Structures and function of a tailoring oxidase in complex with a nonribosomal peptide synthetase module

Nonribosomal peptide synthetases (NRPSs) are large modular enzymes that synthesize secondary metabolites and natural product therapeutics. Most NRPS biosynthetic pathways include an NRPS and additional proteins that introduce chemical modifications before, during or after assembly-line synthesis. The bacillamide biosynthetic pathway is a common, three-protein system, with a decarboxylase that prepares an NRPS substrate, an NRPS, and an oxidase. Here, the pathway is reconstituted in vitro. The oxidase is shown to perform dehydrogenation of the thiazoline in the peptide intermediate while it is covalently attached to the NRPS, as the penultimate step in bacillamide D synthesis. Structural analysis of the oxidase reveals a dimeric, two-lobed architecture with a remnant RiPP recognition element and a dramatic wrapping loop. The oxidase forms a stable complex with the NRPS and dimerizes it. We visualized co-complexes of the oxidase bound to the elongation module of the NRPS using X-ray crystallography and cryo-EM. The three active sites (for adenylation, condensation/cyclization, and oxidation) form an elegant arc to facilitate substrate delivery. The structures enabled a proof-of-principle bioengineering experiment in which the BmdC oxidase domain is embedded into the NRPS.


REVIEWER COMMENTS Reviewer #1 (Remarks to the Author):
Most NRPS biosynthetic pathways include an NRPS and additional proteins that introduce chemical modifications before, during or after assembly-line synthesis. For example, bacillamide biosynthetic pathway is a three-protein BGC that contains genes for BmdA, BmdB, an NRPS and BmdC. BmdC appeared likely to oxidize the thiazoline ring to the thiazole, but there is no indication of the timing of this oxidation. In this study, the bacillamide pathway is reconstituted in vitro by the authors. The oxidase is shown to perform dehydrogenation of the thiazoline in the peptide intermediate while it is covalently attached to the NRPS, as the penultimate step in bacillamide D synthesis. Structural analysis of the oxidase using X-ray crystallography and cryo-EM reveals a dimeric, two-lobed architecture with a remnant RiPP recognition element and a dramatic wrapping loop. The oxidase forms a stable complex with the NRPS and dimerizes it. The structures enabled a proof-of-principle bioengineering experiment in which the BmdC oxidase domain is embedded into the NRPS. The experiments were well designed. It is recommended for publication in NC after minor revision, for example: 1.It should be more specific in the novelty description: this study reveals unexpected biochemical aspects and elegant structural features previously undescribed for any nonribosomal peptide synthesis (lines 77-79) 2. There are many format problems in the references (lines 729-968), which must be adjusted carefully: (1) some journal names are abbreviated but some are not, e.g. lines 736, 776, 814, 820, 828, 831…… (2) microbial name must be italic, e.

Reviewer #2 (Remarks to the Author):
This manuscript describes the structure and function of an NRPS which synthesizes Bacillamide D. The ternary structure of the complex, the BmdB-BmdC complex, was analyzed by X-ray crystallography and cryo-EM methods. These structures showed that BmdB exists as a BmdCinduced dimer. Most of the experiments are appropriately performed, and the manuscript is clearly written. However, there are several concerns that should be addressed.

