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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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Cavities in DHDPS active sites.(A) Wild-type E. coli DHDPS; (B) L197Y E. coli engineered dimer; (C) Wild-type MRSA DHDPS. Active site cavities are represented as mesh surfaces (yellow) for the last 100 ns of dim-A, tet-1 and mrsa-1.
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pcbi-1002537-g009: Cavities in DHDPS active sites.(A) Wild-type E. coli DHDPS; (B) L197Y E. coli engineered dimer; (C) Wild-type MRSA DHDPS. Active site cavities are represented as mesh surfaces (yellow) for the last 100 ns of dim-A, tet-1 and mrsa-1.

Mentions: The mutant dimer L197Y was crystallized in the absence of the substrate pyruvate, with a molecule of α-ketoglutarate trapped in its active site [6]. The latter was not added in the crystallization conditions but rather captured from the expression system. The repositioning of Y107 side chain observed in the L197Y E. coli DHDPS dimer is associated with an enlargement of the active site pocket (Figure 9A and 9B). We propose that the widening of the pocket in the mutant dimer is responsible for allowing the substrate analogue α-ketoglutarate, which is larger than the natural substrate pyruvate, to bind K161 and form a Schiff base before cyclisation, as observed in the crystalline state [6]. This newly formed covalent species acts as a stable inhibitory adducts towards pyruvate, thus explaining the loss of specificity and affinity measured [6]. Following this hypothesis originally formulated by Griffin et al. (2008) [6], in MRSA DHDPS the relatively stable positions of all active site residues would prohibit binding and perhaps entry of α-ketoglutarate in the active site. This is reflected by similar affinity for pyruvate and enzymatic activity in both MRSA and wild-type E. coli DHDPS [8].


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)

Cavities in DHDPS active sites.(A) Wild-type E. coli DHDPS; (B) L197Y E. coli engineered dimer; (C) Wild-type MRSA DHDPS. Active site cavities are represented as mesh surfaces (yellow) for the last 100 ns of dim-A, tet-1 and mrsa-1.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1002537-g009: Cavities in DHDPS active sites.(A) Wild-type E. coli DHDPS; (B) L197Y E. coli engineered dimer; (C) Wild-type MRSA DHDPS. Active site cavities are represented as mesh surfaces (yellow) for the last 100 ns of dim-A, tet-1 and mrsa-1.
Mentions: The mutant dimer L197Y was crystallized in the absence of the substrate pyruvate, with a molecule of α-ketoglutarate trapped in its active site [6]. The latter was not added in the crystallization conditions but rather captured from the expression system. The repositioning of Y107 side chain observed in the L197Y E. coli DHDPS dimer is associated with an enlargement of the active site pocket (Figure 9A and 9B). We propose that the widening of the pocket in the mutant dimer is responsible for allowing the substrate analogue α-ketoglutarate, which is larger than the natural substrate pyruvate, to bind K161 and form a Schiff base before cyclisation, as observed in the crystalline state [6]. This newly formed covalent species acts as a stable inhibitory adducts towards pyruvate, thus explaining the loss of specificity and affinity measured [6]. Following this hypothesis originally formulated by Griffin et al. (2008) [6], in MRSA DHDPS the relatively stable positions of all active site residues would prohibit binding and perhaps entry of α-ketoglutarate in the active site. This is reflected by similar affinity for pyruvate and enzymatic activity in both MRSA and wild-type E. coli DHDPS [8].

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