase (29 kDa); 3, ovalbumin (45 kDa); 4, BSA (66 kDa); and 5,

aldolase (158 kDa) Inset, elution profile of FabG.

ation constant of 0.40 lm)1 with n¼ 1 The affinity

(Ka¼ 1.1 lm)1) and the number of binding sites

increased to two for ACP in the presence of NADPH

FabG activity was monitored spectrophotometri-cally at 340 nm in the presence of NADPH and ACP The maximum activity was observed when the

Fig 2 Emission spectra for the intrinsic protein fluorescence (k max 334 nm) of b-ketoacyl-acyl carrier protein reductase (FabG) and increase

in fluorescence of NADPH (kmax456 nm) upon titration of the enzyme with NADPH at 20
C Aliquots of 3 lL of NADPH from stock solu-tions of 5 m M were added to 1 mL of FabG (2 l M tetramer in 3 m M Hepes, pH 7.5, 100 m M NaCl, 2 m M b-mercaptoethenol and 10% gly-cerol) and the changes in fluorescence intensities were monitored between 300 and 500 nm Samples were excited at 280 nm A fluorescence spectrum with a maximum at 334 nm is caused by the fluorescence of tryptophan in the protein In addition, there was acquisi-tion of the fluorescence by NADPH, with a maximum at 456 nm, upon the binding of NADPH to the enzyme, as a consequence of energy transfer between the lone tryptophan (k max 334 nm) in the protein and bound NADPH (k max 456 nm) (A) Quenching of tryptophan fluores-cence of FabG (300–400 nm range; kmax334 nm) occurred as a function of increasing concentration of the reduced cofactor The arrow indi-cates the direction in which the change in tryptophan fluorescence (quenching) occurs with an increase in NADPH concentration There is

an increase in NADPH fluorescence (400–500 nm range; k max 456 nm) as a function of increasing concentration of NADPH The upward direction of the arrow indicates that the cofactor fluorescence (k max 456 nm) increases as a function of its concentration (B) The fractional fluorescence changes at 334 nm (d) and 456 nm (s) are plotted versus the varying concentrations of NADPH Inset: quenching of trypto-phan fluorescence at 334 nm (d) and the enhancement of NADPH fluorescence at 456 nm (s) as a function of increasing concentrations of NADPH (C) Hill plot of the data obtained as a result of the changes in fluorescence intensity of tryptophan of the enzyme at 334 nm, as a function of NADPH concentration, with a Hill constant (nH) of 0.8 (s) In the presence of acyl carrier protein (ACP), the nH¼ 0.5 (d) (D) Fit-ting with the Adair equation corresponding to four sites (dotted line) for NADPH yields the best r 2 value of 0.949, with an excellent fit of the experimental values (d) Fit with one site (thin line) diverges significantly from the experimental points (d) Error bars for the fit with n ¼ 1 are not shown in the figure in the interest of clarity, but are shown in Fig S1 Likewise, fits with n ¼ 2 and n ¼ 3, together with the associ-ated error bars, are shown in Fig S1.

cofactor was preincubated with the enzyme before

adding acetoacetyl-CoA and ACP The enzyme was

catalytically less efficient in the presence of ACP

(Table 2)

Binding of acetoacetyl-CoA and

b-hydroxybutyryl-CoA to FabG

The association constant for the substrate,

acetoace-tyl-CoA, is 12.3 lm)1 with four equivalent and

independent sites In the presence of NADPH

(Fig S2A), the Ka for acetoacetyl-CoA is increased

by 16-fold to 189.2 lm)1 (Table 1) Thus,

acetoace-tyl-CoA now has a larger number of favorable

inter-actions at the active site of the enzyme in the

presence of NADPH b-hydroxybutyryl-CoA, the

product of the reaction, has affinity (23.2 lm)1)

(Fig S2B) comparable with that of acetoacetyl-CoA

(18.8 lm)1) in the absence of NADPH Binding of

b-hydroxybutyryl-CoA in the presence of NADP+ is

enhanced by 1.7-fold

Effect of the cofactor and acetoacetyl-CoA on the far-UV CD spectrum of FabG

The presence of NADPH has a considerable effect on the conformation of FabG While the helicity of the protein increased from 30 to 35%, the b-sheet content increased from 27 to 33%, as evident by the CD

