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Crystal structure of PksH

The crystal structure of the dehydratase PksH (PDB 3HP0) has been determined to 2.3 Å resolution. Although deposited in the PDB, this structure is yet to be published. In light of this, we first undertook a detailed analysis of the PksH structure in an effort to establish a putative reaction mechanism for this enzyme. The final model of PksH describes an asymmetric unit (AU) comprised of six protein chains. There are no significant structural differences between each of the six chains that form the AU, with a maximum Cα r.m.s.d between individual chains of 0.48 Å. PksH is trimeric (Fig. 2A), with dimensions of 66 Å × 75 Å × 32 Å. Each trimer is formed across a crystal symmetry axis. A total of 17% (1,207 Å2) of the solvent-accessible surface area of each PksH monomer is buried at the trimer interface indicative that this oligomeric state is obligatory. As would be expected, the buried trimer interface is dominated by hydrophobic amino acids. The oligomeric state of PksH observed in the crystal structure is consistent with our hydrodynamic analyses that suggest that this enzyme is trimeric both in solution and in crystallo (Fig. 2B).

Figure 2

Structural studies of PksH and PksI. (A) X-ray crystal structures of PksH and PksI. Individual monomers comprising the PksH and PksI trimers are differentially coloured with colour match circles indicating the location of individual active sites within each monomer. (B) Chromatogram showing the elution profiles of PksH (green) and PksI (orange) from a Superdex 200 10/60 column (GE Healthcare) pre-equilibrated in 20 mM Tris–HCl, 150 mM NaCl, pH 7.5. 0.5 mg of each protein was loaded onto the column. Standards, in order of elution, are 1. thyroglobulin Ve = 10.1 (Mr = 670,000 Da), 2. γ-globulin Ve = 13.3 (Mr = 158,000 Da), 3. ovalbumin Ve = 15.2 (Mr = 44,000 Da), 4. myoglobin Ve = 16.8 (Mr = 17,000 Da) and 5. vitamin B12 Ve = 19.6 (Mr = 1,350 Da). (C) X-ray crystal structure of a single PksH monomer. (D) X-ray crystal structure of a single PksI monomer. (E) Details of the PksH active site with key amino acids and secondary structure elements highlighted. (F) Details of the PksI active site in both “closed” and “open” states, with key amino acids and secondary structure elements highlighted.

Each monomer within the PksH trimer possesses a single active site and these are positioned equidistant ~ 57 Å apart within the trimer. Each active site of PksH sits at the base of a solvent exposed channel positioned on the surface of the molecule (Fig. 2C). The fold of PksH is consistent with other CS members, with a characteristic ββα fold, consisting of two perpendicular β-sheets flanked by α-helices30. PksH contains a total of 8 β-strands (β1–β8), organized into a pair of β-sheets. The first of these is formed by strands β1, β2, β3, β5 and β7, the second by β4, β6, and β8 (Fig. 2C).

Active architecture of site of PksH

Despite sharing < 20% amino acid sequence identity with other structurally characterized CS members, PksH retains the distinctive protein fold common to these polypeptides. Superposition of the structure of PksH with those of related homologues thus enabled the unambiguous identification of the active site of the enzyme, along with a number of critical elements therein. These include the active site access tunnel, a putative oxyanion hole, and to a lesser extent, tentative assignment of catalytic residues within the enzyme active site. The identification of these critical features is complicated by the highly diverse sequences of CS proteins. This translates into divergent amino acid composition within the active sites of different superfamily members. Despite this, structural alignment of PksH with other CS polypeptides clearly indicates that the backbone amides of residues Ala67 and Gly114 form the oxyanion hole in this enzyme (Fig. 2E), which in the PksH structure is occupied by a single water molecule. The active site is comprised of the residues Asp68, Asn232, Gln86, Glu137, Ala67 and Gly114, of which Asp68 and Glu137 appear to be optimally positioned to contact the 3-hydroxy-3-methylglutaryl group of the authentic HMG-ACP substrate. This is based on both their respective locations within the enzyme active site and their proximity to the oxyanion hole.

