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. 2001 Jun;10(6):1137-49.
doi: 10.1110/ps.52501.

Biochemical and X-ray crystallographic studies on shikimate kinase: the important structural role of the P-loop lysine

T Krell  1 , J Maclean, D J Boam, A Cooper, M Resmini, K Brocklehurst, S M Kelly, N C Price, A J Lapthorn, J R Coggins
Affiliations

Biochemical and X-ray crystallographic studies on shikimate kinase: the important structural role of the P-loop lysine

T Krell et al. Protein Sci. 2001 Jun.

Abstract

Shikimate kinase, despite low sequence identity, has been shown to be structurally a member of the nucleoside monophosphate (NMP) kinase family, which includes adenylate kinase. In this paper we have explored the roles of residues in the P-loop of shikimate kinase, which forms the binding site for nucleotides and is one of the most conserved structural features in proteins. In common with many members of the P-loop family, shikimate kinase contains a cysteine residue 2 amino acids upstream of the essential lysine residue; the side chains of these residues are shown to form an ion pair. The C13S mutant of shikimate kinase was found to be enzymatically active, whereas the K15M mutant was inactive. However, the latter mutant had both increased thermostability and affinity for ATP when compared to the wild-type enzyme. The structure of the K15M mutant protein has been determined at 1.8 A, and shows that the organization of the P-loop and flanking regions is heavily disturbed. This indicates that, besides its role in catalysis, the P-loop lysine also has an important structural role. The structure of the K15M mutant also reveals that the formation of an additional arginine/aspartate ion pair is the most likely reason for its increased thermostability. From studies of ligand binding it appears that, like adenylate kinase, shikimate kinase binds substrates randomly and in a synergistic fashion, indicating that the two enzymes have similar catalytic mechanisms.

