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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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Structural comparison of class IIb FBAsubtypes. (Top) Cartoonrepresentations of the crystal structures for class IIb-i FBAs from H. pylori (PDB Code: 3N9S_A), G. lamblia (3GAK_B), T.aquaticus (1RV8_A), and T. caldophilus (2FJK_A). (Bottom) Cartoonrepresentations of the crystal structures for class IIb-iv FBAs from B. anthracis (3Q94_A), C. immitis (3PM6_A), and S. aureus (PDB code: 4TO8_A). Arrows point to the loop and αhelix seen in class IIb-i FBAs due to the 21 amino acid insertion.
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fig6: Structural comparison of class IIb FBAsubtypes. (Top) Cartoonrepresentations of the crystal structures for class IIb-i FBAs from H. pylori (PDB Code: 3N9S_A), G. lamblia (3GAK_B), T.aquaticus (1RV8_A), and T. caldophilus (2FJK_A). (Bottom) Cartoonrepresentations of the crystal structures for class IIb-iv FBAs from B. anthracis (3Q94_A), C. immitis (3PM6_A), and S. aureus (PDB code: 4TO8_A). Arrows point to the loop and αhelix seen in class IIb-i FBAs due to the 21 amino acid insertion.

Mentions: Similar to MtFBA, the SaFBA structure appears to havethe same clarifying effect for class IIb FBAs. Previously, Plaumannet al. proposed possible functional roles to delineate class IIb FBAsubtypes, while Henze et al. further outlined at least four classIIb FBA subtypes (i-iv).40,41 Due to the limiteddata at the time neither group could definitively assemble each putativeclass IIb FBA studied into an appropriate subtype. Regrettably, subsequentreported structures of class IIb FBA structures from H. pylori and Giardia lamblia (GlFBA)provided only a little additional clarity. Although their sequencealignment score as determined by ClustalW was 0.41, analysis of thestructures by the Proteins, Interfaces, Structures and Assemblies(PISA) server revealed a score of 0.80 indicating that they likelybelong to a single subtype.41,67 Intriguingly, whenSaFBA was run through the PISA server it was shown to be most similarto the FBA from Bacillus anthracis (BaFBA) with ascore of 0.88 and when compared to GlFBA both SaFBA and BaFBA scoredroughly 0.70 implying that SaFBA and BaFBA likely fall into theirown subtype. Using SaFBA along with the recent deposition of classIIb FBAs from B. anthracis (PDB Code: 3Q94) and Coccoidioidesimmitis (PDB Code: 3PM6) into the PDB, an updated categorization of classIIb subtypes can be envisioned (Figure 6).42 Specifically, the class II FBAs from G. lamblia and H. pylori contain an insertionof ∼21 residues that does not align with SaFBA or other classIIb FBAs such as BaFBA (Figure 1). The resultof this insert is an additional α-helix seen in several classIIb FBAs but missing in others (Figure 6).The additional helix seen in the likes of GlFBA and HpFBA confirmthe assertion by Henze et al. that they belong to the same subtype,class IIb-i. Although neither SaFBA nor BaFBA were previously analyzedfor a class IIb subtype, both share >79% similarity and >80%identitywith Bacillus subtilis which was categorized as classIIb-iv.41 Furthermore, the nearly completelyconserved crystal structures of SaFBA and the currently unreportedBaFBA implicate that they belong to the same subtype. It remains tobe determined structurally whether other subtypes exist as previouslyreported or what, if any, the functional differences between eachsubtype may be.40,41 Therefore, SaFBA can most accuratelybe classified as a class IIb-iv. It is interesting to note that theadditional helix in class IIb-i FBAs is positioned very near the Z-loop,and the composition of this helix may play a role in substrate binding.In the case of GlFBA, a shift in this loop was noticed when comparingthe unbound structure to that of GlFBA with FBP bound.44 Only with further mutagenesis and enzymatictesting will reveal the function and significance of this discrepancybetween class IIb FBAs be elucidated.


