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Mycocerosic acid synthase exemplifies the architecture of reducing polyketide synthases

Nature volume 531, pages 533–537 (2016)Cite this article

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A Corrigendum to this article was published on 11 May 2016

This article has been updated

Abstract

Polyketide synthases (PKSs) are biosynthetic factories that produce natural products with important biological and pharmacological activities1,2,3. Their exceptional product diversity is encoded in a modular architecture. Modular PKSs (modPKSs) catalyse reactions colinear to the order of modules in an assembly line3, whereas iterative PKSs (iPKSs) use a single module iteratively as exemplified by fungal iPKSs (fiPKSs)3. However, in some cases non-colinear iterative action is also observed for modPKSs modules and is controlled by the assembly line environment4,5. PKSs feature a structural and functional separation into a condensing and a modifying region as observed for fatty acid synthases6. Despite the outstanding relevance of PKSs, the detailed organization of PKSs with complete fully reducing modifying regions remains elusive. Here we report a hybrid crystal structure of Mycobacterium smegmatis mycocerosic acid synthase based on structures of its condensing and modifying regions. Mycocerosic acid synthase is a fully reducing iPKS, closely related to modPKSs, and the prototype of mycobacterial mycocerosic acid synthase-like7,8 PKSs. It is involved in the biosynthesis of C20–C28 branched-chain fatty acids, which are important virulence factors of mycobacteria9. Our structural data reveal a dimeric linker-based organization of the modifying region and visualize dynamics and conformational coupling in PKSs. On the basis of comparative small-angle X-ray scattering, the observed modifying region architecture may be common also in modPKSs. The linker-based organization provides a rationale for the characteristic variability of PKS modules as a main contributor to product diversity. The comprehensive architectural model enables functional dissection and re-engineering of PKSs.

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Figure 1: Domain organization, condensing region, and dimeric DH domain of MAS.
Figure 2: Crystal structure of the dimeric MAS modifying region.
Figure 3: Linker-based organization of the MAS modifying region.
Figure 4: Hybrid model of a dynamic MAS dimer.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the reported crystal structures have been deposited in the Protein Data Bank under accession numbers 5BP1, 5BP2, 5BP3, 5BP4.

Change history

  • 23 March 2016

    Figure 4b was corrected to include a rotation axis line.

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Acknowledgements

We acknowledge F. Widdel and J. Zedelius for providing gammaproteobacterium HdN1, P. Leadlay and L. Betancor for providing plasmid pETcoco-2A-L1SL2, and EMBL Heidelberg for providing the pETG-10A vector; J. Missimer and A. Menzel for support in SAXS data acquisition and raw data processing; T. Sharpe for analytical ultracentrifugation, A. Mazur for SAXS refinement, and M. Bertoni for support of the homology-based assignment of the oligomeric state of MAS KS–AT. Data were collected at beamlines PXI, PXIII, and cSAXS of PSI; we acknowledge support from the beamline teams. This work was supported by the Swiss National Science Foundation project grants 125357, 138262, 159696 and R’equip grant 145023. D.A.H. acknowledges a fellowship by the Werner-Siemens Foundation.

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Author notes

  1. Franziska Zähringer

    Present address: † Present address: F. Hoffmann-La Roche AG, Grenzacherstrasse 124, 4070 Basel, Switzerland.,

  2. Dominik A. Herbst and Roman P. Jakob: These authors contributed equally to this work.

Authors and Affiliations

  1. Department Biozentrum, University of Basel, Klingelbergstrasse 50/70, Basel, 4056, Switzerland

    Dominik A. Herbst, Roman P. Jakob, Franziska Zähringer & Timm Maier

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Contributions

R.P.J. expressed, purified and crystallized MAS, obtained the crystal structure of the condensing region, collected SAXS data and cloned constructs. F.Z. cloned constructs and purified MAS, GpEryA and MsPks. D.A.H. purified MAS, optimized MAS crystallization, determined the structure of the isolated DH domains and the modifying region, collected SAXS data, analysed the data, performed homology modelling, cloned constructs, and wrote the manuscript. T.M. designed and guided research, analysed data, contributed to crystallographic analysis and wrote the manuscript.

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Correspondence to Timm Maier.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Reconstruction of the dimeric KS–AT di-domain and DH dimer organization.

