Figure 1.
The product and substrates of the VldE and OtsA catalyzed reactions are shown. Note the considerable similarity between the ligands of VldE and OtsA, and the conservation of the anomeric centers. The remainders of both biosynthetic pathways are then drawn to completion. VldE catalyzes the formation of validoxylamine A 7′-phosphate via a non-glycosidic C-N bond between GDP-valienol and validamine 7-phosphate. After the validoxylamine A 7′-phosphate has been produced; VldH and VldK complete the catalytic synthesis of Validamycin A. OtsA catalyzes the formation of trehalose 6-phosphate via a glycosidic bond between UDP-glucose and glucose 6-phosphate. OtsB dephosphorylates trehalose 6-phosphate to produce trehalose.
Figure 2.
An Sequence Alignment of VldE and OtsA.
Shown is the protein sequence alignment of VldE and OtsA as generated by ClustalW2 [54]. Residues whose side-chains are involved in ligand binding are indicated with shaded boxes. Conserved residues are indicated with unfilled boxes. The two-dimensional secondary structure of VldE and OtsA are also illustrated next to the corresponding sequence with α-helixes represented by spirals and β-strands represented by arrows. Numerical values for helices and strands are assigned.
Figure 3.
Shown is the overall fold of VldE as well as comparison to OtsA by superimposition. (A) The overall fold of the monomeric VldE is represented in a ribbon diagram. The monomer is rendered with the N-terminal domain in red, and the C-terminal domain in blue. The α-helical C-terminus, which stretches back across both domains, is rendered in green. VldE consists of twin Rossman-like β/α/β domains in a GT-B configuration with the catalytic site marked by VDO and GDP at the interface of the two domains. (B) To compare the overall fold of VldE (green) to OtsA (gray), the folding patterns, which were represented by a tracing of Cα, were superimposed. (C) Shown is a topology diagram of VldE with β-strands and α-helices labeled. Blue boxes identify the core β-sheets of the N- and C-terminal Rossman domains as well as the unique β-hairpin motif.
Figure 4.
Shown is a comparison of the VldE and OtsA catalytic sites in ribbon diagrams. Residues/molecules of interest are represented in stick models. The dotted lines mark hydrogen bonds and ionic interactions. The preferential binding of GDP by VldE is demonstrated by comparing (A) the protein-ligand interactions within the VldE active site (green) with (B) the protein-ligand interactions between OtsA and UDP within the OtsA active site (gray). The protein interactions with ribose and phosphate are conserved between VldE and OtsA. However, there are differing interactions with the nucleotide base groups. The large purine makes interactions with the residues Arg321, Asn323, Asn361, and Thr366. The ribose and phosphate moieties interact with Arg290, Lys295, Leu387, Ser388, and Glu391. Within OtsA, Leu344, Ile295, Pro297, and His338 only allow for the binding of the smaller pyrimidine. The ribose and phosphate moieties make interactions with residues Arg262, Lys267, Leu365, and Glu369. The mesh represents the |Fo| − |Fc| electron density omit map of the GDP binding site. The map is contoured at 3.0σ levels. (C) Shown is a superimposition of the VldE cyclitol binding sites in the presence (yellow) and absence (green) of validoxylamine A 7′-phosphate. The mesh represents the |Fo| − |Fc| electron density omit map of the VDO binding site. The map is contoured at 3.0σ levels. VDO makes interactions with residues the side-chains of residues Asp158, His182, Arg12, Asn325, Arg326, and Asp383. VDO also makes interactions with the backbones of residues 384–387. Binding of the acceptor cyclitol is recognized by conformation changes by the side-chains of residues Asp158, Tyr159, and Arg326. (D) Shown is a comparison by superimposition of VDO binding within the catalytic sites of VldE (yellow) and OtsA (gray). Note the strong conservation of residue and ligand positions.
Table 1.
Statistics of reflection data and structure refinements.
Figure 5.
Shown in this figure is the hydrogen bond network between VldE and (A) GDP and then (B) VDO.
Table 2.
Notable interactions between VldE and ligands.
Figure 6.
Trehalose within the Catalytic Site.
Shown is a trehalose (TRE) within the VldE cyclitol binding site in ribbon diagrams. Residues/molecules of interest are represented in stick models. The dotted lines mark hydrogen bonds. (A) The mesh represents the |Fo|−|Fc| electron density omit map of trehalose within the VldE catalytic site (pink). The map is contoured at 3.0σ levels. Trehalose makes interactions with the backbone amides of residues Gly384, Gln385, Asn386, and Leu387 as well with the side-chains of Asp383, His182 and Arg290. (B) Shown is a superimposition of the catalytic sites of VldE·GDP·VDO model (yellow) and the VldE·GDP·TRE model (pink) in ribbon diagrams. Trehalose does not assume a binding pose comparable to VDO. This is most likely to do the absence of a phosphoryl group. Due to the absence of the phosphate moiety, Arg326 and Asn325 also swing out of the catalytic site.
Figure 7.
Shown are ribbon diagrams of the three conformers of VldE modeled using the VldE·GDP·TRE crystallographic data. Residues/molecules of interest are represented in stick models. Arrows indicate the direction of residue movement. Ligands bound within each conformer are shown to provide a point of reference for comparison. (A) The global conformation of each conformer (gray) is shown with the areas of conformational change highlighted by coloring (pink, blue, cyan). (B) Residues 11–50 for each conformer is shown. Note that this region of residues is capable of 10.9 Å shift towards the catalytic center and that strands of the β-hairpin motif (β2 and β3) move simultaneously with strands β6 and β5 to extend the core β-sheet from ten to twelve strands. (C) A view of helix α3 for each conformer is shown. Note that residues at the kink of the helix are capable of reorganizing.
Figure 8.
Shown is a figure comparing the SNi mechanism of (A) OtsA to the proposed SNi mechanism of (B) VldE. The olefinic moiety of GDP-valienol plays a critical role in facilitating the coupling reaction through a mechanism similar to the formation of an oxonium ion-like transition state upon the detachment of the nucleotide phosphate within OtsA. Both transition states are formed by the coordination of the allylic carbon on the donated group, the bridging nucleophile of the acceptor group and the leaving oxygen of the donor diphosphate-nucleotide.
Table 3.
DALI Server Results.