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Structures of Saccharomyces cerevisiae D-arabinose dehydrogenase Ara1 and its complex with NADPH: implications for cofactor-assisted substrate recognition.

Hu XQ, Guo PC, Ma JD, Li WF - Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. (2013)

Bottom Line: The primary role of yeast Ara1, previously mis-annotated as a D-arabinose dehydrogenase, is to catalyze the reduction of a variety of toxic α,β-dicarbonyl compounds using NADPH as a cofactor at physiological pH levels.Ara1 exists as a homodimer, each subunit of which adopts an (α/β)8-barrel structure and has a highly conserved cofactor-binding pocket.Structural comparison revealed that induced fit upon NADPH binding yielded an intact active-site pocket that recognizes the substrate.

View Article: PubMed Central - HTML - PubMed

Affiliation: College of Life and Environment Science, Huangshan University, Huangshan, Anhui 245041, People's Republic of China.

ABSTRACT
The primary role of yeast Ara1, previously mis-annotated as a D-arabinose dehydrogenase, is to catalyze the reduction of a variety of toxic α,β-dicarbonyl compounds using NADPH as a cofactor at physiological pH levels. Here, crystal structures of Ara1 in apo and NADPH-complexed forms are presented at 2.10 and 2.00 Å resolution, respectively. Ara1 exists as a homodimer, each subunit of which adopts an (α/β)8-barrel structure and has a highly conserved cofactor-binding pocket. Structural comparison revealed that induced fit upon NADPH binding yielded an intact active-site pocket that recognizes the substrate. Moreover, the crystal structures combined with computational simulation defined an open substrate-binding site to accommodate various substrates that possess a dicarbonyl group.

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NADPH-binding site. (a) Interactions between NADPH and Ara1. (b) Induced fit upon NADPH binding. Ara1 is shown in cyan and the Ara1–NADPH complex is shown in orange. NADPH is shown in green lines and the interacting residues are shown as sticks.
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fig2: NADPH-binding site. (a) Interactions between NADPH and Ara1. (b) Induced fit upon NADPH binding. Ara1 is shown in cyan and the Ara1–NADPH complex is shown in orange. NADPH is shown in green lines and the interacting residues are shown as sticks.

Mentions: The structure of the Ara1–NADPH complex showed that a molecule of NADPH binds at the carboxyl edge of the β-strands of the barrel in an extended conformation (Figs. 1 ▶c and 1 ▶d). In detail, the adenine ring of NADPH is stabilized by the main chains of Ala249 and Ser250 and the side chains of Ala248, Leu251, Asn268 and Arg291 via van der Waals interactions. The phosphate group of the adenosine ribose is fixed by Ser286 Oγ, Leu287 N and Arg291 Nη1 and the hydroxyl group of the adenosine ribose is stabilized by Arg285 Nη1 through hydrogen bonds. The pyrophosphate is threaded through a short tunnel, with one side occupied by Ser241–His246. The other side is lined with Ile283, Pro284 and Arg285. The pyrophos­phate group forms two hydrogen bonds to Ser241 N and Oγ. One hydroxyl group of the nicotinamide ribose makes a hydrogen bond to Ala41 N. The nicotinamide moiety accommodates a wider cavity and forms hydrogen bonds to Gln214 O∊1 and Ser192 Oγ (Fig. 2 ▶a). Superposition of apo-form Ara1 on the the Ara1–NADPH complex yields an r.m.s.d. of 0.25 Å over 297 Cα atoms. The major conformational change results from the induced fit upon NADPH binding. In order to accommodate the cofactor, loop B (Tyr240–Pro249) and a short segment (Ile283–Arg291) near the C-terminal end shift towards each other and lead to a narrower cleft (Fig. 2 ▶b). For example, Leu243, Ser245, His246 and Ala248 shift by 1.8, 1.0, 1.3 and 1.0 Å, respectively, whereas the phenolic ring of Tyr240 and the hydroxyl group of Ser241 shift by about 0.7 and 0.9 Å, respectively, leaving space for the NADPH nicotinamide moiety. On the other side, Arg285, Ser286 and Leu287 also shift by about 1.1, 0.7 and 0.3 Å, respectively, to stabilize the adenosine ribose of NADPH (Fig. 2 ▶b).


