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Structural and dynamic requirements for optimal activity of the essential bacterial enzyme dihydrodipicolinate synthase.

Reboul CF, Porebski BT, Griffin MD, Dobson RC, Perugini MA, Gerrard JA, Buckle AM - PLoS Comput. Biol. (2012)

Bottom Line: DHDPS from E. coli is a homotetramer consisting of a 'dimer of dimers', with the catalytic residues found at the tight-dimer interface.Crystallographic and biophysical evidence suggest that the dimers associate to stabilise the active site configuration, and mutation of a central dimer-dimer interface residue destabilises the tetramer, thus increasing the flexibility and reducing catalytic efficiency and substrate specificity.These reveal a striking contrast between the dynamics of tetrameric and dimeric forms.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.

ABSTRACT
Dihydrodipicolinate synthase (DHDPS) is an essential enzyme involved in the lysine biosynthesis pathway. DHDPS from E. coli is a homotetramer consisting of a 'dimer of dimers', with the catalytic residues found at the tight-dimer interface. Crystallographic and biophysical evidence suggest that the dimers associate to stabilise the active site configuration, and mutation of a central dimer-dimer interface residue destabilises the tetramer, thus increasing the flexibility and reducing catalytic efficiency and substrate specificity. This has led to the hypothesis that the tetramer evolved to optimise the dynamics within the tight-dimer. In order to gain insights into DHDPS flexibility and its relationship to quaternary structure and function, we performed comparative Molecular Dynamics simulation studies of native tetrameric and dimeric forms of DHDPS from E. coli and also the native dimeric form from methicillin-resistant Staphylococcus aureus (MRSA). These reveal a striking contrast between the dynamics of tetrameric and dimeric forms. Whereas the E. coli DHDPS tetramer is relatively rigid, both the E. coli and MRSA DHDPS dimers display high flexibility, resulting in monomer reorientation within the dimer and increased flexibility at the tight-dimer interface. The mutant E. coli DHDPS dimer exhibits disorder within its active site with deformation of critical catalytic residues and removal of key hydrogen bonds that render it inactive, whereas the similarly flexible MRSA DHDPS dimer maintains its catalytic geometry and is thus fully functional. Our data support the hypothesis that in both bacterial species optimal activity is achieved by fine tuning protein dynamics in different ways: E. coli DHDPS buttresses together two dimers, whereas MRSA dampens the motion using an extended tight-dimer interface.

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Network of essential active site interactions over the course of the simulations.(A) Electrostatic interactions and residues considered: Wild-type E. coli tetramer (dark blue), E. coli L197 mutant dimer (light blue), MRSA wild-type dimer (green). Only Wild-type E. Coli interactions (dashed lines) are shown for clarity. (B) Distance between T44-Oγ and Y133-Oç. (C) Distance between T44-Oγ and Y107-Oç. (D) Distance between K161-Nζ and Y133-Oç. Equivalent MRSA residues are T46, Y135, Y109 and K163.
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pcbi-1002537-g007: Network of essential active site interactions over the course of the simulations.(A) Electrostatic interactions and residues considered: Wild-type E. coli tetramer (dark blue), E. coli L197 mutant dimer (light blue), MRSA wild-type dimer (green). Only Wild-type E. Coli interactions (dashed lines) are shown for clarity. (B) Distance between T44-Oγ and Y133-Oç. (C) Distance between T44-Oγ and Y107-Oç. (D) Distance between K161-Nζ and Y133-Oç. Equivalent MRSA residues are T46, Y135, Y109 and K163.

Mentions: To gain more insight into the potential changes occurring in the active sites we focused on the conserved network of hydrogen bonds present in the catalytic site (Figure 7A). This network is formed by 2 hydrogen bonds between the hydroxyl groups of T44 and Y133 (E. coli numbering), and between the hydroxyl groups of T44 and Y107. Point mutation of any of these 3 residues that constitute the catalytic triad results in severely reduced activity [3]. Distances between donor and acceptor atoms were monitored throughout simulations (Figure 7). We find that atoms T44-Oγ/Y133-Oç (Figure 7B) remain in reasonably close contact at a similar average distance of 5.4±1.3 Å and 5.6±1.3 Å in the E. coli and the MRSA dimers respectively. The hydrogen bond is only transiently formed regardless of the species and broken upon flipping of the T44 side chain. In contrast the distance between T44-Oγ/Y107-Oç shows a marked difference (Figure 7C) following the repositioning of Y107 in the E. coli dimer associated with monomer re-arrangement and shown here with a large increase. The relative positions of both side chains seem affected to a smaller extent by rotation in the MRSA dimer (average distance is 5.7±1.3 Å) with a small distance increase suggesting weak electrostatic interaction between the hydroxyl groups.


