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Crystal Structures of a Hyperthermophilic Archaeal Homoserine Dehydrogenase Suggest a Novel Cofactor Binding Mode for Oxidoreductases.

Hayashi J, Inoue S, Kim K, Yoneda K, Kawarabayasi Y, Ohshima T, Sakuraba H - Sci Rep (2015)

Bottom Line: The structure of a newly identified archaeal homoserine dehydrogenase showed this enzyme to have a strong preference for NADP.However, NADP did not act as a cofactor with this enzyme, but as a strong inhibitor of NAD-dependent homoserine oxidation.This observation suggests this enzyme exhibits a new variation on cofactor binding to a dehydrogenase: very strong NADP binding that acts as an obstacle to NAD(P)-dependent dehydrogenase catalytic activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Ikenobe 2393, Miki-cho, Kagawa 761-0795, Japan.

ABSTRACT
NAD(P)-dependent dehydrogenases differ according to their coenzyme preference: some prefer NAD, others NADP, and still others exhibit dual cofactor specificity. The structure of a newly identified archaeal homoserine dehydrogenase showed this enzyme to have a strong preference for NADP. However, NADP did not act as a cofactor with this enzyme, but as a strong inhibitor of NAD-dependent homoserine oxidation. Structural analysis and site-directed mutagenesis showed that the large number of interactions between the cofactor and the enzyme are responsible for the lack of reactivity of the enzyme towards NADP. This observation suggests this enzyme exhibits a new variation on cofactor binding to a dehydrogenase: very strong NADP binding that acts as an obstacle to NAD(P)-dependent dehydrogenase catalytic activity.

No MeSH data available.


Cofactor binding site.a, Comparison of the nucleotide-binding site structures in P. horikoshii HseDH (green and black labels) and S. cerevisiae HseDH (cyan and red labels) (stereo representation). b, Comparison of the nucleotide-binding site structures in P. horikoshii HseDH (green and black labels) and T. thermophilus HseDH (cyan and red labels) (stereo representation). NADPH in P. horikoshii HseDH is shown in magenta. Atoms are colored as in Fig. 3.
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f4: Cofactor binding site.a, Comparison of the nucleotide-binding site structures in P. horikoshii HseDH (green and black labels) and S. cerevisiae HseDH (cyan and red labels) (stereo representation). b, Comparison of the nucleotide-binding site structures in P. horikoshii HseDH (green and black labels) and T. thermophilus HseDH (cyan and red labels) (stereo representation). NADPH in P. horikoshii HseDH is shown in magenta. Atoms are colored as in Fig. 3.

