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Crystal structure and substrate specificity of D-galactose-6-phosphate isomerase complexed with substrates.

Jung WS, Singh RK, Lee JK, Pan CH - PLoS ONE (2013)

Bottom Line: Due to the proximity of the Arg-134 residue and backbone Cα of the α6 helix in LacA to the last Asp-172 residue of LacB with a hydrogen bond, a six-carbon sugar-phosphate can bind in the larger pocket of LacAB, compared with RpiB.Two rare sugar substrates, D-psicose and D-ribulose, show optimal binding in the LacAB-substrate complex.These findings were supported by the results of LacA activity assays.

View Article: PubMed Central - PubMed

Affiliation: Functional Food Center, Korea Institute of Science and Technology Gangneung Institute, Gangneung, Korea.

ABSTRACT
D-Galactose-6-phosphate isomerase from Lactobacillus rhamnosus (LacAB; EC 5.3.1.26), which is encoded by the tagatose-6-phosphate pathway gene cluster (lacABCD), catalyzes the isomerization of D-galactose-6-phosphate to D-tagatose-6-phosphate during lactose catabolism and is used to produce rare sugars as low-calorie natural sweeteners. The crystal structures of LacAB and its complex with D-tagatose-6-phosphate revealed that LacAB is a homotetramer of LacA and LacB subunits, with a structure similar to that of ribose-5-phosphate isomerase (Rpi). Structurally, LacAB belongs to the RpiB/LacAB superfamily, having a Rossmann-like αβα sandwich fold as has been identified in pentose phosphate isomerase and hexose phosphate isomerase. In contrast to other family members, the LacB subunit also has a unique α7 helix in its C-terminus. One active site is distinctly located at the interface between LacA and LacB, whereas two active sites are present in RpiB. In the structure of the product complex, the phosphate group of D-tagatose-6-phosphate is bound to three arginine residues, including Arg-39, producing a different substrate orientation than that in RpiB, where the substrate binds at Asp-43. Due to the proximity of the Arg-134 residue and backbone Cα of the α6 helix in LacA to the last Asp-172 residue of LacB with a hydrogen bond, a six-carbon sugar-phosphate can bind in the larger pocket of LacAB, compared with RpiB. His-96 in the active site is important for ring opening and substrate orientation, and Cys-65 is essential for the isomerization activity of the enzyme. Two rare sugar substrates, D-psicose and D-ribulose, show optimal binding in the LacAB-substrate complex. These findings were supported by the results of LacA activity assays.

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Substrate-specific binding of D-psicose and D-ribulose to LacAB.A and D, The binding of D-psicose and D-ribulose, respectively, at the active site of LacAB is shown, including the amino acid residues and water molecules (red spheres). B and E, The final 2Fo-Fc electron density maps contoured at 0.8σ are overlaid on the models for D-psicose and D-ribulose. C and F, The binding modes of D-psicose and D-ribulose. The substrates are shown in blue. Dashed lines indicate hydrogen bondings and polar interactions, which are labeled with the interatomic distances in Å. Decorated arcs represent van der Waals interactions of less than 5.0 Å. Water molecules are shown as red circles.
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pone-0072902-g005: Substrate-specific binding of D-psicose and D-ribulose to LacAB.A and D, The binding of D-psicose and D-ribulose, respectively, at the active site of LacAB is shown, including the amino acid residues and water molecules (red spheres). B and E, The final 2Fo-Fc electron density maps contoured at 0.8σ are overlaid on the models for D-psicose and D-ribulose. C and F, The binding modes of D-psicose and D-ribulose. The substrates are shown in blue. Dashed lines indicate hydrogen bondings and polar interactions, which are labeled with the interatomic distances in Å. Decorated arcs represent van der Waals interactions of less than 5.0 Å. Water molecules are shown as red circles.

Mentions: The structures of the LacAB complex with D-allose and D-ribose revealed that the substrates are converted to D-psicose and D-ribulose, respectively, which bind in the same orientation as Tag6P in the active site pocket (Figure 5A and D). The electron density maps of D-psicose and D-ribulose were unambiguously observed (Figure 5B and E). To identify and confirm the bound substances in LacAB complex, D-psicose and D-ribulose as the products were substituted with D-allose and D-ribose as the substrates, as well as D-allose and D-ribose as the products by ketose conversion, respectively. However, these substitutions were revealed higher B-factors for ligand after refinement. The interactions between LacAB and the bound sugars are shown schematically in Figure 5C and F. The O1, O3, and O4 hydroxyl groups as well as the O2 carbonyl group of the substrates interact with the same LacAB residues as seen in the LacAB-Tag6P complex. Additionally, the imidazole group of His-96 (LacA) directly binds to the O4 hydroxyl group of D-ribulose via hydrogen bonding at a distance of 3.0 Å. In the LacAB-psicose complex, the O6 hydroxyl group is 2.7 Å from the guanidinium group of Arg-134 (LacA).