Enzyme assays and catalytic mechanism of PksH

In an effort to elucidate the catalytic mechanism of PksH, the wild type (WT) enzyme, along with the point mutants PksH-D68A and PksH-E137A were produced recombinantly by overexpression in E. coli and purified to homogeneity using a two-step procedure (Table 1 and Materials and Methods). All proteins used for enzyme assays were of > 95% purity as judged by SDS-PAGE analysis. In the first instance, assays were attempted by monitoring the conversion of commercially sourced HMG-CoA or synthesized racemic HMG-N-acetylcysteamine (HMG-SNAC; see Materials and Methods) to 3-methyl glutaconyl-CoA (3MG-CoA) or its SNAC equivalent by WT PksH, using Liquid Chromatography coupled Mass Spectrometry (LCMS; Fig. 3). Both CoA and SNAC thioesters are frequently employed as surrogates of authentic ACP-bound intermediates. As racemic HMG-SNAC was used in all bioassays, it would be reasonable to expect that only one enantiomer would be recognised for dehydration. There was clear evidence of the PksH catalyzed conversion of CoA and SNAC tethered substrates to their respective glutaconyl products, as indicated by the observation of new peaks in the LCMS (Fig. 3B,C) with masses consistent with the expected products 4 (SI). However, reaction products were only observed following the incubation of assay mixes for > 72 h at 37 °C, incorporating > 5 mg/ml PksH. These data were interpreted to imply that the equilibrium of the PksH catalyzed transformation favors the reverse reaction. In an effort to circumvent this problem, we cloned and recombinantly overexpressed WT PksI in E. coli and purified the resulting protein to homogeneity. Reactions were repeated including recombinant WT PksI in addition to PksH, with both proteins at 1 mg/ml, and assays incubated for 16 h at 37 °C (Fig. 3). LCMS analysis of these coupled assays identified new peaks on the chromatograms, which eluted at either ~ 3.4 min (CoA) or ~ 5.5 min (SNAC) and possessed masses and retention times consistent with those of 3-methyl crotonyl-CoA (3MC-CoA) or 3-methyl crotonyl-SNAC (3MC-SNAC; Figs. 3B,C, and SI). The identities of these compounds were confirmed by comparison with synthetic standards (retention time and mass). Together, these data demonstrate that WT PksH is catalytically competent and also illustrate the value of employing the PksH/PksI coupled assay when assessing the activity of PksH and its mutants. PksH was found to accept and act upon both CoA and SNAC tethered substrates equally well under the assay conditions used. Following the successful demonstration of WT PksH activity, assays were repeated using the two PksH point mutants PksH-D68A and PksH-E137A (Fig. 4A,B). PksH-D68A was found to be active but kinetically impaired with both SNAC and CoA conjugated HMG. There was an almost complete loss of product 5 formation in assays including HMG-SNAC (Fig. 4A), and a modest reduction in product yield in assays incorporating HMG-CoA (Fig. 4B). PksH-E137A was found to be unable to catalyze the conversion of HMG-SNAC to 3-methyl glutaconyl-SNAC (Fig. 4A), and displayed significantly impaired activity with HMG-CoA, with a barely detectable quantity of crotonyl product 5 observed (Fig. 4B). These data imply a critical role for Glu137 in PksH activity. Given that both PksH mutants exhibit secondary structure compositions and hydrodynamic behaviors equivalent to that of the WT enzyme, as established by CD spectroscopy and SEC (data not shown), we conclude that the observed kinetic effects are not a consequence of protein misfolding. Based on these findings, we propose that the PksH catalyzed dehydration reaction occurs via hydrogen bonding of the thioester carbonyl by the backbone amides of Ala67 and Gly114 in the oxyanion hole and deprotonation by Glu137 to give 6 (Fig. 5A). Dehydration then occurs to give a 3-methyl glutaconyl product and water, with Asp68 functioning as a proton donor. The observed impaired activity of PksH-D68A may reflect the ability of an active site water to act as a surrogate proton source in this mutant.

Table 1 Primers used during this study.
Figure 3
figure3

PksH and PksI enzyme activity assays. (A) Reaction scheme for the PksH/PksI conversion of substrate analogues (3) to products (4) and (5). Free acid molecular weights of the SNAC and CoA derivatives of the compounds are shown below each structure. (B) LCMS diode-array detector chromatograms (210–400 nm) of assays for PksH and PksI using HMG-SNAC substrates. (C) LCMS diode-array detector chromatograms (210–400 nm) of assays for PksH and PksI using HMG-CoA. Peaks correspond to HMG (3), 3-MG (4) and 3-MC (5) derivatives. The peak marked * contained free CoA. The chromatogram for synthetic 3-MC SNAC (5) is also shown in (B). Corresponding mass spectra are shown in SI.