Structures of Saccharomyces cerevisiae D-arabinose dehydrogenase Ara1 and its complex with NADPH: implications for cofactor-assisted substrate recognition.

Hu XQ, Guo PC, Ma JD, Li WF - Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. (2013)

NADPH-binding site. (a) Interactions between NADPH and Ara1. (b) Induced fit upon NADPH binding. Ara1 is shown in cyan and the Ara1–NADPH complex is shown in orange. NADPH is shown in green lines and the interacting residues are shown as sticks.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3818031&req=5

fig2: NADPH-binding site. (a) Interactions between NADPH and Ara1. (b) Induced fit upon NADPH binding. Ara1 is shown in cyan and the Ara1–NADPH complex is shown in orange. NADPH is shown in green lines and the interacting residues are shown as sticks.
Mentions: The structure of the Ara1–NADPH complex showed that a molecule of NADPH binds at the carboxyl edge of the β-strands of the barrel in an extended conformation (Figs. 1 ▶c and 1 ▶d). In detail, the adenine ring of NADPH is stabilized by the main chains of Ala249 and Ser250 and the side chains of Ala248, Leu251, Asn268 and Arg291 via van der Waals interactions. The phosphate group of the adenosine ribose is fixed by Ser286 Oγ, Leu287 N and Arg291 Nη1 and the hydroxyl group of the adenosine ribose is stabilized by Arg285 Nη1 through hydrogen bonds. The pyrophosphate is threaded through a short tunnel, with one side occupied by Ser241–His246. The other side is lined with Ile283, Pro284 and Arg285. The pyrophos­phate group forms two hydrogen bonds to Ser241 N and Oγ. One hydroxyl group of the nicotinamide ribose makes a hydrogen bond to Ala41 N. The nicotinamide moiety accommodates a wider cavity and forms hydrogen bonds to Gln214 O∊1 and Ser192 Oγ (Fig. 2 ▶a). Superposition of apo-form Ara1 on the the Ara1–NADPH complex yields an r.m.s.d. of 0.25 Å over 297 Cα atoms. The major conformational change results from the induced fit upon NADPH binding. In order to accommodate the cofactor, loop B (Tyr240–Pro249) and a short segment (Ile283–Arg291) near the C-terminal end shift towards each other and lead to a narrower cleft (Fig. 2 ▶b). For example, Leu243, Ser245, His246 and Ala248 shift by 1.8, 1.0, 1.3 and 1.0 Å, respectively, whereas the phenolic ring of Tyr240 and the hydroxyl group of Ser241 shift by about 0.7 and 0.9 Å, respectively, leaving space for the NADPH nicotinamide moiety. On the other side, Arg285, Ser286 and Leu287 also shift by about 1.1, 0.7 and 0.3 Å, respectively, to stabilize the adenosine ribose of NADPH (Fig. 2 ▶b).

Bottom Line: The primary role of yeast Ara1, previously mis-annotated as a D-arabinose dehydrogenase, is to catalyze the reduction of a variety of toxic α,β-dicarbonyl compounds using NADPH as a cofactor at physiological pH levels.Ara1 exists as a homodimer, each subunit of which adopts an (α/β)8-barrel structure and has a highly conserved cofactor-binding pocket.Structural comparison revealed that induced fit upon NADPH binding yielded an intact active-site pocket that recognizes the substrate.

View Article: PubMed Central - HTML - PubMed

Affiliation: College of Life and Environment Science, Huangshan University, Huangshan, Anhui 245041, People's Republic of China.

ABSTRACT
The primary role of yeast Ara1, previously mis-annotated as a D-arabinose dehydrogenase, is to catalyze the reduction of a variety of toxic α,β-dicarbonyl compounds using NADPH as a cofactor at physiological pH levels. Here, crystal structures of Ara1 in apo and NADPH-complexed forms are presented at 2.10 and 2.00 Å resolution, respectively. Ara1 exists as a homodimer, each subunit of which adopts an (α/β)8-barrel structure and has a highly conserved cofactor-binding pocket. Structural comparison revealed that induced fit upon NADPH binding yielded an intact active-site pocket that recognizes the substrate. Moreover, the crystal structures combined with computational simulation defined an open substrate-binding site to accommodate various substrates that possess a dicarbonyl group.

Show MeSH
Related in: MedlinePlus