Table 1 Binding constants (Ka) of various ligands to b-ketoacyl-acyl

carrier protein reductase (FabG) at 20
C, using the changes in

pro-tein and ⁄ or cofactor fluorescence intensity at 334 and 450 nm,

respectively (Experimental details are provided in the respective

figure legends a ) Apo-PfACP, Plasmodium falciparum acyl carrier

protein (apo form); Holo-PfACP, Plasmodium falciparum-acyl carrier

protein (holo form); n, number of binding sites for the best value of

r 2 ; ND, not determined; SN, serial number.

SN Titrated with ligand

Saturated

Ka (l M )1)b

Ka (l M )1)c

13 b-hydroxybutyryl-CoA NADP+ 4 39.4 ND

a

Table S1 provides the residuals for each value of n, for each

ligand, to support the given value of n chosen by us for

interpret-ation of our data Also, for a given value of n, the resultant values

of association constants are listed in Table S1 A footnote provides

the rationale for the selection of a particular value of K a from these.

b Ka, association constant for the best value of r 2 , determined

using protein fluorescence (334 nm) c K a , association constant for

the best value of r2, determined using cofactor fluorescence

(450 nm).

Fig 3 Negative co-operative binding of NADPH to b-ketoacyl-acyl carrier protein reductase (FabG) (A) Binding of NADPH to FabG (2 l M of the tetramer) was studied in the absence (s), and pres-ence of 20 l M acyl carrier protein (ACP) (d) by monitoring fluores-cence with excitation at 280 nm and emission at 334 nm, as described in the Experimental procedures Relative fluorescence intensity (observed fluorescence intensity minus fluorescence inten-sity at infinite ligand concentration) values are plotted versus NADPH concentration Identical results were obtained when the reaction was monitored by following the changes in the NADPH fluorescence intensity at 456 nm (B) Intrinsic fluorescence quench-ing of FabG by NADPH with a saturatquench-ing concentration of ACP (20 l M ) Relative fluorescence intensity values are plotted versus NADPH concentration A fit according to the Adair equation corres-ponding to two sites (dashed line) for NADPH in the presence of ACP gave the best value for residuals (0.965) Also, for comparison, the fit for one site (thin line) with the original data (d) is also shown.

spectrum of FabG (Fig 4) Interestingly, the negative

gain in ellipticity brought about by NADPH decreased

with the addition of acetoacetyl-CoA

Analyses of the accessibility of the lone

tryptophan of FabG by Stern–Volmer plots

The oxidized cofactor, NADP+, is 20 times weaker as

a ligand than its reduced counterpart (Fig S3) Plots

of F0‚ (F0) F) versus 1 ‚ [Q], for calculating the

accessibility of the fluorescence of the lone tryptophan

in FabG for NADP+ and NADPH, are shown in

Fig 5 as representative examples A cursory

examina-tion of the plots reveal a greater accessibility of the

tryptophan to the quencher when NADPH is bound

to enzyme compared with that in the presence of

NADP+ In Table 2, Stern–Volmer analyses of the data are summarized for the interactions of various ligands with FabG These data for NADPH yield a value of 1.23, indicating that 81% of the total FabG fluorescence is accessible to it, whereas f)1 with NADP+ is 2.37, showing that only 42% of the total fluorescence of the enzyme is accessible to the oxidized cofactor Likewise, binding of other ligands also exert subtle molecular effects on the exposure of the unique tryptophan in FabG (Table 3)

Stern–Volmer analysis of the interaction of ACP with FabG revealed an f)1of 1.35, indicating that 74%

of the total fluorescence of FabG is accessible when

Table 2 Catalytic efficiency of b-ketoacyl-acyl carrier protein

reduc-tase (FabG) with acetoacetyl-CoA as substrate, with varying

con-centrations of acyl carrier protein (ACP) SN, serial number conc.,

concentration.

