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Structural and functional characterization of methicillin-resistant Staphylococcus aureus's class IIb fructose 1,6-bisphosphate aldolase.

Capodagli GC, Lee SA, Boehm KJ, Brady KM, Pegan SD - Biochemistry (2014)

Bottom Line: Regrettably, scarce biochemical data and no structural data are currently available for the class II FBA found in MRSA (SaFBA).Therefore, we elucidated the crystal structure of SaFBA to 2.1 Å allowing for a more direct structural analysis of SaFBA.Furthermore, we determined the KM for one of SaFBA's substrates, fructose 1,6-bisphosphate, as well as performed mode of inhibition studies for an inhibitor that takes advantage of the Z-loop's flexibility.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of Denver , Denver, Colorado 80208, United States.

ABSTRACT
Staphylococcus aureus is one of the most common nosocomial sources of soft-tissue and skin infections and has more recently become prevalent in the community setting as well. Since the use of penicillins to combat S. aureus infections in the 1940s, the bacterium has been notorious for developing resistances to antibiotics, such as methicillin-resistant Staphylococcus aureus (MRSA). With the persistence of MRSA as well as many other drug resistant bacteria and parasites, there is a growing need to focus on new pharmacological targets. Recently, class II fructose 1,6-bisphosphate aldolases (FBAs) have garnered attention to fill this role. Regrettably, scarce biochemical data and no structural data are currently available for the class II FBA found in MRSA (SaFBA). With the recent finding of a flexible active site zinc-binding loop (Z-Loop) in class IIa FBAs and its potential for broad spectrum class II FBA inhibition, the lack of information regarding this feature of class IIb FBAs, such as SaFBA, has been limiting for further Z-loop inhibitor development. Therefore, we elucidated the crystal structure of SaFBA to 2.1 Å allowing for a more direct structural analysis of SaFBA. Furthermore, we determined the KM for one of SaFBA's substrates, fructose 1,6-bisphosphate, as well as performed mode of inhibition studies for an inhibitor that takes advantage of the Z-loop's flexibility. Together the data offers insight into a class IIb FBA from a pervasively drug resistant bacterium and a comparison of Z-loops and other features between the different subtypes of class II FBAs.

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SaFBAkinetic studies. (a) Plot of turnover number (TN) of FBPby SaFBA as the concentration of FBP was varied from 0 to 5000 μM.(b) Lineweaver–Burke plot of inhibition of SaFBA by HCA. Concentrationsof HCA were (●) 0, (○) 1.95, (▼) 3.91, (▽)7.81, (■) 15.6, (□) 31.3, (⧫) 62.5, (◊)125, (▲) 250, and (△) 500 μM. Data were fit globallyto a pure mixed inhibition model. (c) Plot of inhibition of SaFBAas the HCA concentration was increased from 0 to 2 mM. (d) Plot ofinhibition of SaFBA as the citrate concentration was increased from0 to 560 mM. All plots were fit to the Michaelis–Menten equation.
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fig5: SaFBAkinetic studies. (a) Plot of turnover number (TN) of FBPby SaFBA as the concentration of FBP was varied from 0 to 5000 μM.(b) Lineweaver–Burke plot of inhibition of SaFBA by HCA. Concentrationsof HCA were (●) 0, (○) 1.95, (▼) 3.91, (▽)7.81, (■) 15.6, (□) 31.3, (⧫) 62.5, (◊)125, (▲) 250, and (△) 500 μM. Data were fit globallyto a pure mixed inhibition model. (c) Plot of inhibition of SaFBAas the HCA concentration was increased from 0 to 2 mM. (d) Plot ofinhibition of SaFBA as the citrate concentration was increased from0 to 560 mM. All plots were fit to the Michaelis–Menten equation.

Mentions: FBAs catalyze the reversible aldol condensation ofDHAP with G3P to form FBP. To gain insight on the affinity of SaFBAfor FBP, a fluorescence-based assay was employed. Using the initialslopes over a series of FBP concentrations, the KM and kcat values of SaFBAfor FBP were found to be 239 ± 16 μM and 69.5 ± 1.6min–1, respectively (Figure 5a). These values fall within the range of several other class IIFBAs.61−64


Structural and functional characterization of methicillin-resistant Staphylococcus aureus's class IIb fructose 1,6-bisphosphate aldolase.