Structural and dynamic requirements for optimal activity of the essential bacterial enzyme dihydrodipicolinate synthase.

Reboul CF, Porebski BT, Griffin MD, Dobson RC, Perugini MA, Gerrard JA, Buckle AM - PLoS Comput. Biol. (2012)

Network of essential active site interactions over the course of the simulations.(A) Electrostatic interactions and residues considered: Wild-type E. coli tetramer (dark blue), E. coli L197 mutant dimer (light blue), MRSA wild-type dimer (green). Only Wild-type E. Coli interactions (dashed lines) are shown for clarity. (B) Distance between T44-Oγ and Y133-Oç. (C) Distance between T44-Oγ and Y107-Oç. (D) Distance between K161-Nζ and Y133-Oç. Equivalent MRSA residues are T46, Y135, Y109 and K163.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3369909&req=5

pcbi-1002537-g007: Network of essential active site interactions over the course of the simulations.(A) Electrostatic interactions and residues considered: Wild-type E. coli tetramer (dark blue), E. coli L197 mutant dimer (light blue), MRSA wild-type dimer (green). Only Wild-type E. Coli interactions (dashed lines) are shown for clarity. (B) Distance between T44-Oγ and Y133-Oç. (C) Distance between T44-Oγ and Y107-Oç. (D) Distance between K161-Nζ and Y133-Oç. Equivalent MRSA residues are T46, Y135, Y109 and K163.
Mentions: To gain more insight into the potential changes occurring in the active sites we focused on the conserved network of hydrogen bonds present in the catalytic site (Figure 7A). This network is formed by 2 hydrogen bonds between the hydroxyl groups of T44 and Y133 (E. coli numbering), and between the hydroxyl groups of T44 and Y107. Point mutation of any of these 3 residues that constitute the catalytic triad results in severely reduced activity [3]. Distances between donor and acceptor atoms were monitored throughout simulations (Figure 7). We find that atoms T44-Oγ/Y133-Oç (Figure 7B) remain in reasonably close contact at a similar average distance of 5.4±1.3 Å and 5.6±1.3 Å in the E. coli and the MRSA dimers respectively. The hydrogen bond is only transiently formed regardless of the species and broken upon flipping of the T44 side chain. In contrast the distance between T44-Oγ/Y107-Oç shows a marked difference (Figure 7C) following the repositioning of Y107 in the E. coli dimer associated with monomer re-arrangement and shown here with a large increase. The relative positions of both side chains seem affected to a smaller extent by rotation in the MRSA dimer (average distance is 5.7±1.3 Å) with a small distance increase suggesting weak electrostatic interaction between the hydroxyl groups.

Bottom Line: DHDPS from E. coli is a homotetramer consisting of a 'dimer of dimers', with the catalytic residues found at the tight-dimer interface.Crystallographic and biophysical evidence suggest that the dimers associate to stabilise the active site configuration, and mutation of a central dimer-dimer interface residue destabilises the tetramer, thus increasing the flexibility and reducing catalytic efficiency and substrate specificity.These reveal a striking contrast between the dynamics of tetrameric and dimeric forms.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.

ABSTRACT
Dihydrodipicolinate synthase (DHDPS) is an essential enzyme involved in the lysine biosynthesis pathway. DHDPS from E. coli is a homotetramer consisting of a 'dimer of dimers', with the catalytic residues found at the tight-dimer interface. Crystallographic and biophysical evidence suggest that the dimers associate to stabilise the active site configuration, and mutation of a central dimer-dimer interface residue destabilises the tetramer, thus increasing the flexibility and reducing catalytic efficiency and substrate specificity. This has led to the hypothesis that the tetramer evolved to optimise the dynamics within the tight-dimer. In order to gain insights into DHDPS flexibility and its relationship to quaternary structure and function, we performed comparative Molecular Dynamics simulation studies of native tetrameric and dimeric forms of DHDPS from E. coli and also the native dimeric form from methicillin-resistant Staphylococcus aureus (MRSA). These reveal a striking contrast between the dynamics of tetrameric and dimeric forms. Whereas the E. coli DHDPS tetramer is relatively rigid, both the E. coli and MRSA DHDPS dimers display high flexibility, resulting in monomer reorientation within the dimer and increased flexibility at the tight-dimer interface. The mutant E. coli DHDPS dimer exhibits disorder within its active site with deformation of critical catalytic residues and removal of key hydrogen bonds that render it inactive, whereas the similarly flexible MRSA DHDPS dimer maintains its catalytic geometry and is thus fully functional. Our data support the hypothesis that in both bacterial species optimal activity is achieved by fine tuning protein dynamics in different ways: E. coli DHDPS buttresses together two dimers, whereas MRSA dampens the motion using an extended tight-dimer interface.

Show MeSH
Related in: MedlinePlus