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Figures

Fig. 1.
Fig. 1.
pH-dependence of the second-order rate constant (k) for the reactions at 25°C and I 0.1 M in aqueous buffers containing 1 mM EDTA and 2% (v/v) glycerol of 2PDS with (a) wild-type SK, (b) the C162S mutant, and (c) the K15M mutant. The points are each the mean of 3 determinations (SD ± ≤10% the mean). The continuous lines are theoretical for pH-dependent rate equations in a for 1 reactive protonic state and in b and c for 2 reactive protonic states and the following values of the characterizing parameters (pKa values and pH-independent rate constants, k̃, with values of the latter in M−1 sec−1): (a) pKI = 2.45, pKII = 3.9, k̃= 110; (b) pKI = 3.1, pKII = 3.2, pKIII = 3.9, k̃1 = 250, k̃2 = 10; (c) pKI = 3.0, pKII = 3.0, pKIII = 3.9, k̃1 = 240, k̃2 = 50. In b and c the broken lines are theoretical for the components of the pH-dependent rate equation corresponding to terms in k̃1 and k̃2. In a the broken line is coincident with the continuous line.
Fig. 2.
Fig. 2.
Raw DSC data illustrating the heat uptake associated with thermal unfolding of shikimate kinase (1 mg/mL) in the presence and absence of substrate molecules (shikimate, ADP, and ATP were at 2 mM). The increase in Tm in the presence of each substrate is consistent with specific ligand binding to the native state of the enzyme.
Fig. 3.
Fig. 3.
A stereo view of a representative section of the final weighted 2F0–Fc map of the K15M mutant of shikimate kinase contoured at 1 and 2 σ above the mean electron density. The region shown corresponds to amino acid residues 9–17 of the P-loop. The bound phosphate ion, water molecules (red crosses), and a chloride ion (green cross) are also shown.
Fig. 4.
Fig. 4.
The three-dimensional structure of shikimate kinase mutant K15M. (a) A ribbon representation of the enzyme monomer colored according to secondary structure, with the position of residues mutated in this study shown in ball and stick. (b) Ribbon representation of the two independent molecules in the asymmetric unit. The extensive contacts of neighboring lid domains lead to a stabilization of this part of the molecule that is not visible in the native crystal structure. Both diagrams were generated using RIBBONS (Carson 1991).
Fig. 4.
Fig. 4.
The three-dimensional structure of shikimate kinase mutant K15M. (a) A ribbon representation of the enzyme monomer colored according to secondary structure, with the position of residues mutated in this study shown in ball and stick. (b) Ribbon representation of the two independent molecules in the asymmetric unit. The extensive contacts of neighboring lid domains lead to a stabilization of this part of the molecule that is not visible in the native crystal structure. Both diagrams were generated using RIBBONS (Carson 1991).
Fig. 5.
Fig. 5.
Superimposition of wild-type and K15M shikimate kinase after aligning the central sheet of 5 parallel β-strands using the program LSQKAB from the CCP4 suite. (a) A stereo view of the Cα superposition of the native SK structure (blue) and the K15M mutant (green). The ADP (colored according to atom type) and phosphate ion (cyan) of the two respective structures are shown. Key hinge regions in the shikimate-binding domain and lid domain of SK are highlighted with a magenta point and labeled. The key points of movement in the P-loop caused by the K15M mutation are highlighted with a red point and labeled. (b) Differences in Cα positions as a function of the residue number. Residues 113–122 are missing from the wild-type structure, and residues 1, 2, and 171–173 are missing from the mutant structure, owing to poor or ambiguous electron density. (c) View of the P-loop area of wild-type (in green) and K15M (in purple) shikimate kinase generated using SETOR (Evans 1993). The wild-type enzyme contains ADP and Mg2+ (blue sphere); the mutant enzyme contains a bound phosphate ion. K15 and M15 are shown in blue and orange, respectively. Significant hydrogen bonds are shown as dotted lines.
Fig. 5.
Fig. 5.
Superimposition of wild-type and K15M shikimate kinase after aligning the central sheet of 5 parallel β-strands using the program LSQKAB from the CCP4 suite. (a) A stereo view of the Cα superposition of the native SK structure (blue) and the K15M mutant (green). The ADP (colored according to atom type) and phosphate ion (cyan) of the two respective structures are shown. Key hinge regions in the shikimate-binding domain and lid domain of SK are highlighted with a magenta point and labeled. The key points of movement in the P-loop caused by the K15M mutation are highlighted with a red point and labeled. (b) Differences in Cα positions as a function of the residue number. Residues 113–122 are missing from the wild-type structure, and residues 1, 2, and 171–173 are missing from the mutant structure, owing to poor or ambiguous electron density. (c) View of the P-loop area of wild-type (in green) and K15M (in purple) shikimate kinase generated using SETOR (Evans 1993). The wild-type enzyme contains ADP and Mg2+ (blue sphere); the mutant enzyme contains a bound phosphate ion. K15 and M15 are shown in blue and orange, respectively. Significant hydrogen bonds are shown as dotted lines.
Fig. 5.
Fig. 5.
Superimposition of wild-type and K15M shikimate kinase after aligning the central sheet of 5 parallel β-strands using the program LSQKAB from the CCP4 suite. (a) A stereo view of the Cα superposition of the native SK structure (blue) and the K15M mutant (green). The ADP (colored according to atom type) and phosphate ion (cyan) of the two respective structures are shown. Key hinge regions in the shikimate-binding domain and lid domain of SK are highlighted with a magenta point and labeled. The key points of movement in the P-loop caused by the K15M mutation are highlighted with a red point and labeled. (b) Differences in Cα positions as a function of the residue number. Residues 113–122 are missing from the wild-type structure, and residues 1, 2, and 171–173 are missing from the mutant structure, owing to poor or ambiguous electron density. (c) View of the P-loop area of wild-type (in green) and K15M (in purple) shikimate kinase generated using SETOR (Evans 1993). The wild-type enzyme contains ADP and Mg2+ (blue sphere); the mutant enzyme contains a bound phosphate ion. K15 and M15 are shown in blue and orange, respectively. Significant hydrogen bonds are shown as dotted lines.
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