Six enzymes are required for the production of dTDP-desosamine in Streptomyces venezuelae (Scheme 1). As is common in many biosynthetic pathways for unusual sugars, dTDP-desosamine production begins with the attachment of α-d-glucose-1-phosphate to dTMP (in S. venezuelae, this reaction is catalyzed by DesIII). In the next step, the 6'-hydroxyl group of the sugar is removed and the 4'-hydroxyl group is oxidized to a keto-functionality through the action of a dehydratase referred to as DesIV. The focus of this investigation is DesI which catalyzes the next step in the pathway, namely the PLP-dependent amination of the sugar C-4' position. This ping-pong reaction first requires the conversion of PLP to PMP using glutamate as the nitrogen source, followed by amination of the sugar substrate. In the reaction catalyzed by DesI, the resulting amino group lies in the equatorial position. Following this amination by DesI, the next enzyme in the pathway, DesII removes the amino group at the C-4' position and oxidizes the C-3' hydroxyl group to a keto moiety. As indicated in Scheme 1, there is a second PLP-dependent enzyme in the pathway, DesV, that adds an amino group equatorially to C-3'. Not surprisingly, given their similar modes of action and similar substrates, DesI and DesV demonstrate an amino acid sequence identity of 34.2%. From previous work in our laboratory, the three-dimensional folds of both DesIV and DesV are now known (8, 9). The structure of DesV revealed the manner in which it accommodates either the internal aldimine form of PLP or the ketimine intermediate of glutamate. It was not possible, however, to solve the structure of DesV with a bound dTDP-sugar ligand. In the present investigation, crystals of DesI with bound product were obtained and a high-resolution x-ray analysis conducted. This investigation reveals the manner in which the ligand is accommodated within the active site pocket. The only other sugar-modifying aminotransferase whose structure has been solved with a bound sugar ligand is PseC, an enzyme from Helicobacter pylori that is involved in the biosynthesis of pseudaminic acid (10).

In the resting state, the PLP cofactor is attached to DesI via a Schiff base with the ε-amino group of Lys 200 (Scheme 2). This is referred to as the internal aldimine. For the structural analysis presented here, DesI was crystallized in the presence of its product, dTDP-4-amino-4,6-dideoxyglucose. Apparently the reaction is reversible because, as can be seen from the electron density displayed in Figure 3a, the amino group of the product forms a covalent bond with the C-4' atom of PLP. The formation of this covalent bond displaces Lys 200 and yields what is believed to be the external aldimine. It should be noted, however, that the external aldimine can further react to yield the resonance-stabilized quinonoid followed by the ketimine intermediate as highlighted in Scheme 2. The major structural difference between the external aldimine and the quinonoid intermediate occurs at the sugar C-4' where in the former the carbon is sp³ hybridized and in the latter it assumes sp² hybridization. Likewise, the main structural difference between the quinonoid intermediate and the ketimine intermediate is at C-4' of the pyridoxal ring where in the former it is sp² hybridized and in the latter it is sp³ hybridized. While the electron density appears to accommodate the external aldimine intermediate most easily, at a resolution of 2.1 Å, the presence of the ketimine intermediate cannot be ruled out.

To date, four other x-ray crystallographic structures of sugar modifying aminotransferases have been determined: (1) ArnB from Salmonella typhimurium, a 4-amino-4-deoxy-L-arabinose lipopolysaccharide-modifying enzyme (17), (2) PseC, an aminotransferase from H. pylori that is involved in the biosynthesis of pseudaminic acid (10), PglE, an enzyme responsible for amino transfer in the production of UDP-2,4-diacetamido-2,4,6,trideoxy-α-d-glucopyranose in Campylobacter jejuni (18), and (4) DesV, the second aminotransferase in the desosamine biosynthetic pathway (9). The reactions catalyzed by these enzymes are presented in Scheme 3. ArnB, PseC, PglE, and DesI catalyze amination of the sugar C-4' whereas DesV acts on C-3' of the hexose. As might be expected, the overall three-dimensional folds for these enzymes are similar. It is the details in their active site pockets, however, that allow these enzymes to function on different hydroxyl positions and/or to transfer amino groups to either the equatorial or axial positions.

Ribbon representation of DesI. Subunits 1 and 2 in the x-ray coordinate file are color-coded in gold and green, respectively. The bound dTDP-sugar/PLP complexes are displayed in ball-and-stick representations. All figures were prepared with the software package PyMOL (19).

Overall structure of Subunit 1. A stereo ribbon representation of the DesI subunit is displayed with the β-strands numbered and highlighted in red.

The active site of DesI. Electron density corresponding to the external aldimine is shown in (a). The map was calculated with coefficients of the form (F_o - F_c), where F_o was the native structure factor amplitude and F_c was the calculated structure factor amplitude with the atoms corresponding to external aldimine excluded from the calculation. The map was contoured at 3σ. Those amino acid residues located within ∼3.5 Å of the ligand are shown in (b). Residues highlighted in green bonds and marked by an asterisk belong to Subunit 2. Possible hydrogen bonds (3.2 Å) are indicated by the dashed lines. Note that the electron density for Arg 327 was weak, and its position in the active site is tentative. Consequently, it may or may not interact with the α-phosphoryl oxygens of the ligand. The corresponding active site pocket for PseC is shown in (c). Coordinates were obtained from the protein data bank (accession no. 2FNU). The color-coding is the same as described in (b).

Comparison of the active site regions for DesI and PseC. Those residues belonging to DesI are depicted in gold whereas those for PseC are displayed in slate. The top and bottom labels refer to residues in DesI and PseC, respectively.