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Structure, substrate recognition and reactivity of Leishmania major mevalonate kinase.

Sgraja T, Smith TK, Hunter WN - BMC Struct. Biol. (2007)

Bottom Line: The activity of LmMK was significantly reduced compared to MK from other species and we were unable to obtain ATP-binding data.The mevalonate-binding site is highly conserved yet the ATP-binding site is structurally distinct in LmMK.We are unable to provide a definitive explanation for the low activity of recombinant protein isolated from a bacterial expression system compared to material isolated from procyclic-form Trypanosoma brucei.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee, UK. tanja.sgraja@web.de <tanja.sgraja@web.de>

ABSTRACT

Background: Isoprenoid precursor synthesis via the mevalonate route in humans and pathogenic trypanosomatids is an important metabolic pathway. There is however, only limited information available on the structure and reactivity of the component enzymes in trypanosomatids. Since isoprenoid biosynthesis is essential for trypanosomatid viability and may provide new targets for therapeutic intervention it is important to characterize the pathway components.

Results: Putative mevalonate kinase encoding genes from Leishmania major (LmMK) and Trypanosoma brucei (TbMK) have been cloned, over-expressed in and proteins isolated from procyclic-form T. brucei. A highly sensitive radioactive assay was developed and shows ATP-dependent phosphorylation of mevalonate. Apo and (R)-mevalonate bound crystal structures of LmMK, from a bacterial expression system, have been determined to high resolution providing, for the first time, information concerning binding of mevalonate to an MK. The mevalonate binds in a deep cavity lined by highly conserved residues. His25 is key for binding and for discrimination of (R)- over (S)-mevalonate, with the main chain amide interacting with the C3 hydroxyl group of (R)-mevalonate, and the side chain contributing, together with Val202 and Thr283, to the construction of a hydrophobic binding site for the C3 methyl substituent. The C5 hydroxyl, where phosphorylation occurs, points towards catalytic residues, Lys18 and Asp155. The activity of LmMK was significantly reduced compared to MK from other species and we were unable to obtain ATP-binding data. Comparisons with the rat MK:ATP complex were used to investigate how this substrate might bind. In LmMK, helix alpha2 and the preceding polypeptide adopt a conformation, not seen in related kinase structures, impeding access to the nucleotide triphosphate binding site suggesting that a conformational rearrangement is required to allow ATP binding.

Conclusion: Our new structural information, consistent with data on homologous enzymes allows a detailed description of how mevalonate is recognized and positioned for catalysis in MK. The mevalonate-binding site is highly conserved yet the ATP-binding site is structurally distinct in LmMK. We are unable to provide a definitive explanation for the low activity of recombinant protein isolated from a bacterial expression system compared to material isolated from procyclic-form Trypanosoma brucei.

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The substrate-binding site of LmMK. Molecules are depicted in stick representation, all N positions are blue, O red. C atoms are grey except for (R)-MVA and the catalytic residues where they are green and cyan respectively. For the purpose of clarity only selected water molecules (red spheres) and hydrogen bonding interactions (red dashed lines) are shown. An omit difference density map (blue chicken wire) covering the substrate and the water molecule adjacent to the carboxylate group is shown. The map was calculated with coefficients /Fo-Fc/, αcalc and contoured at 1.5 σ. Fo and Fc represent observed and calculated structure factor amplitudes respectively, αcalc phases calculated on the basis of atomic coordinates of the model but not including the substrate or water oxygen.
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Figure 7: The substrate-binding site of LmMK. Molecules are depicted in stick representation, all N positions are blue, O red. C atoms are grey except for (R)-MVA and the catalytic residues where they are green and cyan respectively. For the purpose of clarity only selected water molecules (red spheres) and hydrogen bonding interactions (red dashed lines) are shown. An omit difference density map (blue chicken wire) covering the substrate and the water molecule adjacent to the carboxylate group is shown. The map was calculated with coefficients /Fo-Fc/, αcalc and contoured at 1.5 σ. Fo and Fc represent observed and calculated structure factor amplitudes respectively, αcalc phases calculated on the basis of atomic coordinates of the model but not including the substrate or water oxygen.

