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Structures of alternatively spliced isoforms of human ketohexokinase.

Trinh CH, Asipu A, Bonthron DT, Phillips SE - Acta Crystallogr. D Biol. Crystallogr. (2009)

Bottom Line: The structure of the KHK-A ternary complex revealed an active site with both the substrate fructose and the ATP analogue in positions ready for phosphorylation following a reaction mechanism similar to that of the pfkB family of carbohydrate kinases.Hepatic KHK deficiency causes the benign disorder essential fructosuria.The effects of the disease-causing mutations (Gly40Arg and Ala43Thr) have been modelled in the context of the KHK structure.

View Article: PubMed Central - HTML - PubMed

Affiliation: Astbury Centre for Structural Molecular Biology, Institute of Molecular and Cellular Biology, University of Leeds, Leeds, England.

ABSTRACT
A molecular understanding of the unique aspects of dietary fructose metabolism may be the key to understanding and controlling the current epidemic of fructose-related obesity, diabetes and related adverse metabolic states in Western populations. Fructose catabolism is initiated by its phosphorylation to fructose 1-phosphate, which is performed by ketohexokinase (KHK). Here, the crystal structures of the two alternatively spliced isoforms of human ketohexokinase, hepatic KHK-C and the peripheral isoform KHK-A, and of the ternary complex of KHK-A with the substrate fructose and AMP-PNP are reported. The structure of the KHK-A ternary complex revealed an active site with both the substrate fructose and the ATP analogue in positions ready for phosphorylation following a reaction mechanism similar to that of the pfkB family of carbohydrate kinases. Hepatic KHK deficiency causes the benign disorder essential fructosuria. The effects of the disease-causing mutations (Gly40Arg and Ala43Thr) have been modelled in the context of the KHK structure.

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Comparison between human KHK-A and E. coli ribokinase. Ribbon diagrams are shown of KHK-A, of KHK-C and of E. coli ribokinase in the presence (PDB code 1rkd) and absence (PDB code 1rka) of ribose and ADP. The green subunit is shown in the same orientation in all structures. The alternative splicing of the KHK gene results in a different sequence for a single region of the chain between the two isoforms (residues 72–115), which is shown in red. These figures were generated using PyMOL (DeLano, 2002 ▶).
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fig3: Comparison between human KHK-A and E. coli ribokinase. Ribbon diagrams are shown of KHK-A, of KHK-C and of E. coli ribokinase in the presence (PDB code 1rkd) and absence (PDB code 1rka) of ribose and ADP. The green subunit is shown in the same orientation in all structures. The alternative splicing of the KHK gene results in a different sequence for a single region of the chain between the two isoforms (residues 72–115), which is shown in red. These figures were generated using PyMOL (DeLano, 2002 ▶).

Mentions: Gel filtration and nondenaturing polyacrylamide gel electrophoresis (Bais et al., 1985 ▶; Raushel & Cleland, 1977 ▶) have demonstrated that KHK is a dimer in solution. In the crystal structure KHK-A is an elongated dimer with appropriate dimensions of 44 × 101 × 47 Å. The dimer interface is formed by the extended four-stranded β-sheets (β2, β3b, β6 and β7), which pack approximately orthogonally to form a distorted barrel (Fig. 3 ▶). The conformations of Cys32 and Leu33 disrupt β3, generating a sharp bend that separates it into two parts, β3a and β3b (Fig. 2 ▶). Alignment of various ketohexokinase sequences using ClustalW2 (Larkin et al., 2007 ▶) show that cysteine and leucine residues are highly conserved at these positions throughout evolution (data not shown). β3a interacts with the four-stranded β-sheet from the other subunit, adding a fifth strand to the sheet in an arrangement described in E. coli ribokinase as a β-clasp structure (Sigrell et al., 1998 ▶; Fig. 3 ▶). The KHK-A ternary-complex structure reveals the presence of one active site per subunit; it is located in a cleft between the α/β-domain and the four-stranded β-­sheet that forms the dimer interface such that the β-sheet forms a lid over the active site. The alternatively spliced region contains 44 residues that begin in the middle of α2 and extend through β5 of the central α/β-fold to β6 and β7 of the extended four-stranded β-sheet that forms the dimer interface (Fig. 3 ▶). Superposition of all Cα atoms for the KHK-A and the ternary-complex dimer structures gives a root-mean-square-deviation (r.m.s.d.) of 0.5 Å.


