Table 1.
Data collection and refinement statistics.
Figure 1.
Crystal structure of DndA from Streptomyces lividans.
(A) Overall structure of the DndA dimer. The structure is viewed perpendicular to the two-fold axis of the dimer. The two protomers are shown in magenta and green, respectively. Their bound PLP cofactors are presented as sticks, with carbon atoms yellow, nitrogen atoms blue, oxygen atoms red, and phosphorus atoms orange. (B) Structure of a protomer of DndA. α helices are shown in cyan, β sheets are shown in magenta, and loops are shown in pink. PLP and its covalently linked Lys200 of DndA, as well as the catalytic Cys327 (mutated to serine in our study), are shown in stick representation.
Figure 2.
Structure-based sequence alignment of DndA and related cysteine desulfurases/selenocysteine lyase.
The amino acid sequences of DndA from Streptomyces lividans, IscS from Escherichia coli, NifS from Thermotoga maritima, CsdB from Escherichia coli, and SufS from Synechocystis sp. PCC 6803 are aligned based on their sequence homology and secondary structures. α helices are shaded in cyan, and β sheets are shaded in yellow. Residues conserved in all five proteins are shown in bold letters. Secondary structure elements and residue numbers for DndA are shown above the sequences. The conserved catalytic cysteine residues (Cys327 in DndA) are emphasized with a red box. The conserved lysine residues (Lys200 in DndA) which form Schiff base covalent links with bound PLP's are indicated with a magenta box. The residues which are presumed to recognize the carboxylate group of L-cysteine substrates (Asn150, Gln178, and Arg353 in DndA) are marked with green boxes.
Figure 3.
The binding site of PLP on DndA.
(A) PLP is located in a deep surface pocket on DndA. The two protomers of DndA are shown in surface representation, with only one PLP shown in stick representation. The protomer of DndA harboring this PLP is colored in light grey, whereas the other protomer is colored in dark grey. Blue, red, yellow, and orange represent nitrogen, oxygen, carbon, and phosphorus atoms, respectively. (B) The interaction interface between PLP and DndA. DndA is shown in grey, with carbon atoms of its side chains and PLP shown in green. Blue, red, and orange represent nitrogen, oxygen, and phosphorus atoms, respectively. Hydrogen bonds are represented by magenta dashed lines. The orange circle indicates the presumable location of the carboxylate group of the L-cysteine substrate.
Figure 4.
Specific activities of WT and point mutants of DndA measured using in vitro cysteine desulfurase activity assay.
Assays were performed for five times, and the average values of specific activities along with standard deviations of the measurements were shown.
Figure 5.
Structural comparison of DndA with related cysteine desulfurases/selenocysteine lyase.
(A) Structural superimposition of DndA (red), IscS (green, PDB code 1P3W), NifS (cyan, PDB code 1ECX), CsdB (magenta, PDB code 1C0N), and SufS (blue, PDB code 1T3I). Their bound PLP's are shown as sticks. (B) In DndA, the active site Cys327 is located on a β strand, and its distance from PLP is ∼16 Å. In IscS (C) and NifS (D), the active site cysteines are located on relatively long loops, and are not visible in the crystal structure. Visible residues closest to the catalytic cysteines on the primary sequence are no less than 9 Å from PLP. In CsdB (E) and SufS (F), the active site cysteines are located on relatively short loops, and are ∼7 Å from PLP.