Figure 4
figure4

Enzyme activity assays using PksH and PksI mutants. (A) LCMS chromatograms for PksH/PksI coupled assays incorporating mutants of PksH and WT PksI using the substrate HMG-SNAC. (B) LCMS chromatograms for PksH/PksI coupled assays incorporating WT PksH and mutants of PksI using the substrate HMG-SNAC. (C) LCMS chromatograms for PksH/PksI coupled assays incorporating mutants of PksH and WT PksI using the substrate HMG-CoA. (D) LCMS chromatograms for PksH/PksI coupled assays incorporating WT PksH and mutants of PksI using the substrate HMG-CoA. All chromatograms were recorded in the 210–400 nm range using a diode-array detector. The peak marked * contained free CoA.

Figure 5
figure5

Catalytic mechanisms of PksH and PksI. (A) Proposed catalytic mechanism of PksH. (B) Proposed catalytic mechanism of PksI.

Crystal structure of PksI

To complement our studies of PksH we next turned our attention to its partner decarboxylase PksI. WT PksI was recombinantly overexpressed in E. coli and purified to homogeneity (Table 1). This polypeptide was crystallized and its structure determined using X-ray diffraction methods (Table 2). Two crystal structures of PksI were elucidated; the first to 1.93 Å resolution using a crystal cryoprotected in glycerol (PksI_GOL), and the second to 2.1 Å using a crystal cryoprotected using ethylene glycol (PksI_EG). Both structures describe an asymmetric unit comprised of three protein chains arranged in the form of a trimer (Fig. 2A). This oligomeric state is consistent with that previously reported for CurF29, the equivalent enzyme from the curacin cis-AT PKS pathway, with a Cα r.m.s.d between the two structures of 1.2 Å. SEC analysis of recombinant PksI demonstrates that this protein is trimeric both in solution and in the crystal (Fig. 2B). Each trimer exhibits a discoidal shape analogous to that of PksH, with dimensions of 83 Å × 75 Å × 39 Å. The final model of PksI_GOL comprises residues 1–247 of chain A, residues 4–249 of chain B and residues 3–231 of chain C, 663 water molecules, 6 glycerol molecules and 1 HEPES molecule. The final model of PksI_EG comprises residues 1–248 of chain A, residues 2–248 of chain B, and residues 4–232 of chain C, 417 water molecules, 4 molecules of ethylene glycol and 2 molecules of glycerol. There is a single active site per PksI monomer (Fig. 2A), with each active site positioned on the periphery of the trimer, ~ 43 Å from one other. Akin to PksH, PksI adopts a characteristic CS fold, with a ββα core, that comprises two approximately perpendicular -sheets surrounded by -helices (Fig. 2D).

Table 2 Summary of X-ray data collection and refinement statistics.

Active architecture of site of PksI

The active site of PksI is located at the base of a solvent exposed channel that permits access to the catalytic machinery of the enzyme. The base of the channel houses a single histidine residue, His230, which occupies an equivalent position to His240 in CurF (Figs. 2F and S1). Based on this positioning His230 would be predicted to be the catalytic histidine of PksI. A histidine at this position is universally conserved in all other -methyl branching decarboxylases for which sequences have been reported. In addition to His230 the active site of PksI comprises the residues Phe81, Phe136, Phe234 and His235. In the PksI_GOL structure a single monomer (chain A) within the PksI trimer possess a bound glycerol molecule within its active site. Comparison of the active site architecture of PksI monomers in glycerol bound and free states allows assessment of active site reorganization as a consequence of ligand binding (Fig. 2F). In the unliganded structure, the active site adopts an “closed” conformation, with the side chains of Phe136 and Phe81 pointing directly into the centre of the active site cavity and His230 rotated to face away from the central chamber (Fig. 2F). In the liganded structure, the active site is occupied by the substrate mimic glycerol, whose hydroxyl group can form a hydrogen bond with His230. For glycerol to be accommodated within the active site of PksI, the side chains of residues in this region must be reconfigured accordingly. In this “open” conformation the side chains of Phe136 and Phe81 are seen to flip away from the centre of the active site, which is occupied by the bound glycerol molecule. As a consequence, Phe136 forms a tripartite stacking interaction with Phe234 and His235, which sits beneath the proposed catalytic residue His230, which itself rotates to point directly into the centre of the active site. It is likely that our unliganded structure represents the ground state of the enzyme, with the glycerol bound form equating to a mimic of a PksI reaction intermediate. The observed side chain rearrangements in the glycerol bound structure are essential for allowing the substrate to be accommodated within the catalytic site of the enzyme and may also function to promote active site solvation, consistent with a potential role for water as a proton donor during catalysis. In the PksI_EG structure all the active sites adopt the “closed” ground state conformation.