Mentions: (R)-MVA, binds in the deep cavity formed between the N-terminal and C-terminal domains, surrounded by residues on α1, the N-terminal end of α6, the C-terminal end of β1 and β5 and the α4–α5 loop (Figures 3, 4, 5, 6). The substrate carboxylate interacts with the guanidinium of Arg169, which is held in place by a hydrogen bond with the hydroxyl of Tyr167. This tyrosine also forms a hydrogen bond with the substrate carboxylate and is conserved both in terms of sequence and position in RnMK (Tyr216). Sequence comparisons of MK from other species (not shown) reveal that Arg169, situated on a flexible loop between β5 and β6 is conserved in some species, e.g. in TbMK and TcMK. In other sequences this loop carries asparagine, glutamine, histidine and lysine residues, which would contribute a similar role in substrate binding. The carboxylate of (R)-MVA is also linked to the side chain of Ser152 via a water molecule. The side chains of Ile20, His25, Val27, Val28 and Val202 in LmMK place (R)-MVA in the cavity by forming hydrophobic interactions with the substrate. The C3 hydroxyl group forms an intramolecular hydrogen bond to the carboxylate, which serves to stabilize the conformation of the substrate itself. This hydroxyl group accepts a hydrogen bond donated by the main chain amide of His25, a component of motif 1. The C3 methyl group is directed into a hydrophobic environment formed by the Cα of Thr283, the side chain of His25, Val28 and Val202. These interactions with the C3 substituents serve to discriminate for (R)-MVA over (S)-MVA. The C5 hydroxyl, the site of phosphorylation, is directed towards Lys18 NZ at a distance of approximately 4 Å, in addition a water molecule provides a bridge over to Asp155 and Thr198 (Figure 7).


Structure, substrate recognition and reactivity of Leishmania major mevalonate kinase.

Sgraja T, Smith TK, Hunter WN - BMC Struct. Biol. (2007)

The substrate-binding site of LmMK. Molecules are depicted in stick representation, all N positions are blue, O red. C atoms are grey except for (R)-MVA and the catalytic residues where they are green and cyan respectively. For the purpose of clarity only selected water molecules (red spheres) and hydrogen bonding interactions (red dashed lines) are shown. An omit difference density map (blue chicken wire) covering the substrate and the water molecule adjacent to the carboxylate group is shown. The map was calculated with coefficients /Fo-Fc/, αcalc and contoured at 1.5 σ. Fo and Fc represent observed and calculated structure factor amplitudes respectively, αcalc phases calculated on the basis of atomic coordinates of the model but not including the substrate or water oxygen.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851959&req=5