SN ACP conc (l M ) kcat⁄ K m (s)1Æ M )1)

(92.5)

(63.3)

a The percentage catalytic efficiency is given in parenthesis.

Enzyme activity was monitored spectrophotometrically at 340 nm,

as described in the Experimental procedures.

Fig 4 The far-UV CD spectra of b-ketoacyl-acyl carrier protein

reductase (FabG) The figure shows the CD spectrum of FabG

alone (14 · 10)6molÆl)1(s), the CD spectrum in the presence of

NADPH alone (200 l M ) (.) and the CD spectrum in the presence

of NADPH (200 l M ) and a saturating concentration of

acetoacetyl-CoA (200 l M ) (d).

Fig 5 Fraction of initial fluorescence accessible to NADPH and NADP + Accessibility was calculated by the Stern–Volmer equation b-Ketoacyl-acyl carrier protein reductase (FabG) (0.5 l M , in 3 m M Hepes, pH 7.5) was preincubated for 15 min with aliquots of 5 m M NADPH (n) or 5 m M NADP + (s) in a total volume of 1 mL and was titrated with 10 lL aliquots of 3 M acrylamide solution The fluores-cence intensity was monitored at 334 nm with excitation at 280 nm.

Table 3 Fluorescence quenching parameters determined from modified Stern–Volmer plots of b-ketoacyl-acyl carrier protein reduc-tase (FabG) with acrylamide, at room temperature.aACP, acyl car-rier protein; f, slope of the SV plot; f)1, fluorescence accessibility;

SN, serial number.

10 NADP + b-hydroxybutyryl-CoA 1.10 90.9

a Samples were excited at 280 nm and the fluorescence intensity was monitored at 334 nm Experiments were carried out at 20
C.

ACP is bound to it, which increases to 98% in the

pres-ence of both ACP and NADPH NADPH therefore

increases the affinity of ACP by increasing its

accessi-bility to FabG In the presence of ACP, the

accessibil-ity of the lone tryptophan of FabG for NADPH

binding increases from 81 to 98%, explaining, likewise,

the increase in affinity of NADPH by ACP

Discussion

Our biophysical and biochemical data provide evidence

that the binding of the cofactor NADPH to

Plasmo-dium FabG induces major conformational change in

the enzyme This change promotes ACP binding as

well as negative co-operativity, which forms the basis

of the mechanism for catalytic activation of FabG We

propose a model to illustrate how PfFabG binds the

cofactor, substrate and other ligands The model also

shows the active⁄ inactive status of each monomer with

the number of binding sites for various ligands in

solution FabG, an allosteric enzyme, is a catalytically

nonproductive homotetramer in the absence of

NADPH, as neither acetoacetyl-CoA, nor ACP (the

substrate mimics of the physiological substrate

aceto-acetyl-ACP) can access the active sites completely

(Scheme 1A,D) ACP has a single binding site, whereas

acetoacetyl-CoA has four independent binding sites in

the tetramer in the absence of NADPH (Table 1) The

binding of NADPH, which has four equivalent and

interdependent binding sites in FabG, results in

con-formational changes (Scheme 1B), which improves the

accessibility of ACP and acetoacetyl-CoA to the active

sites (Scheme 1C) While only one molecule of ACP

can bind to FabG at one of the two dimeric interfaces

of the enzyme in the absence of NADPH, the binding

of NADPH to FabG results into two high-affinity sites for ACP and acetoacetyl-CoA (Scheme 1C) Thus, NADPH binding increases the affinity, as well as the number, of binding sites for ACP Analyses of the data obtained from Adair equations and Hill coefficients for the interactions of various ligands with FabG, indi-cate that the binding of ACP not only increases the affinity, but also the negative co-operativity, of NADPH to the enzyme, fine tuning its catalytic mech-anism Once NADPH and ACP bind to FabG, each can access two active sites from the opposite or adja-cent subunits (Scheme 1C) FabG holds NADPH and ACP close to each other in an orientation which stabil-izes the transition state that leads to the substrate delivery across the dimer interface via the pantetheine arm of the ACP This provides an example of catalysis

