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Comparative thermodynamic studies on substrate and product binding of O-acetylserine sulfhydrylase reveals two different ligand recognition modes.

Banerjee S, Ekka MK, Kumaran S - BMC Biochem. (2011)

Bottom Line: Cysteine binding to OASS shows that both enthalpy and entropy contribute significantly to the binding free energy at all temperatures (10-30°C) examined.Our salt dependent ligand binding studies indicate that methionine binding affinity is more sensitive to [NaCl] as compared to cysteine affinity.We speculate that OASS in general, may exhibit two different binding mechanisms for recognizing substrates and products.

View Article: PubMed Central - HTML - PubMed

Affiliation: Council of Scientific and Industrial Research, Institute of Microbial Technology, Chandigarh, India.

ABSTRACT

Background: The importance of understanding the detailed mechanism of cysteine biosynthesis in bacteria is underscored by the fact that cysteine is the only sulfur donor for all cellular components containing reduced sulfur. O-acetylserine sulfhydrylase (OASS) catalyzes this crucial last step in the cysteine biosynthesis and has been recognized as an important gene for the survival and virulence of pathogenic bacteria. Structural and kinetic studies have contributed to the understanding of mechanistic aspects of OASS, but details of ligand recognition features of OASS are not available. In the absence of any detailed study on the energetics of ligand binding, we have studied the thermodynamics of OASS from Salmonella typhimurium (StOASS), Haemophilus influenzae (HiOASS), and Mycobacterium tuberculosis (MtOASS) binding to their substrate O-acetylserine (OAS), substrate analogue (methionine), and product (cysteine).

Results: Ligand binding properties of three OASS enzymes are studied under defined solution conditions. Both substrate and product binding is an exothermic reaction, but their thermodynamic signatures are very different. Cysteine binding to OASS shows that both enthalpy and entropy contribute significantly to the binding free energy at all temperatures (10-30°C) examined. The analyses of interaction between OASS with OAS (substrate) or methionine (substrate analogue) revealed a completely different mode of binding. Binding of both OAS and methionine to OASS is dominated by a favorable entropy change, with minor contribution from enthalpy change (ΔH(St-Met) = -1.5 ± 0.1 kJ/mol; TΔS(St-Met) = 8.2 kJ/mol) at 20°C. Our salt dependent ligand binding studies indicate that methionine binding affinity is more sensitive to [NaCl] as compared to cysteine affinity.

Conclusions: We show that OASS from three different pathogenic bacteria bind substrate and product through two different mechanisms. Results indicate that predominantly entropy driven methionine binding is not mediated through classical hydrophobic binding, instead, may involve desolvation of the polar active site. We speculate that OASS in general, may exhibit two different binding mechanisms for recognizing substrates and products.

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Spectroscopic characterization of OASS and ligand binding. (A) Fluorescence emission of OASS upon excitation at 295 nm and quenching of fluorescence due to ligand binding (down arrow indicates decrease in fluorescence upon addition of ligands). (B) Fluorescence emission of OASS upon excitation at 412 nm (PLP) and change in fluorescence intensity due to methionine binding (upward arrow indicates increase in PLP fluorescence upon addition of ligands). Fluorescence increase at 507 nm was used for determining the extent of binding. (C) Cartoon representation of substrate (OAS), substrate analogue (methionine), and product (cysteine) used in this study.
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Figure 1: Spectroscopic characterization of OASS and ligand binding. (A) Fluorescence emission of OASS upon excitation at 295 nm and quenching of fluorescence due to ligand binding (down arrow indicates decrease in fluorescence upon addition of ligands). (B) Fluorescence emission of OASS upon excitation at 412 nm (PLP) and change in fluorescence intensity due to methionine binding (upward arrow indicates increase in PLP fluorescence upon addition of ligands). Fluorescence increase at 507 nm was used for determining the extent of binding. (C) Cartoon representation of substrate (OAS), substrate analogue (methionine), and product (cysteine) used in this study.

Mentions: The absorbance spectra of OASS from all three species (H. influenza, S. typhimurium, M.tuberculosis) showed two distinct absorption maxima, one around 280 nm and a second maximum at 412 nm indicative of the presence of internal aldimine [17]. Excitation of OASS at 292 nm yields a fluorescence emission 325-350 nm due to Trp emission and excitation at 412 nm gives another emission spectrum in the range 475-540 nm due to PLP fluorescence. Fluorescence properties of OASS were investigated previously and it was observed that excitation at 298 nm generates a large increase in fluorescence between 340-345 nm [24,25]. Similar to earlier observations, excitation at 292 nm yields an emission spectrum centered at 345 nm and fluorescence at 345 nm is quenched upon ligand binding (Figure 1A). We have examined the binding of O-acetyl serine (OAS), L-cysteine, L-methionine, L-serine, and L-isoleucine to OASS by monitoring the changes in fluorescence properties of the protein. Binding of cysteine or methionine to OASS changes both tryptophan and PLP fluorescence spectra (Figure 1A &1B). Ligand binding quenches tryptophan fluorescence observed at 345 nm (Figure 1A), but increases the PLP fluorescence at 507 nm as observed earlier [26]. It was observed that excitation of OASS at 290 nm leads to fluorescence at 345 nm as well as at 500 nm due to energy transfer from tryptophan to PLP. Therefore, we monitored the increase in the PLP fluorescence emission at 507 nm upon excitation at 412 nm for constructing binding isotherms and determining the equilibrium binding parameters. Addition of OAS quenches the PLP fluorescence initially, but fluorescence signal increases as a function of time indicating the existence of a slow kinetic process after initial quenching (Figure 2A). Detailed studies are necessary to understand the molecular origin of this slow kinetic process.