Structures of alternatively spliced isoforms of human ketohexokinase.

Trinh CH, Asipu A, Bonthron DT, Phillips SE - Acta Crystallogr. D Biol. Crystallogr. (2009)

Comparison between human KHK-A and E. coli ribokinase. Ribbon diagrams are shown of KHK-A, of KHK-C and of E. coli ribokinase in the presence (PDB code 1rkd) and absence (PDB code 1rka) of ribose and ADP. The green subunit is shown in the same orientation in all structures. The alternative splicing of the KHK gene results in a different sequence for a single region of the chain between the two isoforms (residues 72–115), which is shown in red. These figures were generated using PyMOL (DeLano, 2002 ▶).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651755&req=5

fig3: Comparison between human KHK-A and E. coli ribokinase. Ribbon diagrams are shown of KHK-A, of KHK-C and of E. coli ribokinase in the presence (PDB code 1rkd) and absence (PDB code 1rka) of ribose and ADP. The green subunit is shown in the same orientation in all structures. The alternative splicing of the KHK gene results in a different sequence for a single region of the chain between the two isoforms (residues 72–115), which is shown in red. These figures were generated using PyMOL (DeLano, 2002 ▶).
Mentions: Gel filtration and nondenaturing polyacrylamide gel electrophoresis (Bais et al., 1985 ▶; Raushel & Cleland, 1977 ▶) have demonstrated that KHK is a dimer in solution. In the crystal structure KHK-A is an elongated dimer with appropriate dimensions of 44 × 101 × 47 Å. The dimer interface is formed by the extended four-stranded β-sheets (β2, β3b, β6 and β7), which pack approximately orthogonally to form a distorted barrel (Fig. 3 ▶). The conformations of Cys32 and Leu33 disrupt β3, generating a sharp bend that separates it into two parts, β3a and β3b (Fig. 2 ▶). Alignment of various ketohexokinase sequences using ClustalW2 (Larkin et al., 2007 ▶) show that cysteine and leucine residues are highly conserved at these positions throughout evolution (data not shown). β3a interacts with the four-stranded β-sheet from the other subunit, adding a fifth strand to the sheet in an arrangement described in E. coli ribokinase as a β-clasp structure (Sigrell et al., 1998 ▶; Fig. 3 ▶). The KHK-A ternary-complex structure reveals the presence of one active site per subunit; it is located in a cleft between the α/β-domain and the four-stranded β-­sheet that forms the dimer interface such that the β-sheet forms a lid over the active site. The alternatively spliced region contains 44 residues that begin in the middle of α2 and extend through β5 of the central α/β-fold to β6 and β7 of the extended four-stranded β-sheet that forms the dimer interface (Fig. 3 ▶). Superposition of all Cα atoms for the KHK-A and the ternary-complex dimer structures gives a root-mean-square-deviation (r.m.s.d.) of 0.5 Å.

Bottom Line: The structure of the KHK-A ternary complex revealed an active site with both the substrate fructose and the ATP analogue in positions ready for phosphorylation following a reaction mechanism similar to that of the pfkB family of carbohydrate kinases.Hepatic KHK deficiency causes the benign disorder essential fructosuria.The effects of the disease-causing mutations (Gly40Arg and Ala43Thr) have been modelled in the context of the KHK structure.

View Article: PubMed Central - HTML - PubMed

Affiliation: Astbury Centre for Structural Molecular Biology, Institute of Molecular and Cellular Biology, University of Leeds, Leeds, England.

ABSTRACT
A molecular understanding of the unique aspects of dietary fructose metabolism may be the key to understanding and controlling the current epidemic of fructose-related obesity, diabetes and related adverse metabolic states in Western populations. Fructose catabolism is initiated by its phosphorylation to fructose 1-phosphate, which is performed by ketohexokinase (KHK). Here, the crystal structures of the two alternatively spliced isoforms of human ketohexokinase, hepatic KHK-C and the peripheral isoform KHK-A, and of the ternary complex of KHK-A with the substrate fructose and AMP-PNP are reported. The structure of the KHK-A ternary complex revealed an active site with both the substrate fructose and the ATP analogue in positions ready for phosphorylation following a reaction mechanism similar to that of the pfkB family of carbohydrate kinases. Hepatic KHK deficiency causes the benign disorder essential fructosuria. The effects of the disease-causing mutations (Gly40Arg and Ala43Thr) have been modelled in the context of the KHK structure.

Show MeSH
Related in: MedlinePlus