a, The condensing region dimer was reconstructed by least-square fitting on DEBS KS5 (ref. 11) and multi-template homology modelling of disordered segments and the active-site loop (gold). Termini of the remodelled segments are indicated by black spheres. A pseudo-continuous β-sheet is formed across the dimer interface. The post-AT linker terminates close to the dimer axis. b, Close-up view on the reconstructed KS dimer with an active-site tunnel spanning both protomers (white), which is enclosed by four remodelled segments (gold). c, The active-site loop containing the catalytic Cys178 is dislocated in the monomeric (orange) form of MAS KS–AT, whereas the active-site His313 and His349 occupy the same position as in the dimeric DEBS KS5-AT5 structure (white-transparent). The canonical conformation of Cys178 observed in dimeric KS domains is restored in the dimeric KS–AT model (gold-transparent) d, MAS KS–AT (coloured, red line) reveals the most linear overall structure (right) of all PKSs/FAS condensing region structures6,11,12,66,67 (corresponding to Extended Data 6e, f). e, The DH active-site residues are located at the interface of the two hot-dog folds (light and dark green; active-site tunnel in white). f, Interdomain angles in DH dimers6,15,16,17. Dimers were superposed onto one protomer (left) of MAS, and the angles between two protomers are compared. For clarity, only MAS DH is shown in green, for other DH domains only one equivalent helix is highlighted in colour. The FAS pseudo-dimeric DH domains (red helix) adopt a V-shaped structure (interdomain angle 96°), while PKS DH dimers (various colours) are almost linear (167–203°). The MAS DH dimer (green) is bent to the opposite direction relative to FAS, and exhibits the largest interdomain angle (222°) (asterisks indicate DHs that are part of fully reducing modifying regions). g, h, Dimer interface of MAS DH (g) and dimer interface of the isolated DH of the CurH15 modPKS (h). Dimerization of MAS and CurH DH are mediated by ‘handshake’ interactions of the N-terminal hot-dog folds. In MAS DH, an N-terminal β-strand extension further contributes to dimerization.

Extended Data Figure 2 Effect of ACP deletion and electron density maps of the MAS modifying region crystal structure.

a, SAXS experiments reveal conserved scattering profiles for the modifying region with ACP (dotted orange) and without ACP (dotted green), which resemble the scattering curve of the SAXS-refined X-ray structure (green). b, c, The experimentally determined interatomic distance distributions are in agreement with the maximum extends of the modifying domain with (b) and without (c) ACP, 250 Å and 201 Å, respectively. In b a set of plausible ACP positions is shown (transparent), on the basis of the length of the KR–ACP linker. d, Unbiased Fobs − Fcalc omit difference map of the modifying region linkers in chain B (contoured at 2.5σ) is shown. e, Unbiased Fobs − Fcalc omit difference map of the post-AT linker helices in chain A and B (contoured at 2.5σ); the helices could be modelled because of stabilizing crystal contacts. f, Electron density maps covering the three different domain types as indicated (left: 2Fobs − Fcalc at 1.0σ; middle: bias-reduced density modified NCS average map at 1.0σ; right: bias-reduced density modified NCS average map at 1.0σ, with additional details revealed by applying a B-sharpening factor of − 80 Å2).

Extended Data Figure 3 Active site and structural comparison of the MAS ER and ΨKR/KR domains.

a, The MAS ER active-site tunnel (white) is lined by an NADP+ cofactor. b, An Fobs − Fcalc shaked omit map (contoured at 3.0σ) is shown for the NADP+ cofactor in chain J. c, The ER domains of FAS6, MAS, and the modPKS PpsC dimerize via continuous β-sheet formation between the nucleotide binding subdomains (ERNB), whereas the SpnB ER was crystallized as monomer and represents a group of isolated ER domains18,19. d, The active site of ΨKR/KR locates to an elongated surface groove, which partly extends to the ΨKR domain and is presumably closed upon ligand binding by a disordered lid region (aa 1948–1960). An Fobs − Fcalc omit map (contoured at 3.0σ) is shown for the partly ordered NADP+ cofactor. Left: surface; right: cartoon representation. e, MAS (pale yellow) features an N-terminal β1–α1–β2–α2 extension of the ΨKR Rossmann-fold, which is commonly found in PKSs (violet: tylosin PKS ΨKR1 (ref. 21)), but absent in FASs (green: porcine FAS (pFAS) ΨKR6). Secondary structure labels refer to MAS ΨKR. f, Average main chain B-factors across all chains reveal distally increasing flexibility with highest B-factors for the ΨKR domain, in particular its β–α–β–α extension, and the C-terminal ACP anchor.

Extended Data Figure 4 Alignment of linker regions of 55 fully reducing modifying regions of PKSs and FASs.

The alignment reveals sequence conservation of the β-sheet B1 (β1 and β2), which is inserted in a surface groove of the ΨKR/KR domain. In MAS, strands β3 and β4 form the second antiparallel β-sheet B2. The ER–KR linker is considerably shorter in a subgroup of modPKSs. Sequence numbers and secondary structure elements correspond to M. smegmatis MAS (MAS (Ms) highlighted in orange). All modules are labelled as protein name (organism abbreviation) Uniprot number. Modules of Msl-PKSs (green text), modPKSs (light green), fiPKSs (blue), and FASs (yellow) are grouped by phylogeny (for details and colour coding see Extended Data Fig. 7). Protein Data Bank accession numbers are indicated in the boxes representing the corresponding domains. Amino acids are shown in clustal colours. (*Diketide synthase; †PKS cluster contains non-colinear iterative modules; ‡modular non-colinear iPKS module; §trans-AT PKS.)