Comparative thermodynamic studies on substrate and product binding of O-acetylserine sulfhydrylase reveals two different ligand recognition modes.

Banerjee S, Ekka MK, Kumaran S - BMC Biochem. (2011)

Spectroscopic characterization of OASS and ligand binding. (A) Fluorescence emission of OASS upon excitation at 295 nm and quenching of fluorescence due to ligand binding (down arrow indicates decrease in fluorescence upon addition of ligands). (B) Fluorescence emission of OASS upon excitation at 412 nm (PLP) and change in fluorescence intensity due to methionine binding (upward arrow indicates increase in PLP fluorescence upon addition of ligands). Fluorescence increase at 507 nm was used for determining the extent of binding. (C) Cartoon representation of substrate (OAS), substrate analogue (methionine), and product (cysteine) used in this study.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3141655&req=5

Figure 1: Spectroscopic characterization of OASS and ligand binding. (A) Fluorescence emission of OASS upon excitation at 295 nm and quenching of fluorescence due to ligand binding (down arrow indicates decrease in fluorescence upon addition of ligands). (B) Fluorescence emission of OASS upon excitation at 412 nm (PLP) and change in fluorescence intensity due to methionine binding (upward arrow indicates increase in PLP fluorescence upon addition of ligands). Fluorescence increase at 507 nm was used for determining the extent of binding. (C) Cartoon representation of substrate (OAS), substrate analogue (methionine), and product (cysteine) used in this study.
Mentions: The absorbance spectra of OASS from all three species (H. influenza, S. typhimurium, M.tuberculosis) showed two distinct absorption maxima, one around 280 nm and a second maximum at 412 nm indicative of the presence of internal aldimine [17]. Excitation of OASS at 292 nm yields a fluorescence emission 325-350 nm due to Trp emission and excitation at 412 nm gives another emission spectrum in the range 475-540 nm due to PLP fluorescence. Fluorescence properties of OASS were investigated previously and it was observed that excitation at 298 nm generates a large increase in fluorescence between 340-345 nm [24,25]. Similar to earlier observations, excitation at 292 nm yields an emission spectrum centered at 345 nm and fluorescence at 345 nm is quenched upon ligand binding (Figure 1A). We have examined the binding of O-acetyl serine (OAS), L-cysteine, L-methionine, L-serine, and L-isoleucine to OASS by monitoring the changes in fluorescence properties of the protein. Binding of cysteine or methionine to OASS changes both tryptophan and PLP fluorescence spectra (Figure 1A &1B). Ligand binding quenches tryptophan fluorescence observed at 345 nm (Figure 1A), but increases the PLP fluorescence at 507 nm as observed earlier [26]. It was observed that excitation of OASS at 290 nm leads to fluorescence at 345 nm as well as at 500 nm due to energy transfer from tryptophan to PLP. Therefore, we monitored the increase in the PLP fluorescence emission at 507 nm upon excitation at 412 nm for constructing binding isotherms and determining the equilibrium binding parameters. Addition of OAS quenches the PLP fluorescence initially, but fluorescence signal increases as a function of time indicating the existence of a slow kinetic process after initial quenching (Figure 2A). Detailed studies are necessary to understand the molecular origin of this slow kinetic process.

Bottom Line: Cysteine binding to OASS shows that both enthalpy and entropy contribute significantly to the binding free energy at all temperatures (10-30°C) examined.Our salt dependent ligand binding studies indicate that methionine binding affinity is more sensitive to [NaCl] as compared to cysteine affinity.We speculate that OASS in general, may exhibit two different binding mechanisms for recognizing substrates and products.

View Article: PubMed Central - HTML - PubMed

Affiliation: Council of Scientific and Industrial Research, Institute of Microbial Technology, Chandigarh, India.

ABSTRACT

Background: The importance of understanding the detailed mechanism of cysteine biosynthesis in bacteria is underscored by the fact that cysteine is the only sulfur donor for all cellular components containing reduced sulfur. O-acetylserine sulfhydrylase (OASS) catalyzes this crucial last step in the cysteine biosynthesis and has been recognized as an important gene for the survival and virulence of pathogenic bacteria. Structural and kinetic studies have contributed to the understanding of mechanistic aspects of OASS, but details of ligand recognition features of OASS are not available. In the absence of any detailed study on the energetics of ligand binding, we have studied the thermodynamics of OASS from Salmonella typhimurium (StOASS), Haemophilus influenzae (HiOASS), and Mycobacterium tuberculosis (MtOASS) binding to their substrate O-acetylserine (OAS), substrate analogue (methionine), and product (cysteine).

Results: Ligand binding properties of three OASS enzymes are studied under defined solution conditions. Both substrate and product binding is an exothermic reaction, but their thermodynamic signatures are very different. Cysteine binding to OASS shows that both enthalpy and entropy contribute significantly to the binding free energy at all temperatures (10-30°C) examined. The analyses of interaction between OASS with OAS (substrate) or methionine (substrate analogue) revealed a completely different mode of binding. Binding of both OAS and methionine to OASS is dominated by a favorable entropy change, with minor contribution from enthalpy change (ΔH(St-Met) = -1.5 ± 0.1 kJ/mol; TΔS(St-Met) = 8.2 kJ/mol) at 20°C. Our salt dependent ligand binding studies indicate that methionine binding affinity is more sensitive to [NaCl] as compared to cysteine affinity.

Conclusions: We show that OASS from three different pathogenic bacteria bind substrate and product through two different mechanisms. Results indicate that predominantly entropy driven methionine binding is not mediated through classical hydrophobic binding, instead, may involve desolvation of the polar active site. We speculate that OASS in general, may exhibit two different binding mechanisms for recognizing substrates and products.

Show MeSH
Related in: MedlinePlus