X-ray structure of ornithine decarboxylase from Trypanosoma brucei: The native structure and the structure in complex with α difluoromethylornithine

Nick V. Grishin, Andrei L. Osterman, Harold B. Brooks, Margaret A. Phillips, Elizabeth J. Goldsmith

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Abstract

Ornithine decarboxylase (ODC) is a pyridoxal 5'-phosphate (PLP) dependent homodimeric enzyme. It is a recognized drug target against African sleeping sickness, caused by Trypanosoma brucei. One of the currently used drugs, α-difluoromethylornithine (DFMO), is a suicide inhibitor of ODC. The structure of the T. brucei ODC (TbODC) mutant K69A bound to DFMO has been determined by X-ray crystallography to 2.0 Å resolution. The protein crystallizes in the space group P21 (a = 66.8 Å, b = 154.5 Å, c = 77.1 Å, β = 90.58°), with two dimers per asymmetric unit. The initial phasing was done by molecular replacement with the mouse ODC structure. The structure of wild-type uncomplexed TbODC was also determined to 2.9 Å resolution by molecular replacement using the TbODC DFMO-bound structure as the search model. The N-terminal domain of ODC is a β/α-barrel, and the C-terminal domain of ODC is a modified Greek key β-barrel. In comparison to structurally related alanine racemase, the two domains are rotated 27°relative to each other. In addition, two of the β-strands in the C-terminal domain have exchanged positions in order to maintain the location of essential active site residues in the context of the domain rotation. In ODC, the contacts in the dimer interface are formed primarily by the C-terminal domains, which interact through six aromatic rings that form stacking interactions across the domain boundary. The PLP binding site is formed by the C-termini of β-strands and loops in the β/α-barrel. In the native structure Lys69 forms a Schiff base with PLP. In both structures, the phosphate of PLP is bound between the seventh and eighth strands forming interactions with Arg277 and a Gly loop (residues 235-237). The pyridine nitrogen of PLP interacts with Glu274. DFMO forms a Schiff base with PLP and is covalently attached to Cys360. It is bound at the dimer interface and the σ-carbon amino group of DFMO is positioned between Asp361 of one subunit and Asp332 of the other. In comparison to the wild-type uncomplexed structure, Cys-360 has rotated 145°toward the active site in the DFMO-bound structure. No domain, subunit rotations, or other significant structural changes are observed upon ligand binding. The structure offers insight into the enzyme mechanism by providing details of the enzyme/inhibitor binding site and allows for a detailed comparison between the enzymes from the host and parasite which will aid in selective inhibitor design.

Original languageEnglish (US)
Pages (from-to)15174-15184
Number of pages11
JournalBiochemistry
Volume38
Issue number46
DOIs
StatePublished - Nov 16 1999

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Eflornithine
Trypanosoma brucei brucei
Pyridoxal Phosphate
Ornithine Decarboxylase
X-Rays
X rays
Dimers
Schiff Bases
Catalytic Domain
Alanine Racemase
Enzymes
Binding Sites
African Trypanosomiasis
X ray crystallography
X Ray Crystallography
Enzyme Inhibitors
Pharmaceutical Preparations
Suicide
Parasites
Phosphates

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{a3797cbabfcc488f9dbe8ad7a036e3e4,
title = "X-ray structure of ornithine decarboxylase from Trypanosoma brucei: The native structure and the structure in complex with α difluoromethylornithine",
abstract = "Ornithine decarboxylase (ODC) is a pyridoxal 5'-phosphate (PLP) dependent homodimeric enzyme. It is a recognized drug target against African sleeping sickness, caused by Trypanosoma brucei. One of the currently used drugs, α-difluoromethylornithine (DFMO), is a suicide inhibitor of ODC. The structure of the T. brucei ODC (TbODC) mutant K69A bound to DFMO has been determined by X-ray crystallography to 2.0 {\AA} resolution. The protein crystallizes in the space group P21 (a = 66.8 {\AA}, b = 154.5 {\AA}, c = 77.1 {\AA}, β = 90.58°), with two dimers per asymmetric unit. The initial phasing was done by molecular replacement with the mouse ODC structure. The structure of wild-type uncomplexed TbODC was also determined to 2.9 {\AA} resolution by molecular replacement using the TbODC DFMO-bound structure as the search model. The N-terminal domain of ODC is a β/α-barrel, and the C-terminal domain of ODC is a modified Greek key β-barrel. In comparison to structurally related alanine racemase, the two domains are rotated 27°relative to each other. In addition, two of the β-strands in the C-terminal domain have exchanged positions in order to maintain the location of essential active site residues in the context of the domain rotation. In ODC, the contacts in the dimer interface are formed primarily by the C-terminal domains, which interact through six aromatic rings that form stacking interactions across the domain boundary. The PLP binding site is formed by the C-termini of β-strands and loops in the β/α-barrel. In the native structure Lys69 forms a Schiff base with PLP. In both structures, the phosphate of PLP is bound between the seventh and eighth strands forming interactions with Arg277 and a Gly loop (residues 235-237). The pyridine nitrogen of PLP interacts with Glu274. DFMO forms a Schiff base with PLP and is covalently attached to Cys360. It is bound at the dimer interface and the σ-carbon amino group of DFMO is positioned between Asp361 of one subunit and Asp332 of the other. In comparison to the wild-type uncomplexed structure, Cys-360 has rotated 145°toward the active site in the DFMO-bound structure. No domain, subunit rotations, or other significant structural changes are observed upon ligand binding. The structure offers insight into the enzyme mechanism by providing details of the enzyme/inhibitor binding site and allows for a detailed comparison between the enzymes from the host and parasite which will aid in selective inhibitor design.",
author = "Grishin, {Nick V.} and Osterman, {Andrei L.} and Brooks, {Harold B.} and Phillips, {Margaret A.} and Goldsmith, {Elizabeth J.}",
year = "1999",
month = "11",
day = "16",
doi = "10.1021/bi9915115",
language = "English (US)",
volume = "38",
pages = "15174--15184",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "46",

}

TY - JOUR

T1 - X-ray structure of ornithine decarboxylase from Trypanosoma brucei

T2 - The native structure and the structure in complex with α difluoromethylornithine

AU - Grishin, Nick V.

