Figure 1.
The proposed reactions catalyzed by SiaM.
SiaM is proposed to reduce the β-ketoacyl intermediates with various lengths. It is used iteratively along the biosynthetic pathway of polyketide dimer SIA7248 [17].
Table 1.
Data collection and refinement statistics.
Figure 2.
The overall structures of several polyketide KRs.
The ribbon diagrams are colored by secondary structures. The helices are in cyan; the sheets are in magenta and the loops are in orange. (a) SiaM from Streptomyces spp. A7248; (b) ActKR from Streptomyces coelicolor; (c) TylKR1 from Streptomyces fradiae; (d) EryKR1 from Streptomyces erythraea.
Figure 3.
Structure-based sequence alignment of KRs from different sources.
The sequence of SiaM is aligned with sequences of ActKR from Streptomyces coelicolor, TylKR1 from Streptomyces fradiae, EryKR1 from Streptomyces erythraea and AmpKR2 from Streptomyces nodosus. The sequences are annotated with corresponding secondary structures in SiaM. Arrows represent β-strands and helices represent α-helices. The conserved residues are colored in red. The LDD motif is marked by asterisks.
Figure 4.
The comparison of LDD and LDD-like motifs in KRs.
The LDD and LDD-like motifs are shown as sticks and colored by elements. The rest of the molecules are shown as ribbons and colored in green. (a) The IRD motif in SiaM. The IRD motif is a degenerate form of LDD motif, with a hydrophobic residue occupying the first position and an aspartic acid occupying the third position. (b) The LGG motif in ActKR. (c) The LDD motif in TylKR1. (d) The LDD motif in EryKR1.
Figure 5.
The catalytic triad and the NADPH binding site.
The NADPH binding site is predicted by overlapping the structure of SiaM and the structure of ActKR/NADPH complex. The NADPH molecule and the catalytic triad (Ser146, Tyr159, Lys163) are shown in sticks. The rest of SiaM is displayed as surface and colored in light gray.
Figure 6.
The composition of the SiaM tetramer.
The SiaM tetramer is constructed through symmetry operations. (a) The square-shaped SiaM tetramer. Each protomer is colored in red, yellow, green and cyan separately. (b) The aromatic stacking interactions in the N-terminal interface. The Phe123, Tyr111 in one protomer and Phe123′, Tyr111′ in the other protomer are shown as sticks and colored in red. The rest of the protomers are colored in yellow and green separately. The aromatic residues form T-shaped aromatic stacking interactions. (c) The aromatic stacking interaction in the C-terminal interface. The Phe227, Tyr235 in one protomer and Phe227′, Tyr235′ in the other protomer are shown as sticks and colored in red. The rest of the protomers are colored in green and cyan separately. The Phe227 and Phe227′ form a parallel-displaced aromatic stacking interaction. (d) The interface formation around Tyr235. The Tyr235 residue is colored in red and shown as sticks. The rest of the molecule is shown in ribbon. The neighboring protomer is shown as surface. A deep cavity is clearly visible at the Tyr235 binding site. The residues in the cavity are shown as sticks.
Figure 7.
The theoretical scattering data were calculated based on the coordinates of the SiaM monomer, dimer1, dimer2 and tetramer. The theoretical data were compared with experimental SAXS data using program CRYSOL. The fitting curves of monomer (brown), dimer1 (green), dimer2 (blue) and tetramer (red) are superposed with the SiaM scattering data (black).
Figure 8.
Fluorescence scans of wild-type SiaM and the mutants.
Fluorescence scans were used to monitor the tertiary structures of SiaM and its mutants. The fluorescence intensity is shown in arbitrary units. (a) Spectra of the mutants at F123. (b) Spectra of the mutants at Y111. (c) Spectra of the mutants at F227. (d) Spectra of the mutants at Y235.
Figure 9.
Enzymatic activity assays of wild-type SiaM and its mutants.
The assays were performed with the substrate mimic β-keto-butanoyl-SNAC. The product and substrate were separated with HPLC and monitored at UV 240 nm. (a) Assays with mutants at F123. (b) Assays with mutants at Y111. (c) Assays with mutants at F227. (d) Assays with mutants at Y235.