Enzyme assays and catalytic mechanism of PksI

To extend our structural studies of PksI, we next sought to elucidate the catalytic mechanism of this enzyme. Kinetic assays were performed using the same coupled assay system as that developed to study PksH, employing either commercially sourced HMG-CoA or synthesized racemic HMG-SNAC as substrates (Fig. 3). Assays were conducted using WT PksI, along with the point mutants PksI-H230A, PksI-H235A, PksI-K80A, PksI-K232A. Although our structural data strongly implied that His230 was the catalytic histidine in the PksI reaction, the positioning of His235 on the periphery of the enzyme active site meant that a role for this residue in catalysis could not be discounted. The two lysine mutants were generated in an effort to identify the proton donor for the PksI catalyzed reaction. In the proposed decarboxylation mechanism of CurF, an active site lysine was shown to be critical for enzyme activity29. Based on our available crystal structures both Lys80 and Lys232 were identified as potential candidates for this role in PksI. WT and mutant proteins were produced recombinantly by overexpression in E. coli and purified to homogeneity using a two-step procedure. All proteins used for enzyme assays were of > 95% purity as judged by SDS-PAGE analysis. As reported above PksH/PksI coupled assays gave reaction products with retention properties and masses consistent with the predicted crotonyl products 5 (Fig. 3 and SI). In all cases HMG-CoA was found to be a better starting substrate for PksI (WT and mutants) than HMG-SNAC, implying a preference for longer chain substrate mimetics (Fig. 3B,C).

Of the four PksI mutants investigated only PksI-H230A was found to be catalytically compromised (Fig. 4C,D), with barely detectable activity for 3-methyl glutaconyl-SNAC and significantly impaired activity for 3-methyl glutaconyl-CoA. This observation is consistent with the proposed key role of this amino acid in the decarboxylation reaction. Given that PksI-H235A showed in vitro activity equivalent to that of the WT enzyme, a role for this residue in PksI catalysis can be discounted. Surprisingly, PksI-K80A and PksI-K232A both display catalytic activities akin to that of WT-PksI, with no evidence of functional impairment. Together, these data imply a deviant mechanism of catalysis in PksI distinct from that previously reported for CurF. This is unsurprising given the disparities between the different active site architectures of these two enzymes. In the absence of any other potential proton donors within the PksI active site, we thus propose a variant mechanism of decarboxylation in which His230 functions as both an acid and a base at physiological pH (Fig. 5B). It is unclear at this stage as to the origin of PksI-H230A activity for the CoA tethered substrate, with the observed turnover (albeit reduced) likely arising from the presence of an active site water molecule.

In tandem with kinetic assays, the crystal structures of PksI-K80A, PksI-K232A and H230A were determined (Table 2). The active sites of each of these enzymes displays the ground state “closed” conformation as observed in the WT enzyme, with no evidence of significant structural perturbations within the active site. These data thus imply that the observed kinetic effects are not attributable to global or local structural reorganization within the enzyme as a consequence of the amino acid substitutions made.

PksH and PksI do not form heterotrimeric complexes

Given their significant structural identities we hypothesized that PksH and PksI may co-assemble to form heterotrimeric complexes. Such an organisation would provide an elegant solution to achieving spatial colocation of dehydration and decarboxylation activities during β-methyl branch processing. In a bid to establish if PksH and PksI were able to form heterotrimers, these proteins were co-expressed in E. coli and the resulting material purified to homogeneity. The expression system used was chosen to enable the selection of dual pksI/pksH transformants, using the antibiotics carbenicillin (pksI::pOPINF) and kanamycin (pksH::pET28A), followed by recombinant co-expression and protein purification. Although both constructs encode N-terminally hexa-his tagged proteins, these tags may be cleaved using different proteases; PksH thrombin cleavable, PksI C3 cleavable. It is therefore possible to selectively cleave the his-tag from either protein while leaving an intact tag fused to the other, thus enabling the identification and recovery of heterotrimeric complexes using nickel affinity chromatography.

