Limits...
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.

Show MeSH

Related in: MedlinePlus

Secondary structurecomparisons of Class II FBAs. (a) Surface renderingof the SaFBA dimer found in the asymmetric unit with one protomercolored tan, the other colored gray, citrate is shown as sticks andcolored yellow, and Zn(II) ion is shown as a black sphere. (b) Cartoonrendering of the SaFBA protomer. Helical regions are represented ascylinders with β-strands as arrows. Helices and loops are coloredtan with β-strands colored teal, citrate and Zn(II) are as in(a). (c) SaFBA the same as in (b) but overlaid with MtFBA-PGH (PDBcode: 4DEL)shown in gray. The β5α5 loop (green) and the β6α6loop (blue) for which is density is observed in the MtFBA-PGH structurebut only partially in the SaFBA structure. (d) SaFBA same as in (b)but overlaid with HpFBA-TD4 (PDB code: 3N9S_A) shown in maroon. The β5α5loop (green) and the β6α6 loop (blue) for which is densitywas observed in the HpFBA-TD4 structure and only partially in theSaFBA structure.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Secondary structurecomparisons of Class II FBAs. (a) Surface renderingof the SaFBA dimer found in the asymmetric unit with one protomercolored tan, the other colored gray, citrate is shown as sticks andcolored yellow, and Zn(II) ion is shown as a black sphere. (b) Cartoonrendering of the SaFBA protomer. Helical regions are represented ascylinders with β-strands as arrows. Helices and loops are coloredtan with β-strands colored teal, citrate and Zn(II) are as in(a). (c) SaFBA the same as in (b) but overlaid with MtFBA-PGH (PDBcode: 4DEL)shown in gray. The β5α5 loop (green) and the β6α6loop (blue) for which is density is observed in the MtFBA-PGH structurebut only partially in the SaFBA structure. (d) SaFBA same as in (b)but overlaid with HpFBA-TD4 (PDB code: 3N9S_A) shown in maroon. The β5α5loop (green) and the β6α6 loop (blue) for which is densitywas observed in the HpFBA-TD4 structure and only partially in theSaFBA structure.

Mentions: SaFBA was observed as a dimer in the asymmetricunit with the secondary structure of each monomer resembling the TIMbarrel fold of other class II FBAs, such as MtFBA (Figure 2a,b). The dimeric nature of SaFBA in the asymmetricunit is consistent with the oligomeric state suggested by size exclusionchromatography (data not shown). Of the SaFBA’s 295 amino acids,all showed good electron density except for residues 1, 144, 145,183–187, and 288–295. The structure is comprised of12 α-helices and 8 β-sheets. Comparison of SaFBA and MtFBAimmediately highlighted the differences between class IIb and classIIa FBAs. Several helices such as α3, α5, and α6are shifted considerably, and helices α8b, α8c, and a310 in MtFBA are not present in SaFBA (Figure 2c). This is reflective of the additional lengththat class IIa FBAs possess over their class IIb counterparts.


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)

Secondary structurecomparisons of Class II FBAs. (a) Surface renderingof the SaFBA dimer found in the asymmetric unit with one protomercolored tan, the other colored gray, citrate is shown as sticks andcolored yellow, and Zn(II) ion is shown as a black sphere. (b) Cartoonrendering of the SaFBA protomer. Helical regions are represented ascylinders with β-strands as arrows. Helices and loops are coloredtan with β-strands colored teal, citrate and Zn(II) are as in(a). (c) SaFBA the same as in (b) but overlaid with MtFBA-PGH (PDBcode: 4DEL)shown in gray. The β5α5 loop (green) and the β6α6loop (blue) for which is density is observed in the MtFBA-PGH structurebut only partially in the SaFBA structure. (d) SaFBA same as in (b)but overlaid with HpFBA-TD4 (PDB code: 3N9S_A) shown in maroon. The β5α5loop (green) and the β6α6 loop (blue) for which is densitywas observed in the HpFBA-TD4 structure and only partially in theSaFBA structure.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Secondary structurecomparisons of Class II FBAs. (a) Surface renderingof the SaFBA dimer found in the asymmetric unit with one protomercolored tan, the other colored gray, citrate is shown as sticks andcolored yellow, and Zn(II) ion is shown as a black sphere. (b) Cartoonrendering of the SaFBA protomer. Helical regions are represented ascylinders with β-strands as arrows. Helices and loops are coloredtan with β-strands colored teal, citrate and Zn(II) are as in(a). (c) SaFBA the same as in (b) but overlaid with MtFBA-PGH (PDBcode: 4DEL)shown in gray. The β5α5 loop (green) and the β6α6loop (blue) for which is density is observed in the MtFBA-PGH structurebut only partially in the SaFBA structure. (d) SaFBA same as in (b)but overlaid with HpFBA-TD4 (PDB code: 3N9S_A) shown in maroon. The β5α5loop (green) and the β6α6 loop (blue) for which is densitywas observed in the HpFBA-TD4 structure and only partially in theSaFBA structure.
Mentions: SaFBA was observed as a dimer in the asymmetricunit with the secondary structure of each monomer resembling the TIMbarrel fold of other class II FBAs, such as MtFBA (Figure 2a,b). The dimeric nature of SaFBA in the asymmetricunit is consistent with the oligomeric state suggested by size exclusionchromatography (data not shown). Of the SaFBA’s 295 amino acids,all showed good electron density except for residues 1, 144, 145,183–187, and 288–295. The structure is comprised of12 α-helices and 8 β-sheets. Comparison of SaFBA and MtFBAimmediately highlighted the differences between class IIb and classIIa FBAs. Several helices such as α3, α5, and α6are shifted considerably, and helices α8b, α8c, and a310 in MtFBA are not present in SaFBA (Figure 2c). This is reflective of the additional lengththat class IIa FBAs possess over their class IIb counterparts.

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