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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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Cartoon representations of DHDPS crystallographic structures.(A) Wild-type E. coli; (B) E. coli L197Y mutant dimer; (C) wild-type MRSA dimer. The arrows indicate locations of the active sites (1 per monomer) and tight-dimer interfaces; (D) Active site alignments of tetramer and dimers. Wild-type E. coli tetramer (dark blue), E. coli L197 mutant dimer (light blue), MRSA wild-type dimer (green).
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pcbi-1002537-g001: Cartoon representations of DHDPS crystallographic structures.(A) Wild-type E. coli; (B) E. coli L197Y mutant dimer; (C) wild-type MRSA dimer. The arrows indicate locations of the active sites (1 per monomer) and tight-dimer interfaces; (D) Active site alignments of tetramer and dimers. Wild-type E. coli tetramer (dark blue), E. coli L197 mutant dimer (light blue), MRSA wild-type dimer (green).

Mentions: Dihydrodipicolinate synthase (DHDPS) is an essential enzyme involved in the lysine biosynthesis pathway [1]. It is expressed in plants and microorganisms, but not in animals, which makes it a potential target for herbicides and antibiotics [2]. DHDPS from E. coli is a homotetramer consisting of a ‘dimer of dimers’ (Figure 1A). The catalytic residues T44, Y107 and Y133 are found at the tight-dimer interface (Figure 1D), with each tight-dimer containing two complete active sites within the barrel of the monomeric (β/α)8-fold and an allosteric site within a deep cleft between the subunits that binds two (S)-lysine molecules to mediate feedback inhibition [3]. A tyrosine residue (Y107) from one subunit of the tight-dimer protrudes into the active site of the adjacent subunit and forms part of a catalytic triad that is essential for activity [4], [5]. Although this suggests that the tight-dimer contains the minimum requirements for catalysis, mutation of a central residue in the dimer–dimer interface (L197) produced dimeric variants having severely reduced catalytic function (Figure 1B) [6], [7]. Crystallographic, biophysical and Small Angle X-ray Scattering (SAXS) evidence suggest that the dimers associate to stabilise the active site configuration, and removal of this central interface residue destabilises the dimer, thus increasing the flexibility and reducing both catalytic efficiency and substrate specificity. This has led to the hypothesis that the tetramer has evolved to optimise the dynamics within the tight-dimer unit [6].


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)

Cartoon representations of DHDPS crystallographic structures.(A) Wild-type E. coli; (B) E. coli L197Y mutant dimer; (C) wild-type MRSA dimer. The arrows indicate locations of the active sites (1 per monomer) and tight-dimer interfaces; (D) Active site alignments of tetramer and dimers. Wild-type E. coli tetramer (dark blue), E. coli L197 mutant dimer (light blue), MRSA wild-type dimer (green).
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1002537-g001: Cartoon representations of DHDPS crystallographic structures.(A) Wild-type E. coli; (B) E. coli L197Y mutant dimer; (C) wild-type MRSA dimer. The arrows indicate locations of the active sites (1 per monomer) and tight-dimer interfaces; (D) Active site alignments of tetramer and dimers. Wild-type E. coli tetramer (dark blue), E. coli L197 mutant dimer (light blue), MRSA wild-type dimer (green).
Mentions: Dihydrodipicolinate synthase (DHDPS) is an essential enzyme involved in the lysine biosynthesis pathway [1]. It is expressed in plants and microorganisms, but not in animals, which makes it a potential target for herbicides and antibiotics [2]. DHDPS from E. coli is a homotetramer consisting of a ‘dimer of dimers’ (Figure 1A). The catalytic residues T44, Y107 and Y133 are found at the tight-dimer interface (Figure 1D), with each tight-dimer containing two complete active sites within the barrel of the monomeric (β/α)8-fold and an allosteric site within a deep cleft between the subunits that binds two (S)-lysine molecules to mediate feedback inhibition [3]. A tyrosine residue (Y107) from one subunit of the tight-dimer protrudes into the active site of the adjacent subunit and forms part of a catalytic triad that is essential for activity [4], [5]. Although this suggests that the tight-dimer contains the minimum requirements for catalysis, mutation of a central residue in the dimer–dimer interface (L197) produced dimeric variants having severely reduced catalytic function (Figure 1B) [6], [7]. Crystallographic, biophysical and Small Angle X-ray Scattering (SAXS) evidence suggest that the dimers associate to stabilise the active site configuration, and removal of this central interface residue destabilises the dimer, thus increasing the flexibility and reducing both catalytic efficiency and substrate specificity. This has led to the hypothesis that the tetramer has evolved to optimise the dynamics within the tight-dimer unit [6].

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