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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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FBA active site comparison. (a) Wall-eyed stereoview of SaFBA (tan/teal)overlaid with MtFBA (gray; PDB entry 3ELF_A) interacting with FBP (cyan) and Zn(II)(purple) with green dashed lines indicating coordinating bonds. Yellowlabels indicate MtFBA residues. (b) Wall-eyed stereoview of SaFBAcolored as in (a) overlaid with HpFBA (lavender; PDB code: 3N9S_A) interactingwith TD4 (white) and Zn(II) (yellow) with red dashed lines indicatingcoordinating bonds. Black labels indicate HpFBA residues.
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fig4: FBA active site comparison. (a) Wall-eyed stereoview of SaFBA (tan/teal)overlaid with MtFBA (gray; PDB entry 3ELF_A) interacting with FBP (cyan) and Zn(II)(purple) with green dashed lines indicating coordinating bonds. Yellowlabels indicate MtFBA residues. (b) Wall-eyed stereoview of SaFBAcolored as in (a) overlaid with HpFBA (lavender; PDB code: 3N9S_A) interactingwith TD4 (white) and Zn(II) (yellow) with red dashed lines indicatingcoordinating bonds. Black labels indicate HpFBA residues.

Mentions: Unlike the active site loop within class II FBAs, the β6α6loop containing residues 177–191, and previously termed theZ-loop, is almost always well-defined structurally.17,19,20,34,42,44,45,49,56−60 This loop forms part of the substrate pocket as well as containsa histidine residue involved in coordination with the active siteZn(II) ion.46 Between class IIb FBAs, suchas HpFBA, whose structures have been reported, and SaFBA, HpFBA possesstwo additional residues within this region. Additionally, all previouslyreported class IIb FBAs also possess an α-helix located nearthe Z-loop not seen in either SaFBA or MtFBA (Figure 2d). It is unclear whether this helix is influential in theenzyme’s functionality, but it may be part of the reason asto why HpFBA’s Z-loop is two residues longer than that of SaFBA.Interestingly for the SaFBA structure, there was no electron densityobserved for residues 183–187 in its Z-loop, and His181 wasdisplaced from its coordination of the Zn(II) ion. Closer inspectionof this region using a simulated omit map revealed Fo–Fc density of a citratemolecule coordinating the active site Zn(II) in a tridentate manner(Figure 3). This interaction is curiously similarto that observed previously between MtFBA and the Z-loop inhibitorHCA. Naturally, the likely source of this citrate ion is the crystallizationcondition that contained 1.6 M ammonium citrate. Along with the threecoordinating bonds to the citrate ion, the active site Zn(II) alsoforms an additional three coordinating bonds with residues His86 andHis209 and a water molecule. These interactions yield a coordinationnumber of 6 (T6) for the active site Zn(II) similar tothat of the FBP-bound structure of MtFBA and different from the T5 coordination found in the inhibitor-bound structure of HpFBAfor which the entire protein was observed including the active siteloop (Figure 4).17,43 This coordinationstate is also different from the T5 coordination stateobserved in the HCA bound MtFBA structure. Additionally, the citrateion forms hydrogen bonds (H-bonds) with Asn233 and Gly210, as wellas a network of H-bonds through several waters to Glu137, His181,and Asp85 (Figure 3).


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)

FBA active site comparison. (a) Wall-eyed stereoview of SaFBA (tan/teal)overlaid with MtFBA (gray; PDB entry 3ELF_A) interacting with FBP (cyan) and Zn(II)(purple) with green dashed lines indicating coordinating bonds. Yellowlabels indicate MtFBA residues. (b) Wall-eyed stereoview of SaFBAcolored as in (a) overlaid with HpFBA (lavender; PDB code: 3N9S_A) interactingwith TD4 (white) and Zn(II) (yellow) with red dashed lines indicatingcoordinating bonds. Black labels indicate HpFBA residues.
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fig4: FBA active site comparison. (a) Wall-eyed stereoview of SaFBA (tan/teal)overlaid with MtFBA (gray; PDB entry 3ELF_A) interacting with FBP (cyan) and Zn(II)(purple) with green dashed lines indicating coordinating bonds. Yellowlabels indicate MtFBA residues. (b) Wall-eyed stereoview of SaFBAcolored as in (a) overlaid with HpFBA (lavender; PDB code: 3N9S_A) interactingwith TD4 (white) and Zn(II) (yellow) with red dashed lines indicatingcoordinating bonds. Black labels indicate HpFBA residues.
Mentions: Unlike the active site loop within class II FBAs, the β6α6loop containing residues 177–191, and previously termed theZ-loop, is almost always well-defined structurally.17,19,20,34,42,44,45,49,56−60 This loop forms part of the substrate pocket as well as containsa histidine residue involved in coordination with the active siteZn(II) ion.46 Between class IIb FBAs, suchas HpFBA, whose structures have been reported, and SaFBA, HpFBA possesstwo additional residues within this region. Additionally, all previouslyreported class IIb FBAs also possess an α-helix located nearthe Z-loop not seen in either SaFBA or MtFBA (Figure 2d). It is unclear whether this helix is influential in theenzyme’s functionality, but it may be part of the reason asto why HpFBA’s Z-loop is two residues longer than that of SaFBA.Interestingly for the SaFBA structure, there was no electron densityobserved for residues 183–187 in its Z-loop, and His181 wasdisplaced from its coordination of the Zn(II) ion. Closer inspectionof this region using a simulated omit map revealed Fo–Fc density of a citratemolecule coordinating the active site Zn(II) in a tridentate manner(Figure 3). This interaction is curiously similarto that observed previously between MtFBA and the Z-loop inhibitorHCA. Naturally, the likely source of this citrate ion is the crystallizationcondition that contained 1.6 M ammonium citrate. Along with the threecoordinating bonds to the citrate ion, the active site Zn(II) alsoforms an additional three coordinating bonds with residues His86 andHis209 and a water molecule. These interactions yield a coordinationnumber of 6 (T6) for the active site Zn(II) similar tothat of the FBP-bound structure of MtFBA and different from the T5 coordination found in the inhibitor-bound structure of HpFBAfor which the entire protein was observed including the active siteloop (Figure 4).17,43 This coordinationstate is also different from the T5 coordination stateobserved in the HCA bound MtFBA structure. Additionally, the citrateion forms hydrogen bonds (H-bonds) with Asn233 and Gly210, as wellas a network of H-bonds through several waters to Glu137, His181,and Asp85 (Figure 3).

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