Crystal structure and substrate specificity of D-galactose-6-phosphate isomerase complexed with substrates.

Jung WS, Singh RK, Lee JK, Pan CH - PLoS ONE (2013)

Substrate-specific binding of D-psicose and D-ribulose to LacAB.A and D, The binding of D-psicose and D-ribulose, respectively, at the active site of LacAB is shown, including the amino acid residues and water molecules (red spheres). B and E, The final 2Fo-Fc electron density maps contoured at 0.8σ are overlaid on the models for D-psicose and D-ribulose. C and F, The binding modes of D-psicose and D-ribulose. The substrates are shown in blue. Dashed lines indicate hydrogen bondings and polar interactions, which are labeled with the interatomic distances in Å. Decorated arcs represent van der Waals interactions of less than 5.0 Å. Water molecules are shown as red circles.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3755991&req=5

pone-0072902-g005: Substrate-specific binding of D-psicose and D-ribulose to LacAB.A and D, The binding of D-psicose and D-ribulose, respectively, at the active site of LacAB is shown, including the amino acid residues and water molecules (red spheres). B and E, The final 2Fo-Fc electron density maps contoured at 0.8σ are overlaid on the models for D-psicose and D-ribulose. C and F, The binding modes of D-psicose and D-ribulose. The substrates are shown in blue. Dashed lines indicate hydrogen bondings and polar interactions, which are labeled with the interatomic distances in Å. Decorated arcs represent van der Waals interactions of less than 5.0 Å. Water molecules are shown as red circles.
Mentions: The structures of the LacAB complex with D-allose and D-ribose revealed that the substrates are converted to D-psicose and D-ribulose, respectively, which bind in the same orientation as Tag6P in the active site pocket (Figure 5A and D). The electron density maps of D-psicose and D-ribulose were unambiguously observed (Figure 5B and E). To identify and confirm the bound substances in LacAB complex, D-psicose and D-ribulose as the products were substituted with D-allose and D-ribose as the substrates, as well as D-allose and D-ribose as the products by ketose conversion, respectively. However, these substitutions were revealed higher B-factors for ligand after refinement. The interactions between LacAB and the bound sugars are shown schematically in Figure 5C and F. The O1, O3, and O4 hydroxyl groups as well as the O2 carbonyl group of the substrates interact with the same LacAB residues as seen in the LacAB-Tag6P complex. Additionally, the imidazole group of His-96 (LacA) directly binds to the O4 hydroxyl group of D-ribulose via hydrogen bonding at a distance of 3.0 Å. In the LacAB-psicose complex, the O6 hydroxyl group is 2.7 Å from the guanidinium group of Arg-134 (LacA).

Bottom Line: Due to the proximity of the Arg-134 residue and backbone Cα of the α6 helix in LacA to the last Asp-172 residue of LacB with a hydrogen bond, a six-carbon sugar-phosphate can bind in the larger pocket of LacAB, compared with RpiB.Two rare sugar substrates, D-psicose and D-ribulose, show optimal binding in the LacAB-substrate complex.These findings were supported by the results of LacA activity assays.

View Article: PubMed Central - PubMed

Affiliation: Functional Food Center, Korea Institute of Science and Technology Gangneung Institute, Gangneung, Korea.

ABSTRACT
D-Galactose-6-phosphate isomerase from Lactobacillus rhamnosus (LacAB; EC 5.3.1.26), which is encoded by the tagatose-6-phosphate pathway gene cluster (lacABCD), catalyzes the isomerization of D-galactose-6-phosphate to D-tagatose-6-phosphate during lactose catabolism and is used to produce rare sugars as low-calorie natural sweeteners. The crystal structures of LacAB and its complex with D-tagatose-6-phosphate revealed that LacAB is a homotetramer of LacA and LacB subunits, with a structure similar to that of ribose-5-phosphate isomerase (Rpi). Structurally, LacAB belongs to the RpiB/LacAB superfamily, having a Rossmann-like αβα sandwich fold as has been identified in pentose phosphate isomerase and hexose phosphate isomerase. In contrast to other family members, the LacB subunit also has a unique α7 helix in its C-terminus. One active site is distinctly located at the interface between LacA and LacB, whereas two active sites are present in RpiB. In the structure of the product complex, the phosphate group of D-tagatose-6-phosphate is bound to three arginine residues, including Arg-39, producing a different substrate orientation than that in RpiB, where the substrate binds at Asp-43. Due to the proximity of the Arg-134 residue and backbone Cα of the α6 helix in LacA to the last Asp-172 residue of LacB with a hydrogen bond, a six-carbon sugar-phosphate can bind in the larger pocket of LacAB, compared with RpiB. His-96 in the active site is important for ring opening and substrate orientation, and Cys-65 is essential for the isomerization activity of the enzyme. Two rare sugar substrates, D-psicose and D-ribulose, show optimal binding in the LacAB-substrate complex. These findings were supported by the results of LacA activity assays.

Show MeSH
Related in: MedlinePlus