Mycocerosic acid synthase exemplifies the architecture of reducing polyketide synthases

Abstract

regions of MAS are centrally connected by non-conserved linkers, which permit large-scale 10 relative motions in related systems 10 . To obtain a high-quality hybrid model, we divided MAS 11 into its condensing and modifying region, and excluded the flexibly tethered ACP (Fig. 1a). 12Three constructs of staggered C-terminal length were employed to define the length of 13 the condensing region (see Methods). All variants crystallized under the same condition; 14 structure determination mapped the last ordered residue to Glu887. The structure of the most 15 extended variant (1-892) was refined at 2.3 Å resolution (Extended Data Table 1a). MAS KS-AT 16 comprises an α/β-fold linker domain (LD) connecting AT to KS (Fig. 1b) Fig. 1a-c). Four interface segments of 6-19 amino acid (aa) length are disordered 8 (Fig. 1b), while equivalent regions are ordered in dimeric KS domains. connect to the modifying region, are proximal to the two-fold dimer axis above the KS active 15 site, as observed in previous condensing region structures 6,11,12 . 16The DHs connect the modifying region to the post-AT linkers of the condensing region. 17We solved crystal structures of a MAS DH construct (aa 884-1186), which overlaps in sequence 18 with the crystallized KS-AT, in two crystal forms with a total of six protomers arranged into 19 almost identical dimers (Extended Data Table 1a). The DH protomer is composed of two hot-dog 20 folds connected by a 20 aa hot-dog linker (Fig. 1c). A hydrophobic substrate binding tunnel 21 extends over both hot-dog folds with entrances near the C-terminus and at the distal end of hot-22 dog fold 2. Active site residues are contributed by both hot-dog folds and are located close to the Table 2a). In the DH dimer, the two protomers arrange 2 with their lateral ends bent towards the post-AT linkers with an interdomain angle of 222° 3 (Fig. 1c). The MAS DH dimer is distinct from the V-shaped DH arrangement in FAS 6 , which 4 lacks a dimerization interface and is bent into the opposite direction at an angle of 96°. MAS DH 5 rather resembles linear DH dimers of modPKSs with interdomain angles of 167-203°1 5-17 and a 6 common mode of dimerization via "handshake" interactions between β-strands of the N-terminal 7 hot-dog folds (Extended Data Fig. 1f-h). 8To obtain an authentic representation of the MAS modifying region, we crystallized in 9 presence of NADP + the complete DH-ΨKR-ER-KR segment, which is dimeric in solution based 10 on AUC. Based on SAXS, ACP deletion is not affecting the overall structure of this region 11 (Extended Data Fig. 2a-c). The crystallographic asymmetric unit reveals a complex packing of Fig. 3a, b) (aa 1948-1960) remains disordered in the absence of ligand, and 8 concomitantly, the nicotinamide moiety of NADP + is disordered (Extended Data Fig. 3d). The 9 MAS ΨKR exhibits an N-terminal β-α-β-α extension, which is commonly observed in modPKSs, 10but not in FASs 6,23 ; this extension exhibits increased flexibility as indicated by temperature 11 factor distributions (Extended Data ...

The structural organization of substrate loading in iterative polyketide synthases

“…The individual domain structures (Supplementary Table 2a) and the overall arrangement of the KS-LD-MAT domains (Fig. 2a,b) resemble condensing-region structures from mFAS 5 , 15 , fully-reducing iPKS 2 and several modPKS 4 , 13 , 14 , but differ from the architecture of the PikAIII modPKS that was resolved at intermediate resolution by cryo-EM 12 , 26 (Supplementary Fig. 2).…”

“…3a). The two SAT domains form a direct pseudodimer interface of 324 Å 2 that is devoid of hydrogen bonds or salt bridges (Supplementary Table 2b and Supplementary Fig. 3b,c).…”

“…3b,c). The C-terminal end of the SAT-KS linker adds an average interface area of 493 Å 2 by binding to a surface groove of the opposite SAT (Fig. 2c and Supplementary Table 2b).…”

“…PKSs are dimeric multienzymes with structural segregation of condensing and modifying regions 2 , 12 , similar to the mammalian fatty acid synthase (mFAS) 5 . However, the structural integration of dedicated loading modules or specialized substrate-loading domains, as exemplified by the NR-PKS SAT-KS-MAT core region, remains unknown.…”

“…However, the structural integration of dedicated loading modules or specialized substrate-loading domains, as exemplified by the NR-PKS SAT-KS-MAT core region, remains unknown. The KS-MAT didomains of NR-PKSs are homologs of the corresponding condensing regions in other PKSs and mFAS, which have been structurally characterized as isolated dido-mains 2 , 4 , 5 , 13 – 15 . However, in NR-PKSs, soluble expression of KS-MAT is only achieved by including the SAT domain in a tridomain construct 16 , 17 , indicating that SAT, KS, and MAT form an integrated loading/condensing region architecture.…”