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

Analyses of co-operative transitions in Plasmodium falciparum ẞ-ketoacyl acyl carrier protein reductase upon co-factor and acyl carrier protein binding

Tác giả: Krishanpal Karmodiya and Namita Surolia

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

Nội dung tài liệu: Nghiên cứu này tập trung vào enzyme β-ketoacyl-acyl carrier protein reductase (FabG) của ký sinh trùng sốt rét Plasmodium falciparum, một mục tiêu tiềm năng cho thuốc chống sốt rét mới. Các nhà nghiên cứu đã khảo sát tương tác trực tiếp của enzyme này với cofactor NADPH và acyl carrier protein (ACP) bằng phương pháp đo huỳnh quang. Kết quả cho thấy việc gắn kết cofactor NADPH làm tăng hiệu quả xúc tác của enzyme và tăng ái lực gắn kết với cơ chất acetoacetyl-coenzyme A lên 16 lần. Đồng thời, ái lực gắn kết với ACP cũng tăng gấp ba lần. Enzyme thể hiện sự gắn kết âm tính, đồng vận cho NADPH, và hiệu ứng này càng được tăng cường khi có sự hiện diện của ACP. Nghiên cứu này cung cấp cơ sở để khám phá ảnh hưởng của các ligand lên mối quan hệ cấu trúc-hoạt tính của enzyme.

Mục lục chi tiết:

• Analyses of co-operative transitions in Plasmodium falciparum ẞ-ketoacyl acyl carrier protein reductase upon co-factor and acyl carrier protein binding
• Keywords
• Correspondence
• Analyses of co-operative transitions in Plasmodium falciparum ẞ-ketoacyl acyl carrier protein reductase upon co-factor and acyl carrier protein binding
• 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.
• ẞ-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 interactions of Plasmodium ẞ-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 (max 334 nm) of the protein as a result of its lone tryptophan, as well as the fluorescence of NADPH (max 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 ẞ-ketoacyl-acyl carrier protein reductase exhibits negative, homotropic co-operative binding for NADPH, which is enhanced in the presence of acyl carrier protein.
• Acyl carrier protein increases the accessibility of NADPH to ẞ-ketoacyl-acyl carrier protein reductase, as evident from the increase in the accessibility of the tryptophan of ẞ-ketoacyl-acyl carrier protein reductase to acrylamide, from 81 to 98%.
• In the presence of NADP+, the reaction proceeds in the reverse direction (Ka = 23.17 µм¯¹).
• These findings provide impetus for exploring the influence of ligands on the structure-activity relationship of Plasmodium ẞ-ketoacyl-acyl carrier protein reductase.
• 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 falciparum.
• The acquisition of resistance by this parasite to conventional antimalarial drugs, such as chloroquine, is growing at an alarming rate and the increasing burden 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 demonstration of the occurrence of the type II fatty acid synthase (FAS) pathway in the malaria parasite and its inhibition by triclosan, an inhibitor of a key enzyme (enoyl-acyl carrier protein reductase) of the type II FAS
• Abbreviations
• ACP, acyl carrier protein; apo-PfACP, Plasmodium falciparum acyl carrier protein (apo form); holo-PfACP, Plasmodium falciparum acyl carrier protein (holo form); FabG, ẞ-ketoacyl-ACP reductase; FAS, fatty acid synthase, PfFabG, Plasmodium falciparum ẞ-ketoacyl-ACP reductase; SDR, short-chain alcohol dehydrogenase/reductase.
• 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 corresponded 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 homogeneity on a Ni-nitrilotriacetic acid affinity column.
• On SDS/PAGE, the purified protein yielded a monomeric M₁ of 31 000 (Fig. 1A) and on Superdex™ 200 yielded an M. 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 ẞ-mercaptoethanol and 10% glycerol).
• The enzyme has a Km value for the substrate acetoacetyl-CoA of 0.43 ± 0.05 mm and a Km value for NADPH of 42.6 ± 0.05 μμ.
• The specific activity of the enzyme with acetoacetyl-CoA is 59.8 Umg¯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/A340 = 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 (max = 334 nm), decreased when it was titrated with increasing concentrations of NADPH (Fig. 2A,B), with simultaneous appearance of another peak with a max at 456 nm as a result of NADPH.
• The appearance of fluorescence with a max at 456 nm is caused by energy transfer 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
• n = 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 n = 4 (Ka = 40.90 µм¯¹) being closest 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 Ka value for its binding to FabG, with n = 4, was found to be 45.2 µм¯¹.
• Binding of other ligands to the binary complex of NADPH.FabG also alters the cofactor-specific fluorescence intensity (max 456 nm).
• The Ka values for all the ligands that were determined [i.e. acetoacetyl-CoA, Plasmodium falciparum acyl carrier protein (apo form) (apo-PfACP) and Plasmodium 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 tryptophan 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 catalytic mechanism.
• The affinities (Ka) of FabG for its cofactor, NADPH, determined by quenching of the fluorescence of its tryptophan (max 334 nm) in the absence and presence of 20 μμ ACP, were found to be 40.9 µм¯¹ and 48.4 µм¯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, heterotropic 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, n₁ = 0.5) (Fig. 2B).
• In the absence of ACP, as stated earlier, the binding of NADPH exhibited negative, 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 calculations 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 association constant of 0.40 µm¯¹ with n = 1.
• The affinity (Ka = 1.1 μμ¯¹) and the number of binding sites increased to two for ACP in the presence of NADPH.
• FabG activity was monitored spectrophotometrically at 340 nm in the presence of NADPH and ACP.
• The maximum activity was observed when the
• Table 1. Binding constants (Ka) of various ligands to ẞ-ketoacyl-acyl carrier protein reductase (FabG) at 20 °C, using the changes in protein and/or cofactor fluorescence intensity at 334 and 450 nm, respectively.
• (Experimental details are provided in the respective figure legendsª).
• 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 2; ND, not determined; SN, serial number.
• Binding of acetoacetyl-CoA and ẞ-hydroxybutyryl-CoA to FabG
• The association constant for the substrate, acetoacetyl-CoA, is 12.3 µм¯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 µм¯¹ (Table 1).
• Thus, acetoacetyl-CoA now has a larger number of favorable interactions at the active site of the enzyme in the presence of NADPH.
• ẞ-hydroxybutyryl-CoA, the product of the reaction, has affinity (23.2 μμ¯¹) (Fig. S2B) comparable with that of acetoacetyl-CoA (18.8 μμ¯¹) in the absence of NADPH.
• Binding of ẞ-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 ẞ-sheet content increased from 27 to 33%, as evident by the CD 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 examination 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¯¹ of 1.35, indicating that 74% of the total fluorescence of FabG is accessible when
• ACP is bound to it, which increases to 98% in the presence of both ACP and NADPH.
• NADPH therefore increases the affinity of ACP by increasing its accessibility to FabG.
• In the presence of ACP, the accessibility 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 Plasmodium 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 acetoacetyl-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 conformational 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, indicate 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 mechanism.
• Once NADPH and ACP bind to FabG, each can access two active sites from the opposite or adjacent subunits (Scheme 1C).
• FabG holds NADPH and ACP close to each other in an orientation which stabilizes 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′ phosphopantetheine moiety for the binding of holo-PfACP (Table 1).
• As evident from the CD data (Fig. 4), the FabG secondary structure increases