by approximation [17] Our studies also demonstrate that holo-PfACP, as compared with its apo form, binds more strongly to FabG in the presence of NADPH, attesting the importance of the 4¢ phospho-pantetheine moiety for the binding of holo-PfACP (Table 1) As evident from the CD data (Fig 4), the FabG secondary structure increases in the presence of NADPH and decreases in the presence of acetoacetyl-CoA These conformational changes appear to be directly related to the binding and oxidation of NADPH to the enzyme at equilibrium Such a mech-anism seems to be unique to the FabG members of the SDR family and are consistent with B napus and

E coli counterparts, as demonstrated by crystallo-graphic studies [18–21] These conformational changes point to the need for an open FabG active site to accept the acyl-ACP substrates

ACP binding site

Active site Active site

Inactive FabG

homo-tetramer

in the absence

of NADPH

Active FabG tetramer

Conformational change in the active sites in presence of NADPH

ACP binding site

Phospho-pantetheine arm of ACP to which acyl moiety is attached

+NADPH

Scheme 1 Proposed model for b-ketoacyl-acyl carrier protein reductase (FabG) repositioning and allosteric regulation by the binding of NADPH and acyl carrier protein (ACP).

Our studies highlight the importance of PfFabG in

regulating the flux of the substrates during the

elonga-tion phase of FAS in the parasite This phase will

pro-ceed when the NADPH⁄ NADP ratio is high; on the

other hand, when the NADPH⁄ NADP ratio is low,

b-hydroxyacyl-ACPs are converted to b-ketoacyl-ACPs

Hence, b-ketoacyl-ACPs will accumulate and the

elon-gation phase of FAS will become regulated at this step

FabG exhibits negative co-operativity, and the

bind-ing of NADPH to one site increases the affinity at that

site for the ACP-bound substrate and simultaneously

decreases the affinity for cofactor at the other site

This, in turn, indicates a greater sensitivity for the low

ligand (NADPH) concentration and is probably

associ-ated with conformational changes that have occurred

in the transition from one state of co-operativity to the

other Furthermore, the increase in negative

co-opera-tivity for NADPH in the presence of ACP might be

useful, as the concentration of NADPH may change in

the cell as a result of reactions other than FAS, where

FabG is not participating Under such circumstances,

it may be of benefit if the enzyme does not react to

changes in the substrate [22] In summary, the data for

the interactions of PfFabG with the reduced and

oxid-ized cofactor, substrate and product, allow us to

rationalize the importance of this enzyme in regulating

the flux of substrates in the elongation cycle of parasite

FAS Our major focus for future research will be to

identify and compare the various acyl-ACP thioester

intermediates of Plasmodium FAS, as well as to have a

deeper understanding of the regulation of FAS by this

enzyme in P falciparum

Experimental procedures

Materials

Acetoacetyl-CoA, b-hydroxybutyryl-CoA, b-NADPH,

NADP+, imidazole, kanamycin, chloramphenicol and

SDS⁄ PAGE reagents were obtained from Sigma Chemicals

(St Louis, MO, USA) Protein molecular weight markers

were from MBI Fermentas GmbH (St Leon-Rot, Germany)

His-binding resin and anti-His tag horseradish peroxidase

conjugates were obtained from Novagen (Darmstadt,

Ger-many) Media components were obtained from DifcoTM

(Detroit, MI, USA) Hi-Trap desalting and SuperdexTM200

columns were from Amersham Biosciences (Uppsala,

Swe-den) All other chemicals used were of analytical grade

Strains and plasmids

E coli DH5a cells were used for cloning the

b-ketoacyl-ACP reductase The pET-28a (+) vector (Novagen) and

BL21 (DE3) codon plus (Novagen) were used for the expression of FabG

Cloning of PfFabG

Total RNA isolated from asynchronous cultures of P falci-parum, treated for 45 min at 37
C with RNase-free DNase (Promega, Madison, WI, USA; 1 UÆlg)1 RNA), was phe-nol⁄ chloroform extracted and ethanol precipitated, then sub-jected to RT-PCR using a one step RT-PCR kit (Qiagen, Hilden, Germany) The primers were designed to clone the protein (encompassing a 771 bp fragment, starting at posi-tion 132 and ending at posiposi-tion 903) The primers used for