Capodagli GC, Lee SA, Boehm KJ, Brady KM, Pegan SD - Biochemistry (2014)

SaFBAkinetic studies. (a) Plot of turnover number (TN) of FBPby SaFBA as the concentration of FBP was varied from 0 to 5000 μM.(b) Lineweaver–Burke plot of inhibition of SaFBA by HCA. Concentrationsof HCA were (●) 0, (○) 1.95, (▼) 3.91, (▽)7.81, (■) 15.6, (□) 31.3, (⧫) 62.5, (◊)125, (▲) 250, and (△) 500 μM. Data were fit globallyto a pure mixed inhibition model. (c) Plot of inhibition of SaFBAas the HCA concentration was increased from 0 to 2 mM. (d) Plot ofinhibition of SaFBA as the citrate concentration was increased from0 to 560 mM. All plots were fit to the Michaelis–Menten equation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263427&req=5

fig5: SaFBAkinetic studies. (a) Plot of turnover number (TN) of FBPby SaFBA as the concentration of FBP was varied from 0 to 5000 μM.(b) Lineweaver–Burke plot of inhibition of SaFBA by HCA. Concentrationsof HCA were (●) 0, (○) 1.95, (▼) 3.91, (▽)7.81, (■) 15.6, (□) 31.3, (⧫) 62.5, (◊)125, (▲) 250, and (△) 500 μM. Data were fit globallyto a pure mixed inhibition model. (c) Plot of inhibition of SaFBAas the HCA concentration was increased from 0 to 2 mM. (d) Plot ofinhibition of SaFBA as the citrate concentration was increased from0 to 560 mM. All plots were fit to the Michaelis–Menten equation.
Mentions: FBAs catalyze the reversible aldol condensation ofDHAP with G3P to form FBP. To gain insight on the affinity of SaFBAfor FBP, a fluorescence-based assay was employed. Using the initialslopes over a series of FBP concentrations, the KM and kcat values of SaFBAfor FBP were found to be 239 ± 16 μM and 69.5 ± 1.6min–1, respectively (Figure 5a). These values fall within the range of several other class IIFBAs.61−64

Bottom Line: Regrettably, scarce biochemical data and no structural data are currently available for the class II FBA found in MRSA (SaFBA).Therefore, we elucidated the crystal structure of SaFBA to 2.1 Å allowing for a more direct structural analysis of SaFBA.Furthermore, we determined the KM for one of SaFBA's substrates, fructose 1,6-bisphosphate, as well as performed mode of inhibition studies for an inhibitor that takes advantage of the Z-loop's flexibility.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of Denver , Denver, Colorado 80208, United States.

ABSTRACT
Staphylococcus aureus is one of the most common nosocomial sources of soft-tissue and skin infections and has more recently become prevalent in the community setting as well. Since the use of penicillins to combat S. aureus infections in the 1940s, the bacterium has been notorious for developing resistances to antibiotics, such as methicillin-resistant Staphylococcus aureus (MRSA). With the persistence of MRSA as well as many other drug resistant bacteria and parasites, there is a growing need to focus on new pharmacological targets. Recently, class II fructose 1,6-bisphosphate aldolases (FBAs) have garnered attention to fill this role. Regrettably, scarce biochemical data and no structural data are currently available for the class II FBA found in MRSA (SaFBA). With the recent finding of a flexible active site zinc-binding loop (Z-Loop) in class IIa FBAs and its potential for broad spectrum class II FBA inhibition, the lack of information regarding this feature of class IIb FBAs, such as SaFBA, has been limiting for further Z-loop inhibitor development. Therefore, we elucidated the crystal structure of SaFBA to 2.1 Å allowing for a more direct structural analysis of SaFBA. Furthermore, we determined the KM for one of SaFBA's substrates, fructose 1,6-bisphosphate, as well as performed mode of inhibition studies for an inhibitor that takes advantage of the Z-loop's flexibility. Together the data offers insight into a class IIb FBA from a pervasively drug resistant bacterium and a comparison of Z-loops and other features between the different subtypes of class II FBAs.

Show MeSH
Related in: MedlinePlus