Major concerns:
The tertiary structure and biochemical analysis of the BmdB-BmdC complex revealed a part of the functional mechanism of this complex. However, it is still unclear why the oxidation only occurs in the BmdB-BmdC complex. Although the authors discussed oxidase-NRPS interactions with several examples, there are no discussions to explain the results of Figs 3a, b, and c. It is fantastic if there is experimental evidence to explain these results based on 3D structures. At least, the authors should provide some discussions on this point. Figure S5a FSC lines are a bit poor. The authors should give some explanations for these FSC lines. Furthermore, they only provided FSC lines, and it is difficult to validate the results of the SPA analysis. In general, SPA analyses have been published with a flow chart of the SPA analysis, angular distribution, FSC curves(corrected maps, masked maps, unmasked maps, phase randomized masked maps), map-to-model FSC curve, and information of local resolutions. Please add these data to the supplementary information.
Minor concerns: p. 5, line 113 The authors used SEC for analyzing the molecular weight of proteins. However, it is not appropriate to use SEC for the molecular weight analysis. The molecular weight should be analyzed by SEC-MALS. At least, the authors should provide a standard line for SEC with molecular weight markers.
p. 6, line 120 "rmsd values of 2.0-2.9" should be "rmsd values of 2.0-2.9 Å". Furthermore, the number of matched atoms (probably, the number of CA atoms) should be provided.
p. 6, line 141-142 While they described that an FMN cofactor was found in the BmdC, no details are provided about catalytically important atoms in the FMN. They should provide more structural information around the FMN and discuss the catalytic mechanism of oxidation by BmdC.
p.8, line 208 They stated that "with the Ox:A2 interface seen at slightly different angles". It is better to describe this part more quantitatively.
p. 11, line 302 -p. 12, line 329 This part seems to be a review article. I wonder if these descriptions are required for the manuscript. I think this part should be shortened and clarify the point. Figure 1b Colors of the EIC and UV lines are very similar. Please use clearer colors. In addition, the font size of the axis title is too small. Particularly, superscripts are too small to see. The font size used in Sup Figure 1 is also tiny. Since molecular weight information is important, the authors should use a larger font size.

Figure 3d
There are many lines in one SEC chart, and it is hard to distinguish. Line colors are similar to each other. Please combine different colors and different line styles for clarity (Figure 5d is much better). Table S1, the column of PDB:7LY7 The statistics said that B-factors of protein atoms and ligand atoms were 178.71 and 99.39 Å2, respectively. B-factors for the ligand atoms were substantially smaller than those of the protein atoms. However, it is unlikely. Something wrong seems to be going on in the crystallographic refinement of 7LY7. The authors should check the results of the crystallographic refinement carefully. Figure S3b Lines in the graphs are difficult to distinguish. Please use clearer colors or use solid and dashed lines to make the differences clear.

Reviewer #3 (Remarks to the Author):
In this manuscript Fortinez et al describe the structural and functional analysis of a tailoring oxidase involved in the synthesis of the nonribosomal peptide, baccillamide D. Tailoring enzymes play an important role in non-ribosomal peptide synthesis but remain poorly characterized. The authors have demonstrated that the oxidase acts on a substrate tethered to the NRPS enzyme and that the oxidase forms a complex in solution with the NRPS enzyme. They have also used X-ray crystallography and Cryo-EM to determine the structure of the oxidase alone and in complex with the 2nd module of the NRPS enzyme. The structural information provides insights into how the oxidation reaction of the tethered peptide by the oxidase is integrated into peptide synthesis by the NRPS. Similar oxidases are found as domains embedded in NRPS enzymes in nature and the authors have used bioengineering to embed the baccillamide oxidase into the NRPS enzyme and shown that the embedded oxidase remains functional. The manuscript is well written and the conclusions are supported by the data. The methodology is sound and while there are clearly some issues remaining with the accuracy of some of the structural models (indicated by relatively high R-factors and number of geometry outliers) they are adequate for the resolution and likely as accurate as possible with the data available. This is a very interesting paper that significantly contributes to the understanding of non-ribosomal peptide synthesis and should be accepted with minor revisions.
A few suggestions and minor criticisms: Line 176: The authors reference Supplementary Fig. 3c here but I believe they meant to reference 3b iv?
The authors have not described the interaction observed between the A and Cy domains of the BmdB module. I assume it is similar to other NRPS A-C or A-Cy interactions but it would be useful to include a sentence or two to clarify this in the manuscript.
Line 212: At first glance, I found it a bit difficult to understand what the authors meant by "focusing on the BmdC dimer". I suggest rewording and perhaps include a bit more methodological detail here to clarify.
In Fig 4 b The authors have used white to depict the EM structure which is very similar to the colour used for one of the BmdC monomers. I suggest changing the colour of one of these to make the overlay a bit clearer.