Figure 1.
Schematic of the reaction catalyzed by LacAB and alignment of LacA, LacB, and RpiB sequences.
A, Galactose-6-phosphate (Gal6P) is converted to tagatose-6-phosphate (Tag6P) by LacAB during lactose catabolism. B, Multiple sequence alignment of LacA and LacB from Lactobacillus rhamnosus (GenBank accession numbers ZP03210387 and ZP03210388, respectively), EcRpiB from Escherichia coli (PDB ID 1NN4; NP418514), MtRpiB from Mycobacterium tuberculosis (2VVP; YP006515902), and CtRpiB from Clostridium thermocellum (3PH4; YP001038990). The sequences are for precursors, and the numbering is based on LacA. Highly conserved residues are shown in red type and boxed in blue; strictly conserved residues are shown on a red background. Secondary structure elements are indicated in pink for LacA and in green for LacB. Residues interacting directly with bound Taga6P are indicated with a triangle in pink (LacA) and green (LacB). The figure was prepared using ESPript [41].
Figure 2.
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.
Figure 3.
The active site pocket in LacAB.
A, The four active sites in the whole LacAB structure are located at the inside of LacAB homotetramer. B, Based on the structure of the LacAB complex with the product tagatose-6-phosphate (Tag6P), the substrate-binding site of LacAB is at the interface between LacA and LacB subunits. C, A close-up surface representation of the active site at the interface shows a deep, wide-mouthed, trapezoid-shaped cavity formed by the residues Met-92, His-96, Asn-97, Arg-130, His-131, and Arg-134 in LacA, and Asp-8, His-9, Ile-10, Arg-39, Tyr-42, Cys-65, Thr-67, Ile-69, and Thr-73 in LacB. Residue of LacB in other neighboring LacAB is indicated by asterisks.
Figure 4.
The active site in the LacAB-Tag6P complex.
A, Surface representation of the active site showing the binding orientation of tagatose-6-phosphate in the pocket. B, The final 2Fo-Fc electron density map contoured at 1.0 σ and overlaid on the model for tagatose-6-phosphate and water molecules (red spheres) binding in the active site pocket of LacAB. C, Schematic showing the detailed binding mode of tagatose-6-phosphate (blue) in the active site. 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. Residues of LacA are indicated by asterisks.
Figure 5.
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.