Figure 7: The substrate-binding site of LmMK. Molecules are depicted in stick representation, all N positions are blue, O red. C atoms are grey except for (R)-MVA and the catalytic residues where they are green and cyan respectively. For the purpose of clarity only selected water molecules (red spheres) and hydrogen bonding interactions (red dashed lines) are shown. An omit difference density map (blue chicken wire) covering the substrate and the water molecule adjacent to the carboxylate group is shown. The map was calculated with coefficients /Fo-Fc/, αcalc and contoured at 1.5 σ. Fo and Fc represent observed and calculated structure factor amplitudes respectively, αcalc phases calculated on the basis of atomic coordinates of the model but not including the substrate or water oxygen.
Mentions: (R)-MVA, binds in the deep cavity formed between the N-terminal and C-terminal domains, surrounded by residues on α1, the N-terminal end of α6, the C-terminal end of β1 and β5 and the α4–α5 loop (Figures 3, 4, 5, 6). The substrate carboxylate interacts with the guanidinium of Arg169, which is held in place by a hydrogen bond with the hydroxyl of Tyr167. This tyrosine also forms a hydrogen bond with the substrate carboxylate and is conserved both in terms of sequence and position in RnMK (Tyr216). Sequence comparisons of MK from other species (not shown) reveal that Arg169, situated on a flexible loop between β5 and β6 is conserved in some species, e.g. in TbMK and TcMK. In other sequences this loop carries asparagine, glutamine, histidine and lysine residues, which would contribute a similar role in substrate binding. The carboxylate of (R)-MVA is also linked to the side chain of Ser152 via a water molecule. The side chains of Ile20, His25, Val27, Val28 and Val202 in LmMK place (R)-MVA in the cavity by forming hydrophobic interactions with the substrate. The C3 hydroxyl group forms an intramolecular hydrogen bond to the carboxylate, which serves to stabilize the conformation of the substrate itself. This hydroxyl group accepts a hydrogen bond donated by the main chain amide of His25, a component of motif 1. The C3 methyl group is directed into a hydrophobic environment formed by the Cα of Thr283, the side chain of His25, Val28 and Val202. These interactions with the C3 substituents serve to discriminate for (R)-MVA over (S)-MVA. The C5 hydroxyl, the site of phosphorylation, is directed towards Lys18 NZ at a distance of approximately 4 Å, in addition a water molecule provides a bridge over to Asp155 and Thr198 (Figure 7).

Bottom Line: The activity of LmMK was significantly reduced compared to MK from other species and we were unable to obtain ATP-binding data.The mevalonate-binding site is highly conserved yet the ATP-binding site is structurally distinct in LmMK.We are unable to provide a definitive explanation for the low activity of recombinant protein isolated from a bacterial expression system compared to material isolated from procyclic-form Trypanosoma brucei.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee, UK. tanja.sgraja@web.de <tanja.sgraja@web.de>

ABSTRACT

Background: Isoprenoid precursor synthesis via the mevalonate route in humans and pathogenic trypanosomatids is an important metabolic pathway. There is however, only limited information available on the structure and reactivity of the component enzymes in trypanosomatids. Since isoprenoid biosynthesis is essential for trypanosomatid viability and may provide new targets for therapeutic intervention it is important to characterize the pathway components.

Results: Putative mevalonate kinase encoding genes from Leishmania major (LmMK) and Trypanosoma brucei (TbMK) have been cloned, over-expressed in and proteins isolated from procyclic-form T. brucei. A highly sensitive radioactive assay was developed and shows ATP-dependent phosphorylation of mevalonate. Apo and (R)-mevalonate bound crystal structures of LmMK, from a bacterial expression system, have been determined to high resolution providing, for the first time, information concerning binding of mevalonate to an MK. The mevalonate binds in a deep cavity lined by highly conserved residues. His25 is key for binding and for discrimination of (R)- over (S)-mevalonate, with the main chain amide interacting with the C3 hydroxyl group of (R)-mevalonate, and the side chain contributing, together with Val202 and Thr283, to the construction of a hydrophobic binding site for the C3 methyl substituent. The C5 hydroxyl, where phosphorylation occurs, points towards catalytic residues, Lys18 and Asp155. The activity of LmMK was significantly reduced compared to MK from other species and we were unable to obtain ATP-binding data. Comparisons with the rat MK:ATP complex were used to investigate how this substrate might bind. In LmMK, helix alpha2 and the preceding polypeptide adopt a conformation, not seen in related kinase structures, impeding access to the nucleotide triphosphate binding site suggesting that a conformational rearrangement is required to allow ATP binding.

Conclusion: Our new structural information, consistent with data on homologous enzymes allows a detailed description of how mevalonate is recognized and positioned for catalysis in MK. The mevalonate-binding site is highly conserved yet the ATP-binding site is structurally distinct in LmMK. We are unable to provide a definitive explanation for the low activity of recombinant protein isolated from a bacterial expression system compared to material isolated from procyclic-form Trypanosoma brucei.

Show MeSH