In the first instance control experiments were conducted to demonstrate that selective his-tag cleavage could be achieved. Treatment of recombinant his-tagged PksH with thrombin results in a small mass change observable by SDS-PAGE consistent with tag removal, with analogous behavior observed upon treatment of PksI with C3 protease (Fig. 6A). Treatment of PksH with C3 protease and PksI with thrombin, results in no mass change consistent with no tag cleavage (Fig. 6A). These data illustrate target specific activity for the proteases employed in his study. Next, protease treated samples were subjected to HisPur Ni–NTA Spin Column repurification in an effort to establish their capacity to bind immobilized nickel ions. Both untreated and thrombin treated PksI bound to the Ni–NTA column matrix and showed no reduction in molecular weight (Fig. 6B). In contrast, C3 treated PksI was not retained on the column and exhibited a reduction in mass consistent with his-tag removal. Equivalent results were obtained when this experiment was repeated substituting PksI for PksH (Fig. 6C). In this instance, his-tag cleavage and his-load buffer elution is only observed for protein samples pretreated with thrombin. Co-expressed and purified PksH/PksI was subjected to the same protease digestion regime as outlined above (Fig. 6D). In the absence of protease both polypeptides are observed in the column eluent only. When incubated with C3 protease a clear reduction in mass is observed consistent with tag cleavage from PksI, but not from PksH (Fig. 6A). When this material is subjected to HisPur Ni–NTA repurification a clear band is observed in the column flow through, consistent with non-retention of PksI (Fig. 6D). Following treatment with elution buffer, however, a single band is observed by SDS-PAGE in the eluate with a molecular weight consistent with that of PksH (Fig. 6D). There is no evidence of PksI in this sample, and by extension, no evidence of PksH/PksI heterotrimer formation. Treatment of co-expressed and purified PksH/PksI with thrombin results in a clear reduction in the mass of PksH (Fig. 6A). As his-tag cleaved PksH possesses the same molecular weight as uncleaved PksI a single band is observed on the gel. Repurification of this material gave a single species with a molecular weight consistent with that of tag cleaved PksH, implying that this protein is not retained on the column following thrombin treatment. SDS-PAGE analysis of the eluted sample reveals a single species with a molecular weight consistent with non-tag cleaved PksI. Given that it is not possible to distinguish between tag cleaved PksH and uncleaved PksI this band was subjected to tryptic digest MALDI MS and was identified as PksI only (Figures S2 and S3). Together, these data demonstrate that PksH and PksI do not form heterotrimeric complexes, even when co-expressed and purified.

Figure 6
figure6

Co-expression and pull-down assays for PksH/PksI heterotrimer formation. Co-expressed and purified His6PksH and His6PksI were treated with C3 protease (to cleave His6PksI) or thrombin protease (to cleave His6PksH), then repurified by Ni2+-affinity. (A) SDS-PAGE gel showing the test digestion of PksH, PksI and coexpressed PksH/PksI with 3C protease and thrombin. + / − signs indicate the presence or absence of the respective protease. (B) SDS-PAGE gel showing PksI protease digests and subsequent Ni2+-affinity repurification. (C) SDS-PAGE gel showing PksH protease digests and subsequent Ni2+-affinity repurification. (D) SDS-PAGE gel showing protease digests and subsequent Ni2+-affinity repurification coexpressed PksH-PksI. The band marked * was analyzed by tryptic digest MALDI MS and was identified as PksI only. Labels correspond to the following; total protein loaded (T), column flow-through (FT), column wash (W) and column eluate (E). All gel images were collected using a BioDoc-It imaging system (UVP Ltd.) with integrated Doc-It LS Analysis software version 1 (https://www.uvp.com/) applying the system’s default imaging settings.

Discussion

In this study we present a structural and functional description of the bacillaene synthase β-branching enzymes PksH and PksI. These biocatalysts are responsible for the final two steps in β-methyl branch incorporation in this PKS/NRPS pathway. Crystals structures of both enzymes, in combination with in vitro enzyme assays using synthesized substrate analogues and mutagenesis studies, have provided insight into the catalytic mechanisms of these two enzymes. Our findings constitute the first reported molecular description of a HCS cassette dehydratase, and identify a deviant catalytic mechanism in PksI, which is distinct from that previously reported in other HCS cassette decarboxylases.

Our structural studies demonstrate that both PksH and PksI adopt distinctive trimeric CS folds, both in vitro and in crystallo. The active sites of both enzymes (one per monomer) are situated in equivalent locations on the periphery of their respective oligomers. The chemically disparate nature of the dehydration and decarboxylation reactions catalyzed by these polypeptides serves to further highlight the diverse array of chemistries that can be supported by CS members. Intriguingly, despite possessing analogous folds and trimeric architectures, we have been unable to provide any evidence that these polypeptides can co-assemble to form heterotrimeric complexes, even when heterologously co-expressed in E. coli. This is likely to be a direct consequence of the incompatibility of the amino acid residues that occupy the trimer interfaces of either protein. Significantly, in these regions the two polypeptides share < 10% primary amino acid sequence identity. This reflects the disparate residue composition of both proteins at their respective trimer interfaces and offers an explanation for their inability to co-assemble into a single functional unit.