Extended Data Figure 5 Helical organization of central linking segments in MAS and modPKSs.

a, Assembly of the MAS central linking region from authentic crystal structures of the condensing and modifying regions. The two structures overlap in sequence by four residues (blue). b, Hybrid model based on the homology completed KS dimer and reconnected helical linkers. Ends of loops defined by the KS–AT crystal structure are indicated by black spheres. Disordered segments in the dimeric condensing region are reconstructed by multi-template homology modelling (gold); colour coding is as in a. c, d, Helix formation in sequence regions corresponding to central linkers are also observed in the isolated crystal structure of the modPKS DH domain of the fully reducing DEBS module 4 (ref. 16) (c), RifDH10 (ref. 17) (not shown), and in the crystal structure of the RhiE KS-B di-domain24 (d), where a KS domain is connected directly to a DH homologous domain, the B domain.

Extended Data Figure 6 Analysis of structural variability in the modifying and condensing regions of MAS and related multienzymes.

a–d, Analysis of interdomain conformational variability between the 18 protein chains in the MAS modifying region crystal structure. a, b, Variability of ER positioning relative to DH from two perspectives reveals a screw axis motion combining translation of up to 8.5 Å with rotation of up to 13.6°. c, d, Variability of ΨKR/KR domain orientation relative to DH (c) and ER (d), respectively, reveals a hinge located in the interdomain linker region. e, f, Top and front view of six overlayed KS–AT di-domain structures6,11,12,66,67 as indicated and the derived rotational distance of AT positioning around a common hinge in the LD. a–f, Relative locations of individual structures are highlighted by representative coloured helices. Translational components are indicated with an arrow on the rotation axes with signs indicated on the principle axis (thick, coloured according to the moving domain). All structures are aligned to a MAS reference domain (coloured ribbon). Rotation axes are shown for rotations larger than 6° and arrows are shown for translations larger than 1 Å.

Extended Data Figure 7 A comprehensive phylogenetic analysis classifies MAS into the branch of modPKSs

. Phylogenetic trees for 55 fully reducing MASs/PKSs/FASs modules were constructed on the basis of only KS domains (a), complete condensing regions (b), the ER domain (c), or all catalytic domains (d). M. smegmatis MAS (MAS (Ms), bold, italic) and Msl-PKSs (italic) are more closely related to modPKSs (light green) and distinct from fiPKSs (blue) and animal FASs (yellow). All modules are labelled as protein name (organism abbreviation) Uniprot number. Units are given as amino-acid substitutions per site. Indices correspond to Extended Data Fig. 5.

Extended Data Figure 8 SAXS analysis supports a MAS-like organization of PKS modifying regions.

Models (left) of modifying region organization and their respective theoretical and experimental scattering curves as well as pair–distance distributions (right) are shown. a, b, As proposed in ref. 19, the intact SpnB modifying region was modelled on the basis of the domain-swapped SpnB ER–ΨKR/KR structure, using either the structure of FAS (a) or of the MAS modifying region (b) as a guide for positioning KR relative to DH. The SpnB DH structure was generated by homology modelling. c, Model of the intact SpnB modifying region with dimeric DH and ER based on the structure of the intact MAS modifying region. d, Crystal structure of MAS before and after fitting to experimental SAXS data. A good fit (χ = 1.79) is obtained by fitting SAXS data with a single model corresponding to an average conformation of the MAS structure. e, Sequence organization of two authentic modPKS modifying regions of similar ER–KR linker length to SpnB (left), together with experimental SAXS scattering data (right). The data closely match calculated scattering curves for a MAS-like architecture, but disagree with models based on a monomeric ER as suggested for SpnB.

Extended Data Table 1 X-ray data collection and processing table
Full size table
Extended Data Table 2 Structural comparison and interface analysis
Full size table

Supplementary information

Conformational variability and coupling in the MAS modifying region

All dimeric modifying regions of the MAS crystal structure were aligned to the DH domains and combined into one animation. The ER dimer moves in a screw motion with a lateral translation of up to 8.5Å and a rotation of up to 13.6° on the dimeric DH platform. The ER motion is linked to a rotation of the laterally double-tethered ΨKR/KR domains and couples the conformations of the ΨKR/KR across the MAS modifying region dimer. The maximum observed rotation of the ΨKR/KR domains (40.4°) causes an active site distance shift of 10 Å (euclidean space) relative to the DH domain. (MOV 18497 kb)

Condensing region conformations in crystal structures of PKSs and FASs

Structures of the homologous condensing regions are aligned and animated from MAS over PDB: 2QO3, 2HG4, 4MZ0, 2VZ9 to 3HHD. The superposition indicates a common hinge for rotational mapping of the AT domain positions located in the linker domain (grey). MAS adopts the most linear arrangement of all AT domains, which differs by a rotation of 43.2° around the common hinge from those observed in the crystal structure of human FAS (PDB: 3HHD). (MOV 13319 kb)

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Herbst, D., Jakob, R., Zähringer, F. et al. Mycocerosic acid synthase exemplifies the architecture of reducing polyketide synthases. Nature 531, 533–537 (2016). https://doi.org/10.1038/nature16993

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