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)

Structural comparison of class IIb FBAsubtypes. (Top) Cartoonrepresentations of the crystal structures for class IIb-i FBAs from H. pylori (PDB Code: 3N9S_A), G. lamblia (3GAK_B), T.aquaticus (1RV8_A), and T. caldophilus (2FJK_A). (Bottom) Cartoonrepresentations of the crystal structures for class IIb-iv FBAs from B. anthracis (3Q94_A), C. immitis (3PM6_A), and S. aureus (PDB code: 4TO8_A). Arrows point to the loop and αhelix seen in class IIb-i FBAs due to the 21 amino acid insertion.
© Copyright Policy
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4263427&req=5

fig6: Structural comparison of class IIb FBAsubtypes. (Top) Cartoonrepresentations of the crystal structures for class IIb-i FBAs from H. pylori (PDB Code: 3N9S_A), G. lamblia (3GAK_B), T.aquaticus (1RV8_A), and T. caldophilus (2FJK_A). (Bottom) Cartoonrepresentations of the crystal structures for class IIb-iv FBAs from B. anthracis (3Q94_A), C. immitis (3PM6_A), and S. aureus (PDB code: 4TO8_A). Arrows point to the loop and αhelix seen in class IIb-i FBAs due to the 21 amino acid insertion.
Mentions: Similar to MtFBA, the SaFBA structure appears to havethe same clarifying effect for class IIb FBAs. Previously, Plaumannet al. proposed possible functional roles to delineate class IIb FBAsubtypes, while Henze et al. further outlined at least four classIIb FBA subtypes (i-iv).40,41 Due to the limiteddata at the time neither group could definitively assemble each putativeclass IIb FBA studied into an appropriate subtype. Regrettably, subsequentreported structures of class IIb FBA structures from H. pylori and Giardia lamblia (GlFBA)provided only a little additional clarity. Although their sequencealignment score as determined by ClustalW was 0.41, analysis of thestructures by the Proteins, Interfaces, Structures and Assemblies(PISA) server revealed a score of 0.80 indicating that they likelybelong to a single subtype.41,67 Intriguingly, whenSaFBA was run through the PISA server it was shown to be most similarto the FBA from Bacillus anthracis (BaFBA) with ascore of 0.88 and when compared to GlFBA both SaFBA and BaFBA scoredroughly 0.70 implying that SaFBA and BaFBA likely fall into theirown subtype. Using SaFBA along with the recent deposition of classIIb FBAs from B. anthracis (PDB Code: 3Q94) and Coccoidioidesimmitis (PDB Code: 3PM6) into the PDB, an updated categorization of classIIb subtypes can be envisioned (Figure 6).42 Specifically, the class II FBAs from G. lamblia and H. pylori contain an insertionof ∼21 residues that does not align with SaFBA or other classIIb FBAs such as BaFBA (Figure 1). The resultof this insert is an additional α-helix seen in several classIIb FBAs but missing in others (Figure 6).The additional helix seen in the likes of GlFBA and HpFBA confirmthe assertion by Henze et al. that they belong to the same subtype,class IIb-i. Although neither SaFBA nor BaFBA were previously analyzedfor a class IIb subtype, both share >79% similarity and >80%identitywith Bacillus subtilis which was categorized as classIIb-iv.41 Furthermore, the nearly completelyconserved crystal structures of SaFBA and the currently unreportedBaFBA implicate that they belong to the same subtype. It remains tobe determined structurally whether other subtypes exist as previouslyreported or what, if any, the functional differences between eachsubtype may be.40,41 Therefore, SaFBA can most accuratelybe classified as a class IIb-iv. It is interesting to note that theadditional helix in class IIb-i FBAs is positioned very near the Z-loop,and the composition of this helix may play a role in substrate binding.In the case of GlFBA, a shift in this loop was noticed when comparingthe unbound structure to that of GlFBA with FBP bound.44 Only with further mutagenesis and enzymatictesting will reveal the function and significance of this discrepancybetween class IIb FBAs be elucidated.

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