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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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Sequence alignment of class II FBAs. FBAsare from Staphylococcusaureus (S. aureus; Q5HE75), Bacillus anthracis (B. anthra; PDB entry 3Q94_A), Giardialamblia (G. lamblia; 3GAK_B), Helicobacterpylori (H. pylori;3N9S_A), Mycobacteriumtuberculosis (M. tuber; NP_334786), and Escherichia coli (E. coli; PDB entry 1DOS_A). FBA namesare colored based on class II grouping: tan denotes class IIb-i, purpledenotes class IIb-iv, and black denotes class IIa. Secondary structureof SaFBA according to Defined Secondary Structure of Proteins (DSSP)is shown as tan cylinders (helical regions), teal arrows (β-sheets),and tan lines (loops). Hashed lines represent residues for which electrondensity was not defined in the crystal structure. Green line representsthe active site loop. Cyan line represents the Z-loop. Breaks denoteregions were SaFBA does not have residues. Black brackets indicateresidues that are part of SaFBA’s active site. Amino acidsare color-coded with respect to being acidic (red), basic (blue),polar uncharged (yellow), and hydrophobic nonpolar (green).
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fig1: Sequence alignment of class II FBAs. FBAsare from Staphylococcusaureus (S. aureus; Q5HE75), Bacillus anthracis (B. anthra; PDB entry 3Q94_A), Giardialamblia (G. lamblia; 3GAK_B), Helicobacterpylori (H. pylori;3N9S_A), Mycobacteriumtuberculosis (M. tuber; NP_334786), and Escherichia coli (E. coli; PDB entry 1DOS_A). FBA namesare colored based on class II grouping: tan denotes class IIb-i, purpledenotes class IIb-iv, and black denotes class IIa. Secondary structureof SaFBA according to Defined Secondary Structure of Proteins (DSSP)is shown as tan cylinders (helical regions), teal arrows (β-sheets),and tan lines (loops). Hashed lines represent residues for which electrondensity was not defined in the crystal structure. Green line representsthe active site loop. Cyan line represents the Z-loop. Breaks denoteregions were SaFBA does not have residues. Black brackets indicateresidues that are part of SaFBA’s active site. Amino acidsare color-coded with respect to being acidic (red), basic (blue),polar uncharged (yellow), and hydrophobic nonpolar (green).

Mentions: ClassII FBAs can be categorized as class IIa or class IIb on thebasis of sequence homology. Although class IIa FBAs were traditionallythought to be all dimers, recent findings have shown that there existcertain exceptions, such as the class IIa FBA from Mycobacteriumtuberculosis (MtFBA), which forms a tetramer.17 Meanwhile, class IIb FBAs can range from dimersto tetramers to even octomers and tend to be shorter in amino acidlength than their class IIa counterparts.17,19,20,39,40 Furthermore, sequence alignments of class IIb FBAssuggest that this group might be divided into at least four additionalsubtypes (i-iv) although certain discrepancies exist within the purelysequence-based categorization.40,41 To date, only the structuresof class IIb-i FBAs have been studied in regard to their enzymaticcapacities.20,42−45 Based on sequence composition,SaFBA appears to belong to the class IIb-iv of FBAs; however, no structureof either SaFBA, or a proposed class IIb-iv FBA, has yet been reported(Figure 1).


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)

Sequence alignment of class II FBAs. FBAsare from Staphylococcusaureus (S. aureus; Q5HE75), Bacillus anthracis (B. anthra; PDB entry 3Q94_A), Giardialamblia (G. lamblia; 3GAK_B), Helicobacterpylori (H. pylori;3N9S_A), Mycobacteriumtuberculosis (M. tuber; NP_334786), and Escherichia coli (E. coli; PDB entry 1DOS_A). FBA namesare colored based on class II grouping: tan denotes class IIb-i, purpledenotes class IIb-iv, and black denotes class IIa. Secondary structureof SaFBA according to Defined Secondary Structure of Proteins (DSSP)is shown as tan cylinders (helical regions), teal arrows (β-sheets),and tan lines (loops). Hashed lines represent residues for which electrondensity was not defined in the crystal structure. Green line representsthe active site loop. Cyan line represents the Z-loop. Breaks denoteregions were SaFBA does not have residues. Black brackets indicateresidues that are part of SaFBA’s active site. Amino acidsare color-coded with respect to being acidic (red), basic (blue),polar uncharged (yellow), and hydrophobic nonpolar (green).
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Related In: Results  -  Collection

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fig1: Sequence alignment of class II FBAs. FBAsare from Staphylococcusaureus (S. aureus; Q5HE75), Bacillus anthracis (B. anthra; PDB entry 3Q94_A), Giardialamblia (G. lamblia; 3GAK_B), Helicobacterpylori (H. pylori;3N9S_A), Mycobacteriumtuberculosis (M. tuber; NP_334786), and Escherichia coli (E. coli; PDB entry 1DOS_A). FBA namesare colored based on class II grouping: tan denotes class IIb-i, purpledenotes class IIb-iv, and black denotes class IIa. Secondary structureof SaFBA according to Defined Secondary Structure of Proteins (DSSP)is shown as tan cylinders (helical regions), teal arrows (β-sheets),and tan lines (loops). Hashed lines represent residues for which electrondensity was not defined in the crystal structure. Green line representsthe active site loop. Cyan line represents the Z-loop. Breaks denoteregions were SaFBA does not have residues. Black brackets indicateresidues that are part of SaFBA’s active site. Amino acidsare color-coded with respect to being acidic (red), basic (blue),polar uncharged (yellow), and hydrophobic nonpolar (green).
Mentions: ClassII FBAs can be categorized as class IIa or class IIb on thebasis of sequence homology. Although class IIa FBAs were traditionallythought to be all dimers, recent findings have shown that there existcertain exceptions, such as the class IIa FBA from Mycobacteriumtuberculosis (MtFBA), which forms a tetramer.17 Meanwhile, class IIb FBAs can range from dimersto tetramers to even octomers and tend to be shorter in amino acidlength than their class IIa counterparts.17,19,20,39,40 Furthermore, sequence alignments of class IIb FBAssuggest that this group might be divided into at least four additionalsubtypes (i-iv) although certain discrepancies exist within the purelysequence-based categorization.40,41 To date, only the structuresof class IIb-i FBAs have been studied in regard to their enzymaticcapacities.20,42−45 Based on sequence composition,SaFBA appears to belong to the class IIb-iv of FBAs; however, no structureof either SaFBA, or a proposed class IIb-iv FBA, has yet been reported(Figure 1).

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