Mentions: The electron density corresponding to the NADPH bound within the nucleotide-binding site of P. horikoshii HseDH was very clear, enabling us to place the ligand with reasonable accuracy (Fig. 3). Three hydrogen-bonding interactions are made between the nicotinamide amide group (O7N and N7N) and the backbone nitrogen of Gly296, the side-chain of Thr300 and the nicotinamide phosphate (O2N). Via a water molecule (W1), the C2 and C3 hydroxyl groups (O2D and O3D) of the nicotinamide ribose interact with the backbone nitrogen of Lys116 and the side chain of Asn115. The O2D and O3D also interact with O4 of the MPD molecule and the backbone O atom of Ser92, respectively. The nicotinamide phosphate (O1N) interacts with the backbone N atoms of Thr12 and Val13, and via a water molecule (W2), the O1N also interacts with the backbone O atom of Val91 and the backbone N atoms of Gly11 and Gly14. Also via a water molecule (W3), the O2N of the nicotinamide phosphate interacts with the side chain of Thr12. The O2A of the adenine phosphate forms a hydrogen bond with W3, in addition to interactions with the backbone N atom and side chain of Thr12. The C3 hydroxyl group (O3B) of the adenine ribose interacts with the backbone N atom of Phe10 and the C2 phosphate group (O1X) of the adenine ribose. No hydrogen bonding interactions are made between the adenine base and the enzyme. By contrast, the C2 phosphate group (O1X, O2X, and O3X) of the adenine ribose is tightly held at its position through interactions with the side chain and backbone N atom of Arg40, the side chain of Lys57, and via a water molecule (W4), the backbone O atom of Gly61. Most of the interactions between the enzyme and the NADH moiety of the cofactor are conserved in the NAD-bound S. cerevisiae HseDH structure (1EBF-A), although Thr12, Phe10, Val91 and Ser92 in P. horikoshii HseDH are replaced by Val15, Ala13, Asn92 and Thr93, respectively, in S. cerevisiae HseDH (Fig. 4a). By contrast, the residues interacting with the C2 phosphate group of the adenine ribose in P. horikoshii HseDH are not conserved in S. cerevisiae HseDH. Arg40 in the former is replaced with Ala41 in the latter. In addition, Lys57 and Gly61 in P. horikoshii HseDH are absent from S. cerevisiae HseDH, because both residues belong to the C-terminal part of the helix α2, which specifically extends toward the cofactor-binding site in P. horikoshii HseDH. On the other hand, comparison of the cofactor-binding sites between P. horikoshii HseDH and substrate/cofactor-free T. thermophilus HseDH indicates that the residues interacting with the cofactor in the P. horikoshii enzyme are likely more strictly conserved in the T. thermophilus enzyme, though Phe10, Val91 and Ser92 in P. horikoshii HseDH are respectively replaced by Gly11, Ala73 and Met74 in T. thermophilus HseDH (Fig. 4b). In contrast to the situation in S. cerevisiae HseDH, the two residues (Arg44 and Arg50) that respectively correspond to Arg40 and Lys57 in P. horikoshii HseDH are observed in T. thermophilus HseDH. These residues belong to L1, which extends toward the cofactor-binding site in T. thermophilus HseDH, and their side chains are thought to be situated at positions where they can interact with the C2 phosphate group of the adenine ribose. That said, superposition of T. thermophilus HseDH onto P. horikoshii HseDH showed that Arg44 of T. thermophilus HseDH would sterically hinder the binding of NADP(H) (Fig. 4b), which means structural changes would occur upon NADP(H) binding to the T. thermophilus HseDH. Thus, the cofactor-bound structure of T. thermophilus HseDH will be necessary for further analysis of the cofactor-enzyme interactions of this enzyme.


Crystal Structures of a Hyperthermophilic Archaeal Homoserine Dehydrogenase Suggest a Novel Cofactor Binding Mode for Oxidoreductases.

Hayashi J, Inoue S, Kim K, Yoneda K, Kawarabayasi Y, Ohshima T, Sakuraba H - Sci Rep (2015)

Cofactor binding site.a, Comparison of the nucleotide-binding site structures in P. horikoshii HseDH (green and black labels) and S. cerevisiae HseDH (cyan and red labels) (stereo representation). b, Comparison of the nucleotide-binding site structures in P. horikoshii HseDH (green and black labels) and T. thermophilus HseDH (cyan and red labels) (stereo representation). NADPH in P. horikoshii HseDH is shown in magenta. Atoms are colored as in Fig. 3.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4495429&req=5

