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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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Overall structure of LacAB and each subunit.A, A ribbon diagram and transparent surface representation of LacAB as a homotetramer. B, A ribbon diagram of the LacAB monomer showing the secondary structure as defined in Figure 1B. C, A ribbon diagram of a LacA subunit showing the five parallel β-sheets in the center surrounded by five α-helices, with α1, α4, and α5 on the left, and α2 and α3 on the right. The α6 helix is located perpendicular to the α3β4α4 motif. D, A ribbon diagram of the LacB subunit, as described for LacA in Figure 2C. The extra C-terminal α7 helix is located nearly parallel to the α2 helix.
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pone-0072902-g002: Overall structure of LacAB and each subunit.A, A ribbon diagram and transparent surface representation of LacAB as a homotetramer. B, A ribbon diagram of the LacAB monomer showing the secondary structure as defined in Figure 1B. C, A ribbon diagram of a LacA subunit showing the five parallel β-sheets in the center surrounded by five α-helices, with α1, α4, and α5 on the left, and α2 and α3 on the right. The α6 helix is located perpendicular to the α3β4α4 motif. D, A ribbon diagram of the LacB subunit, as described for LacA in Figure 2C. The extra C-terminal α7 helix is located nearly parallel to the α2 helix.

Mentions: In the crystal structure, the two LacABs in asymmetric unit (subunit AB and CD) is related by non-crystallographic symmetry, and the whole LacAB homotetramer is arranged with crystallographic 2-fold symmetry of each two asymmetric units, which is shown in Figure 2A, has dimensions of 108 × 79 × 54 Å (Figure 2A and B). This interface is predicted by PISA [22], leading to stable homotetramer formation of LacAB in solution with a ΔG of dissociation of 7.8 kcal/mol. To confirm the solution state of LacAB, the molar mass was analyzed using an online size exclusion chromatography-fast protein liquid chromatography (SEC-FPLC) coupled with multi-angle light scattering (MALS) and UV detector. Figure S1 shows the UV280nm-LS overlay of LacAB. The column retention time in SEC was 13.62 min, and the MALS estimated molecular weight (Mw) of the major peak was about 135.5 kDa with a polydispersity (Pd) of 1.026.


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)

Overall structure of LacAB and each subunit.A, A ribbon diagram and transparent surface representation of LacAB as a homotetramer. B, A ribbon diagram of the LacAB monomer showing the secondary structure as defined in Figure 1B. C, A ribbon diagram of a LacA subunit showing the five parallel β-sheets in the center surrounded by five α-helices, with α1, α4, and α5 on the left, and α2 and α3 on the right. The α6 helix is located perpendicular to the α3β4α4 motif. D, A ribbon diagram of the LacB subunit, as described for LacA in Figure 2C. The extra C-terminal α7 helix is located nearly parallel to the α2 helix.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3755991&req=5

pone-0072902-g002: Overall structure of LacAB and each subunit.A, A ribbon diagram and transparent surface representation of LacAB as a homotetramer. B, A ribbon diagram of the LacAB monomer showing the secondary structure as defined in Figure 1B. C, A ribbon diagram of a LacA subunit showing the five parallel β-sheets in the center surrounded by five α-helices, with α1, α4, and α5 on the left, and α2 and α3 on the right. The α6 helix is located perpendicular to the α3β4α4 motif. D, A ribbon diagram of the LacB subunit, as described for LacA in Figure 2C. The extra C-terminal α7 helix is located nearly parallel to the α2 helix.
Mentions: In the crystal structure, the two LacABs in asymmetric unit (subunit AB and CD) is related by non-crystallographic symmetry, and the whole LacAB homotetramer is arranged with crystallographic 2-fold symmetry of each two asymmetric units, which is shown in Figure 2A, has dimensions of 108 × 79 × 54 Å (Figure 2A and B). This interface is predicted by PISA [22], leading to stable homotetramer formation of LacAB in solution with a ΔG of dissociation of 7.8 kcal/mol. To confirm the solution state of LacAB, the molar mass was analyzed using an online size exclusion chromatography-fast protein liquid chromatography (SEC-FPLC) coupled with multi-angle light scattering (MALS) and UV detector. Figure S1 shows the UV280nm-LS overlay of LacAB. The column retention time in SEC was 13.62 min, and the MALS estimated molecular weight (Mw) of the major peak was about 135.5 kDa with a polydispersity (Pd) of 1.026.

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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