GTAACAGGTGCAGGA-3¢ (NcoI site underlined) and 5¢-CCGCTCGAGAGGTGATAGTCCACCGTCTATTACG AAAACTCG-3¢ (XhoI site underlined) using Platinum Pfx DNA polymerase (Invitrogen, Carlsbad, CA, USA) The DNA sequence encoding the mature protein was amplified using the above gene-specific primers and cloned in NcoI and XhoI sites of the pET-28a (+) vector (Novagen) The PCR conditions consisted of an initial denaturation at 95
C for

5 min, followed by amplification for 25 cycles (95
C for

1 min, 50
C for 50 s and 72
C for 1 min), followed by a final extension at 72
C for 5 min The clone thus obtained was confirmed by DNA sequencing The protein obtained from this clone showed higher specific activity than that from the earlier clone [23] The specific activity of this protein pre-paration is comparable to that reported by Wickramasinghe

et al [16] and was used all throughout these studies

Expression of FabG

The plasmid construct, pET-28a (+), was transformed into

E coli BL21 (DE3) codon plus competent cells The bac-teria were grown in Luria–Bertani broth, with vigorous shaking (200 r.p.m.) at 25
C, to an attenuance (D) of 0.6 Cells were then induced with 0.2 mm isopropyl thio-b-d-galactoside and further incubated at 15
C for 9 h Cells were harvested at 1500 g for 15 min at 4
C, washed twice with LB broth and the resultant pellet was resuspended in

20 mm sodium phosphate, pH 6.8, containing 0.5 m NaCl and supplemented with a protease inhibitor cocktail tab-let, according to the manufacturer’s instructions (Roche, Mannheim, Germany)

Purification of FabG

The cell suspension was sonicated (Vibra-Cells; Sonics and Materials, Newtown, CT, USA) Cell debris was removed

by centrifugation (10 000 g, 30 min, 4
C) The superna-tant obtained was applied to a Ni-nitrilotriacetic acid metal-affinity column pre-equilibrated with the lysis buffer (the same as the buffer used to resuspend the pellet) The

protein was eluted with a step gradient of 50–500 mm

imi-dazole, and fractions were tested for protein purity by

SDS⁄ PAGE (12% gels) The purified protein fractions were

applied onto a Hi-Trap desalting column to remove

imidaz-ole, followed by concentration of the protein Protein was

determined by the Bradford method [24]

Molecular size and oligomeric status

of P falciparum FabG

The subunit molecular size and oligomeric status of FabG

was determined by SDS⁄ PAGE and gel filtration,

respect-ively Purified FabG (2 mgÆmL)1) was loaded onto a

SuperdexTM 200 (1· 30 cm) AKTATM

column, pre-equili-brated with 3 mm Hepes (pH 7.5), 100 mm NaCl, 2 mm

b-mercaptoethanol and 10% glycerol The flow rate was

maintained at 0.4 mLÆ min)1 The column was calibrated

with a mixture of BSA (66 kDa), aldolase (158 kDa),

cyto-chrome c (12 kDa), carbonic anhydrase (29 kDa) and

chicken ovalbumin (45 kDa) The molecular weight of FabG

was determined by plotting Ve⁄ V0 versus log of molecular

mass of standard proteins Ve corresponds to the elution

vol-ume of the protein and V0represents the void volume of the

column, determined using blue dextran (Mr< 2 000 000)