PksH has been shown herein to catalyze the dehydration of HMG substrates in vitro. However, the low product yield appears to reflect the unfavourable equilibrium of this reaction. Through the development of a PksH/PksI coupled assay, it has proven possible to investigate the catalytic activity of this enzyme and guided by the available crystal structure, in tandem with mutagenesis studies, we have probed the dehydration mechanism of this protein. These data show that catalysis by PksH is dependent on the residue Glu137 and is impacted by substitution of Asp68 with alanine, implying a potential role for this latter amino acid as a proton donor in the PksH catalyzed reaction. In keeping with other CS family members, this mechanism is enabled through the polarization of the substrate thioester carbonyl by an active site oxyanion hole, resulting in formation of intermediate 6 (Fig. 5A).

In addition to our studies of PksH, we have also investigated the structure and mechanism of its partner decarboxylase PksI. The activity of this enzyme has been demonstrated in vitro using the same coupled assay system as that employed for PksH. Structure guided mutagenesis and enzyme assays have been used to delineate the catalytic mechanism of this polypeptide. The PksI reaction is shown herein to be dependent on the single catalytic residue His230, indicating a mechanism distinct from that proposed in the curacin cis-AT branching decarboxylase CurF. In CurF, an active site lysine was identified as the proton donor29. However, in PksI, mutation of the two candidate lysine residues in the active site of this enzyme to alanines yielded proteins with activities equivalent to that of the WT protein. Crystal structures of the mutants PksI_K80A, PksI K232A and PksI H230A revealed no significant changes in active site architecture beyond side chain identity. We thus propose a His230 dependent mechanism wherein this residue has a dual function both catalyzing decarboxylation and to act as a proton donor to the decarboxylated intermediate 7 to give the reaction product (Fig. 5B). Progression of the reaction is facilitated by stabilization of the intermediate by an oxyanion hole formed by the backbone amides of Gly66 and Gly108.

Structural studies of WT PksI have also serendipitously revealed that substrate binding is accompanied by significant reorganisation of the enzyme active site. In the absence of bound ligand, the PksI active site adopts an “closed” conformation, where the side chains of residues Phe81 and Phe136 occupy the central solvent exposed cavity of the enzyme active site. In this state the catalytic residue His230 is found to point away from the centre of the site in an apparent catalytically incompetent conformation. Upon ligand binding, the side chains of Phe81 and Phe136 rotate away to enable substrate to be accommodated in the active site pocket. In this conformation Phe234 forms a π–π stacking interaction with Phe136, and the side chain of His230 rotates to present the imidazole N1 for hydrogen bonding to the substrate terminal hydroxyl. The high proportion of aromatic amino acid content within the PksI active site is not shared in other HCS cassette decarboxylases, which implies that the observed ligand induced active site reorganisation represents a distinct binding mode in this enzyme. Unlike PksH, which displays no preference for CoA over SNAC tethered substrates, or vice versa, PksI shows significantly greater in vitro activity when using HMG-CoA as opposed to HMG-SNAC as the coupled assay starting substrate. This indicates a requirement for longer chain substrates in this enzyme. This is unsurprising, given that the authentic substrate for both the dehydration and decarboxylation reactions would be presented on the phosphopantetheine arm of either of the PksL di-domain ACPs. It is also important to emphasise that in this context protein–protein interactions between both PksH and PksI, and the substrate presenting ACPs, will significantly influence the rate and processivity of methyl branch incorporation. This may act to negate any requirement for the coordinated recruitment of PksH and PksI to the branching ACPs as indicated by their inability to co-assemble into a single functional oligomer.

In summary, the data presented herein reveal the molecular intricacies of the final steps in β-methyl branch incorporation in bacillaene biosynthesis, and further expand the mechanistic diversity of enzyme chemistries that can be supported by CS family polypeptides. It is anticipated that these findings will prove valuable in informing future efforts in the exploitation of PksH/PksI and related biocatalysts for the targeted introduction of methyl branches into unbranched polyketides, further expanding the natural product repertoire and facilitating access to new bioactive scaffolds.



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