AU - Osterman, Andrei L.

AU - Brooks, Harold B.

AU - Phillips, Margaret A.

AU - Goldsmith, Elizabeth J.

PY - 1999/11/16

Y1 - 1999/11/16

N2 - Ornithine decarboxylase (ODC) is a pyridoxal 5'-phosphate (PLP) dependent homodimeric enzyme. It is a recognized drug target against African sleeping sickness, caused by Trypanosoma brucei. One of the currently used drugs, α-difluoromethylornithine (DFMO), is a suicide inhibitor of ODC. The structure of the T. brucei ODC (TbODC) mutant K69A bound to DFMO has been determined by X-ray crystallography to 2.0 Å resolution. The protein crystallizes in the space group P21 (a = 66.8 Å, b = 154.5 Å, c = 77.1 Å, β = 90.58°), with two dimers per asymmetric unit. The initial phasing was done by molecular replacement with the mouse ODC structure. The structure of wild-type uncomplexed TbODC was also determined to 2.9 Å resolution by molecular replacement using the TbODC DFMO-bound structure as the search model. The N-terminal domain of ODC is a β/α-barrel, and the C-terminal domain of ODC is a modified Greek key β-barrel. In comparison to structurally related alanine racemase, the two domains are rotated 27°relative to each other. In addition, two of the β-strands in the C-terminal domain have exchanged positions in order to maintain the location of essential active site residues in the context of the domain rotation. In ODC, the contacts in the dimer interface are formed primarily by the C-terminal domains, which interact through six aromatic rings that form stacking interactions across the domain boundary. The PLP binding site is formed by the C-termini of β-strands and loops in the β/α-barrel. In the native structure Lys69 forms a Schiff base with PLP. In both structures, the phosphate of PLP is bound between the seventh and eighth strands forming interactions with Arg277 and a Gly loop (residues 235-237). The pyridine nitrogen of PLP interacts with Glu274. DFMO forms a Schiff base with PLP and is covalently attached to Cys360. It is bound at the dimer interface and the σ-carbon amino group of DFMO is positioned between Asp361 of one subunit and Asp332 of the other. In comparison to the wild-type uncomplexed structure, Cys-360 has rotated 145°toward the active site in the DFMO-bound structure. No domain, subunit rotations, or other significant structural changes are observed upon ligand binding. The structure offers insight into the enzyme mechanism by providing details of the enzyme/inhibitor binding site and allows for a detailed comparison between the enzymes from the host and parasite which will aid in selective inhibitor design.

AB - Ornithine decarboxylase (ODC) is a pyridoxal 5'-phosphate (PLP) dependent homodimeric enzyme. It is a recognized drug target against African sleeping sickness, caused by Trypanosoma brucei. One of the currently used drugs, α-difluoromethylornithine (DFMO), is a suicide inhibitor of ODC. The structure of the T. brucei ODC (TbODC) mutant K69A bound to DFMO has been determined by X-ray crystallography to 2.0 Å resolution. The protein crystallizes in the space group P21 (a = 66.8 Å, b = 154.5 Å, c = 77.1 Å, β = 90.58°), with two dimers per asymmetric unit. The initial phasing was done by molecular replacement with the mouse ODC structure. The structure of wild-type uncomplexed TbODC was also determined to 2.9 Å resolution by molecular replacement using the TbODC DFMO-bound structure as the search model. The N-terminal domain of ODC is a β/α-barrel, and the C-terminal domain of ODC is a modified Greek key β-barrel. In comparison to structurally related alanine racemase, the two domains are rotated 27°relative to each other. In addition, two of the β-strands in the C-terminal domain have exchanged positions in order to maintain the location of essential active site residues in the context of the domain rotation. In ODC, the contacts in the dimer interface are formed primarily by the C-terminal domains, which interact through six aromatic rings that form stacking interactions across the domain boundary. The PLP binding site is formed by the C-termini of β-strands and loops in the β/α-barrel. In the native structure Lys69 forms a Schiff base with PLP. In both structures, the phosphate of PLP is bound between the seventh and eighth strands forming interactions with Arg277 and a Gly loop (residues 235-237). The pyridine nitrogen of PLP interacts with Glu274. DFMO forms a Schiff base with PLP and is covalently attached to Cys360. It is bound at the dimer interface and the σ-carbon amino group of DFMO is positioned between Asp361 of one subunit and Asp332 of the other. In comparison to the wild-type uncomplexed structure, Cys-360 has rotated 145°toward the active site in the DFMO-bound structure. No domain, subunit rotations, or other significant structural changes are observed upon ligand binding. The structure offers insight into the enzyme mechanism by providing details of the enzyme/inhibitor binding site and allows for a detailed comparison between the enzymes from the host and parasite which will aid in selective inhibitor design.

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