Enzyme assay

The activity of FabG was assayed at 25
C by monitoring the

decrease in absorbance at 340 nm, spectrophotometrically,

as a result of the oxidation of NADPH to NADP+(Jasco

V-530 UV-visible spectrophotometer; Tokyo, Japan) The

standard reaction mixture in a final volume of 100 lL

con-tained 50 mm sodium phosphate buffer, pH 6.8, 0.25 m

NaCl, 200 lm NADPH, 0.5 mm acetoacetyl-CoA and 0.2–

0.8 lg of FabG The assay mixture was preincubated for

5 min at room temperature before initiating the reaction by

the addition of substrate or enzyme The reverse reaction (i.e

oxidation of b-hydroxybutyryl-CoA to acetoacetyl-CoA) was

also characterized by monitoring the reduction of NADP+

to NADPH, by following the increase in absorbance at

340 nm Reactions with appropriate blanks were also

per-formed The kinetic parameters were determined by

non-linear regression analyses The data were also evaluated by

double reciprocal plots The ability of ACP to inhibit FabG

activity in the spectrophotometric assay was tested by

incu-bating varying concentrations of ACP with the protein at

room temperature for 5 min before adding acetoacetyl-CoA

to initiate the reaction

Expression and purification of holo-PfACP and

apo-PfACP

Holo-PfACP and apo-PfACP were obtained as described

previously [25]

Purification of NADPH

In order to obtain an accurate picture of negative co-opera-tivity, NADPH was purified (free of contaminating amounts of NADP+) using a standard protocol [26]

Fluorescence titration of FabG–NADPH binding

Equilibrium binding of ligands to FabG was measured by fluorescence titration at 20
C using a Jobin-Yvon Horiba spectrofluorimeter (Edison, NJ, USA) (band-pass of 3 and

5 nm), for the excitation and emission monochromator, respectively The fluorescence spectrum of FabG was stud-ied by exciting the samples at 280 nm and recording the emission spectrum in the range of 300–500 nm In the absence of NADPH, fluorescence, caused by its lone trypto-phan residue, was observed in the range of 300–400 nm, with a maximum at 334 nm However, in the presence of NADPH, while the fluorescence in the region 300–400 nm declined as a result of the quenching of its tryptophan fluorescence (kmax334 nm), fluorescence in the 400–500 nm range, with a maximum at 456 nm, appeared When the FabG.NADPH complex was excited at 340 nm (excitation maximum of enzyme-bound NADPH), fluorescence in the same range (400–500 nm) was observed; however, its kmax

was 450 nm Aliquots of 3 lL of NADPH (from stock solutions of 2, 100 and 500 lm) were added to 0.5 lm FabG in 3 mm Hepes (pH 7.5), 100 mm NaCl, 2 mm b-mercaptoethenol and 10% glycerol The solution was mixed after the addition of each aliquot, and the fluores-cence intensity in the range of the 300–400 nm region was recorded as the average of three readings Samples were excited at 280 nm The effect of ACP and other ligands on NADPH binding to FabG was studied by titration of NADPH and other ligands (3 lL) into 0.5 lm FabG (3 mm Hepes, pH 7.5, 100 mm NaCl, 2 mm b-mercaptoethenol and 10% glycerol), from a stock solution of 5 mm, and the corresponding decrease in the fluorescence intensity of its lone tryptophan was monitored at 334 nm A double-recip-rocal plot of the fluorescence intensity and ligand concen-tration, from the data obtained by titration of a fixed concentration of FabG with ligand, gave the fluorescence intensity at infinite ligand concentration (Fa)

Correction for the inner filter effect was performed according to the following equation:

FC¼ F antilog½ðAexþ AemÞ
2;

where Fc and F are the corrected and measured fluores-cence intensities, respectively [27], and Aexand Aemare the solution absorbance values at the excitation and emission wavelengths, respectively

The fluorescence data were fitted by the Adair equation [28] with number of sites n¼ 1–4, K being the association constants: for a single site, Y¼ KÆ[X] ⁄ (1 + KÆ[X]); for