f4: Cofactor binding site.a, Comparison of the nucleotide-binding site structures in P. horikoshii HseDH (green and black labels) and S. cerevisiae HseDH (cyan and red labels) (stereo representation). b, Comparison of the nucleotide-binding site structures in P. horikoshii HseDH (green and black labels) and T. thermophilus HseDH (cyan and red labels) (stereo representation). NADPH in P. horikoshii HseDH is shown in magenta. Atoms are colored as in Fig. 3.
Mentions: The electron density corresponding to the NADPH bound within the nucleotide-binding site of P. horikoshii HseDH was very clear, enabling us to place the ligand with reasonable accuracy (Fig. 3). Three hydrogen-bonding interactions are made between the nicotinamide amide group (O7N and N7N) and the backbone nitrogen of Gly296, the side-chain of Thr300 and the nicotinamide phosphate (O2N). Via a water molecule (W1), the C2 and C3 hydroxyl groups (O2D and O3D) of the nicotinamide ribose interact with the backbone nitrogen of Lys116 and the side chain of Asn115. The O2D and O3D also interact with O4 of the MPD molecule and the backbone O atom of Ser92, respectively. The nicotinamide phosphate (O1N) interacts with the backbone N atoms of Thr12 and Val13, and via a water molecule (W2), the O1N also interacts with the backbone O atom of Val91 and the backbone N atoms of Gly11 and Gly14. Also via a water molecule (W3), the O2N of the nicotinamide phosphate interacts with the side chain of Thr12. The O2A of the adenine phosphate forms a hydrogen bond with W3, in addition to interactions with the backbone N atom and side chain of Thr12. The C3 hydroxyl group (O3B) of the adenine ribose interacts with the backbone N atom of Phe10 and the C2 phosphate group (O1X) of the adenine ribose. No hydrogen bonding interactions are made between the adenine base and the enzyme. By contrast, the C2 phosphate group (O1X, O2X, and O3X) of the adenine ribose is tightly held at its position through interactions with the side chain and backbone N atom of Arg40, the side chain of Lys57, and via a water molecule (W4), the backbone O atom of Gly61. Most of the interactions between the enzyme and the NADH moiety of the cofactor are conserved in the NAD-bound S. cerevisiae HseDH structure (1EBF-A), although Thr12, Phe10, Val91 and Ser92 in P. horikoshii HseDH are replaced by Val15, Ala13, Asn92 and Thr93, respectively, in S. cerevisiae HseDH (Fig. 4a). By contrast, the residues interacting with the C2 phosphate group of the adenine ribose in P. horikoshii HseDH are not conserved in S. cerevisiae HseDH. Arg40 in the former is replaced with Ala41 in the latter. In addition, Lys57 and Gly61 in P. horikoshii HseDH are absent from S. cerevisiae HseDH, because both residues belong to the C-terminal part of the helix α2, which specifically extends toward the cofactor-binding site in P. horikoshii HseDH. On the other hand, comparison of the cofactor-binding sites between P. horikoshii HseDH and substrate/cofactor-free T. thermophilus HseDH indicates that the residues interacting with the cofactor in the P. horikoshii enzyme are likely more strictly conserved in the T. thermophilus enzyme, though Phe10, Val91 and Ser92 in P. horikoshii HseDH are respectively replaced by Gly11, Ala73 and Met74 in T. thermophilus HseDH (Fig. 4b). In contrast to the situation in S. cerevisiae HseDH, the two residues (Arg44 and Arg50) that respectively correspond to Arg40 and Lys57 in P. horikoshii HseDH are observed in T. thermophilus HseDH. These residues belong to L1, which extends toward the cofactor-binding site in T. thermophilus HseDH, and their side chains are thought to be situated at positions where they can interact with the C2 phosphate group of the adenine ribose. That said, superposition of T. thermophilus HseDH onto P. horikoshii HseDH showed that Arg44 of T. thermophilus HseDH would sterically hinder the binding of NADP(H) (Fig. 4b), which means structural changes would occur upon NADP(H) binding to the T. thermophilus HseDH. Thus, the cofactor-bound structure of T. thermophilus HseDH will be necessary for further analysis of the cofactor-enzyme interactions of this enzyme.

Bottom Line: The structure of a newly identified archaeal homoserine dehydrogenase showed this enzyme to have a strong preference for NADP.However, NADP did not act as a cofactor with this enzyme, but as a strong inhibitor of NAD-dependent homoserine oxidation.This observation suggests this enzyme exhibits a new variation on cofactor binding to a dehydrogenase: very strong NADP binding that acts as an obstacle to NAD(P)-dependent dehydrogenase catalytic activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Ikenobe 2393, Miki-cho, Kagawa 761-0795, Japan.

ABSTRACT
NAD(P)-dependent dehydrogenases differ according to their coenzyme preference: some prefer NAD, others NADP, and still others exhibit dual cofactor specificity. The structure of a newly identified archaeal homoserine dehydrogenase showed this enzyme to have a strong preference for NADP. However, NADP did not act as a cofactor with this enzyme, but as a strong inhibitor of NAD-dependent homoserine oxidation. Structural analysis and site-directed mutagenesis showed that the large number of interactions between the cofactor and the enzyme are responsible for the lack of reactivity of the enzyme towards NADP. This observation suggests this enzyme exhibits a new variation on cofactor binding to a dehydrogenase: very strong NADP binding that acts as an obstacle to NAD(P)-dependent dehydrogenase catalytic activity.

No MeSH data available.