Avalilação PREreview de A PLP-Dependent Decarboxylative Mannich Reaction Initiates Construction of the Nonpeptidic Scaffold of Kaitocephalin

In this manuscript, Noguchi et al. reported the characterization of the PLP-dependent enzyme KpbH from the biosynthetic cluster (BGC) of the fungal neuroactive natural product kaitocephalin. The authors demonstrate the enzyme catalyzes the decarboxylative Mannich-type C-C bond formation between aspartate and pyrroline-5-carboxylate (P5C) to afford ACPCA. A series of LC-MS and NMR experiments validated the in vitro production of ACPCA catalyzed by KpbH. Isotopic labelling experiments provided insights into the mechanism of KpbH catalysis. Feeding experiments supported ACPCA as an on-pathway intermediate toward kaitocephalin biosynthesis.

This work expands the catalytical diversity of PLP-dependent enzymes with another native Mannichase, and sheds light on the early biosynthetic pathway toward kaitocephalin. Overall, this manuscript is well-written and the conclusions are supported by experimental evidence. I provide a few minor suggestions that might further improve the manuscript:

1. The authors may consider adding a scheme showing the characterized KpbIBM pathway in Figure S1, which is not given in the manuscript nor SI. This will give the readers a better overview of the current knowledge of kaitocephalin biosynthesis, and recognize the unknowns in this pathway.

2. On page 5, the authors described the identification of an ornithine aminotransferase encoded outside of the kpb cluster that supplement P5C for KpbH catalysis. However, another ornithine aminotransferase, KpbB is encoded in the BGC and was confirmed to utilize ornithine (ref 7). Other than Kaito hydroxypyruvate, can KpbB also accept 2-oxoglutarate as co-substrate to efficiently produce P5C? The authors may want to comment on this option before moving on to complementary ornithine aminotransferases outside of the cluster, as with the presence of arginase KpbG, readers might expect this cluster to be self-sufficient on P5C production.

3. In figure 4, the third and fourth structures on top are resonance contributors of the same species, therefore a double-headed resonance arrow (↔) would better communicate the relationship between them than a reaction arrow (→).

4. On page 10, the authors reported that feeding of the D₂O-prepared reaction mixture increased the M+1/M+0 peak-area ratio of protonated KCP ions to 26.8%, exceeding the calculated natural-abundance ratio of 19.5%. Figure S10 showed that the authors did a very thorough job repeating the experiment several times. I suggest the authors to report the 26.8% value as mean ± SD in main text, which would indicate that this observation is outside of experimental error and therefore reliable.

I reviewed this preprint as part of the Transparent and Accountable Peer Review training program. This report was written with the support of my research mentor.

Competing interests

The authors declare that they have no competing interests.

Use of Artificial Intelligence (AI)

The authors declare that they did not use generative AI to come up with new ideas for their review.