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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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Cα-trace overlay for part of the N-terminal domains of the LmMK and RnMK structures. The Cα trace and labels for LmMK are gray, and for RnMK red. The substrate, (R)-MVA is from the LmMK structure and shown as sticks colored green for C, red for O. The ATP (also in stick-mode, colored C yellow, N blue, O red, P purple) is from the RnMK structure. Selected elements of secondary structure are labeled and colored according to the structure, grey LmMK, red RnMK.
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Figure 6: Cα-trace overlay for part of the N-terminal domains of the LmMK and RnMK structures. The Cα trace and labels for LmMK are gray, and for RnMK red. The substrate, (R)-MVA is from the LmMK structure and shown as sticks colored green for C, red for O. The ATP (also in stick-mode, colored C yellow, N blue, O red, P purple) is from the RnMK structure. Selected elements of secondary structure are labeled and colored according to the structure, grey LmMK, red RnMK.

Mentions: A most striking difference in the arrangement of secondary structure elements in LmMK compared to other GHMP kinases occurs in the N-terminal domain adjacent to the ATP binding site. This is best illustrated by the overlay of LmMK and RnMK, part of which is shown in Figure 6. The first four elements of LmMK secondary structure (assigned as β1, α1, β2, β3) align well on the corresponding structural features of RnMK (assigned by Fu et al., [9] as β1 and β2, α1, β3 and β4, β5). The structures then diverge as the mammalian MK sequence carries an insert forming a β-strand (β6) anti-parallel to β5, then an extended helix-loop-helix structure of α2, a disordered flexible segment and α3. A tight turn, in the vicinity of where adenine binds, then leads to α4. In LmMK there is no strand equivalent to β6 (RnMK). Strand β3, which is equivalent to β5 in RnMK, is followed by a tight turn into α2. The helices α4 (RnMK) and α2 (LmMK) overlay well though in the parasite protein this helix is extended by two turns at the N-terminal end. The replacement of the insert and two helical segments in the rat enzyme with the short loop connecting β3-α2 in LmMK results in a polypeptide conformation, not observed in structures of GHMP kinases, that lies across and restricts access to the ATP binding cavity (Figure 6). The detailed conformation of the β3-α2 loop in LmMK may be influenced by contacts between symmetry related molecules. This loop is beside and forming hydrogen-bonding interactions with residues at the N-terminal end of β1, the C-terminal end of α3 and the α3–α4 loop of a symmetry-related molecule (not shown). Once into α2/α4 the LmMK and RnMK structures align well and then form a strand-loop-helix structure (motif 2, discussed earlier) that serves to create the base of the ATP-binding site (Figure 5). In LmMK this is β4-loop-α3, in RnMK β7-loop-α5.


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

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

Cα-trace overlay for part of the N-terminal domains of the LmMK and RnMK structures. The Cα trace and labels for LmMK are gray, and for RnMK red. The substrate, (R)-MVA is from the LmMK structure and shown as sticks colored green for C, red for O. The ATP (also in stick-mode, colored C yellow, N blue, O red, P purple) is from the RnMK structure. Selected elements of secondary structure are labeled and colored according to the structure, grey LmMK, red RnMK.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Cα-trace overlay for part of the N-terminal domains of the LmMK and RnMK structures. The Cα trace and labels for LmMK are gray, and for RnMK red. The substrate, (R)-MVA is from the LmMK structure and shown as sticks colored green for C, red for O. The ATP (also in stick-mode, colored C yellow, N blue, O red, P purple) is from the RnMK structure. Selected elements of secondary structure are labeled and colored according to the structure, grey LmMK, red RnMK.
Mentions: A most striking difference in the arrangement of secondary structure elements in LmMK compared to other GHMP kinases occurs in the N-terminal domain adjacent to the ATP binding site. This is best illustrated by the overlay of LmMK and RnMK, part of which is shown in Figure 6. The first four elements of LmMK secondary structure (assigned as β1, α1, β2, β3) align well on the corresponding structural features of RnMK (assigned by Fu et al., [9] as β1 and β2, α1, β3 and β4, β5). The structures then diverge as the mammalian MK sequence carries an insert forming a β-strand (β6) anti-parallel to β5, then an extended helix-loop-helix structure of α2, a disordered flexible segment and α3. A tight turn, in the vicinity of where adenine binds, then leads to α4. In LmMK there is no strand equivalent to β6 (RnMK). Strand β3, which is equivalent to β5 in RnMK, is followed by a tight turn into α2. The helices α4 (RnMK) and α2 (LmMK) overlay well though in the parasite protein this helix is extended by two turns at the N-terminal end. The replacement of the insert and two helical segments in the rat enzyme with the short loop connecting β3-α2 in LmMK results in a polypeptide conformation, not observed in structures of GHMP kinases, that lies across and restricts access to the ATP binding cavity (Figure 6). The detailed conformation of the β3-α2 loop in LmMK may be influenced by contacts between symmetry related molecules. This loop is beside and forming hydrogen-bonding interactions with residues at the N-terminal end of β1, the C-terminal end of α3 and the α3–α4 loop of a symmetry-related molecule (not shown). Once into α2/α4 the LmMK and RnMK structures align well and then form a strand-loop-helix structure (motif 2, discussed earlier) that serves to create the base of the ATP-binding site (Figure 5). In LmMK this is β4-loop-α3, in RnMK β7-loop-α5.

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