two sites equivalent and independent, Y¼ (K1Æ[X] + 2K1Æ

K2Æ[X]2)⁄ (1 + K1Æ[X] + K1ÆK2Æ[X]2); for two sites equivalent

and interdependent, Y¼ (2K1ÆX + K1ÆK2Æ[X]2)⁄ (1 + K1ÆX +

K1ÆK2Æ[X]2); for three sites equivalent and independent,

Y¼ (K1Æ[X] + 2K1ÆK2Æ[X]2+ 3K1ÆK2ÆK3Æ[X]3)⁄ (1 + K1Æ[X] +

K1ÆK2Æ[X2] + K1ÆK2ÆK3Æ[X]3); for three sites equivalent and

interdependent, Y¼ (3K1Æ[X] + 2K1ÆK2Æ[X]2+ K1ÆK2ÆK3Æ[X]3)⁄

(1 + K1Æ[X] + K1ÆK2Æ[X]2+ K1ÆK2ÆK3Æ[X]3); for four sites

equivalent and independent, (K1Æ[X] + 2K1ÆK2Æ[X]2+

3K1ÆK2ÆK3Æ[X]3+ 4K1ÆK2ÆK3ÆK4Æ[X]4)⁄ (1 + K1Æ[X] + K1ÆK2Æ[X]2+

K1ÆK2ÆK3Æ[X]3+ K1ÆK2ÆK3ÆK4Æ[X]4); and for four sites

equiv-alent and interdependent, Y¼ (4K1Æ[X] + 3K1ÆK2Æ[X]2+

2K1ÆK2ÆK3Æ[X]3+ K1ÆK2ÆK3ÆK4Æ[X]4)⁄ (1 + K1Æ[X] + K1ÆK2Æ[X]2+

K1ÆK2ÆK3Æ[X]3+ K1ÆK2ÆK3ÆK4Æ[X]4) All calculations were

car-ried out with sigmaplot 2000 software (Systat Software Inc.,

CA, USA)

The measure of co-operativity for cofactor binding to

FabG was calculated with the Hill equation, as follows:

logðY=1  YÞ ¼ nHlog½S  log Kd;

where Y is the fraction of the enzyme with the bound

cofactor, Y⁄ 1) Y is the fraction of binding sites that are

occupied for an enzyme-binding substrate, Kdis the

dissoci-ation constant, [S] is the cofactor concentrdissoci-ation and nH is

the Hill coefficient

Fluorescence quenching

Quenching of the fluorescence of FabG by acrylamide was

monitored at 334 nm Samples were excited at 280 nm A

fresh 3 m acrylamide (14.2%) solution was made in 3 mm

Hepes (pH 7.5), 100 mm NaCl, 2 mm b-mercaptoethenol

and 10% glycerol Protein (2 lm) in 3 mm Hepes (pH 7.5),

100 mm NaCl, 2 mm b-mercaptoethenol and 10% glycerol,

was titrated with 10 lL aliquots of acrylamide from a 3 m

stock Corrections were made [29], especially applicable to

proteins containing a single tryptophan [30]:

Fo

Fo F¼

1 fK½Qþ

1 f Where F0 and F are the initial fluorescence and the

observed fluorescence in the presence of the given

concen-tration of the quencher, respectively, K is the Stern–Volmer

quenching constant, [Q] is the concentration of the

acryl-amide (quencher) and f is the fraction of initial fluorescence

which is accessible to the quencher The plot of F0⁄ (F0–F)

versus 1⁄ [Q] yields f)1as the intercept

CD measurements

CD measurements were performed on a JASCO J-810

(Tokyo, Japan) spectropolarimeter at 20
C using a 0.1 cm

path length quartz cuvette, with FabG (14 · 10) 6

molÆl) 1), NADPH (200 lm) and acetoacetyl-CoA

(200 lm) Each spectrum was an accumulation of four to

six consecutive scans over a wavelength range of

200) 250 nm (2 nm band-pass) Results are expressed as molar ellipticity (h) in deg cm2Ædmol) 1 The a-helical and b-sheet content of FabG were calculated from the [h] value

at 208 nm and 217 nm, respectively, using the following equation [31]:

Percentage helicity¼ fð½h208 4000Þ
ð33 000  4000Þg  100:

Acknowledgements

We thank the Department of Science and Technology, India, for their financial support to N.S., and the Chairman, MBU, Indian Institute of Science, for the use of the Jobin–Yvon Horiba spectrofluorimeter for these studies

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