Investigating the versatility of cytochalasan cytochrome P450 monooxygenases using combinatorial biosynthesis reveals stereochemical restrictions

This study utilizes combinatorial biosynthesis and genome mining to characterize cytochalasan cytochrome P450 monooxygenases, revealing that while these enzymes exhibit intrinsic promiscuity toward structurally related analogues, their activity on non-native substrates is strictly governed by the stereochemistry of functional groups rather than macrocycle size.

The Gist

Imagine a massive, bustling factory inside a fungus. This factory produces a special type of product called cytochalasans. Think of these products as intricate, twisted necklaces made of beads (carbon chains) and a special clasp (an amino acid). These necklaces are famous because they can stop a cell's internal skeleton (actin) from growing, which gives them powerful effects like killing cancer cells or stopping viruses.

However, the factory doesn't just make one type of necklace. It makes thousands of slightly different versions. Some have extra beads, some have different colored beads, and some have little charms attached.

The "assembly line" for the basic necklace is well understood. But the final step—adding those special charms and decorations—is done by a team of P450 enzymes. You can think of these enzymes as highly skilled, magical painters. Their job is to take the plain necklace and paint specific spots with oxygen (adding hydroxyl groups or epoxides) to change the necklace's properties.

The Big Question

The scientists in this paper asked a simple question: How flexible are these magical painters?

If we take a painter from Factory A and give them the necklace from Factory B, will they still know how to paint it? Or are they so picky that they only work on their own specific necklace?

The Experiment: A "Mix-and-Match" Factory

To find out, the researchers used a technique called combinatorial biosynthesis. Imagine taking the instruction manual (genes) for a painter from one fungus and plugging it into the factory of a different fungus.

1. The Host Factory: They used a mutant fungus (Magnaporthe grisea) that was missing its own painter. Because of this, the factory produced a "plain" necklace (pyrichalasin H) that was missing a crucial decoration.
2. The Guest Painters: They introduced 7 different painters (P450 enzymes) from various other fungi, some of which were previously unknown ("cryptic").
3. The Test: They watched to see if the guest painters could fix the plain necklace or create new, decorated versions.

What They Discovered

1. The "Universal" Painter vs. The "Picky" Painter
Some painters were surprisingly versatile. One painter from a fungus called Chaetomium globosum successfully fixed the host's necklace, adding the missing decoration. This showed that some enzymes are like universal adapters—they can work on different shapes as long as the basic structure is similar.

2. The "Stereochemistry" Trap
However, many other painters failed. They looked at the necklace, tried to paint it, and just... stopped. Why?

The researchers found that the shape and orientation of the beads mattered more than the size of the necklace.

• The Analogy: Imagine trying to put a key into a lock. The key might be the right size (the necklace is the right length), but if the teeth on the key are facing the wrong way (the wrong 3D orientation), it won't turn.
• In the fungus world, this is called stereochemistry. The methyl groups (little side-chains on the necklace) can point "up" or "down." If the guest painter expects the side-chain to point "up" but the host's necklace has it pointing "down," the painter gets confused and refuses to work.

3. The Hidden Clue: The "Thioredoxin" Buddy
While scanning the factories, the scientists found a strange, tiny helper enzyme (a thioredoxin-like protein) that always appeared next to a specific type of painter (BVMO). They suspect this helper is like a safety net that catches dangerous sparks (peroxide radicals) released by the painter, preventing the factory from blowing up. They tried to remove this helper to study it, but the fungus fought back, making it hard to delete.

4. A New Product Found
By growing a specific fungus (Aspergillus heteromorphus) in different "diets" (media), they managed to trigger the factory to produce a new, never-before-seen necklace. They couldn't isolate it fully to see its exact shape, but the chemical fingerprints suggested it was a unique variation with a specific arrangement of beads.

5. The Shortcut: "Feeding" the Factory
Finally, they tried a shortcut. Instead of swapping the painters, they took a plain necklace made by the host and literally fed it to the factory. The factory's native enzymes grabbed the necklace and decorated it. This proved that you can take a finished product from one place, drop it into another, and let the local enzymes finish the job. This is a much faster way to make new variations than swapping the whole instruction manual.

The Takeaway

The main lesson from this study is that nature's enzymes are versatile, but they have strict rules.

• Size doesn't matter as much as you think: A painter can handle a slightly longer or shorter necklace.
• Orientation is everything: If the little side-chains on the necklace are facing the wrong way (wrong stereochemistry), the painter won't touch it.

This discovery is huge for drug development. It means scientists can now design better drugs by carefully matching the "necklace" (substrate) with the right "painter" (enzyme). If they get the orientation right, they can create entirely new, powerful medicines that nature hasn't made yet, using these biological tools.

In short: The scientists learned that while these biological painters are talented, they are also very particular about the 3D shape of the object they are painting. Get the shape right, and you can create a whole new world of medicines. Get it wrong, and the painter just walks away.

Technical Summary

1. Problem Statement

Cytochalasans are a diverse family of fungal natural products with significant biological activities (e.g., actin polymerization inhibition, cytotoxicity). Their structural diversity arises from tailoring enzymes, particularly Cytochrome P450 monooxygenases (P450s), which perform oxidative modifications (hydroxylation, epoxidation) on the macrocyclic core.

• The Challenge: While P450s are known to have some substrate promiscuity, the specific rules governing their activity on non-native substrates are unclear.
• The Gap: Previous studies focused on closely related species. It remains unknown whether P450s from cryptic (silent) biosynthetic gene clusters (BGCs) can function on structurally distinct cytochalasan backbones, and what specific structural features (e.g., macrocycle size vs. stereochemistry) restrict their activity.
• Synthesis Difficulty: Chemical synthesis of cytochalasans is arduous due to complex stereochemistry and protection/deprotection steps. Chemoenzymatic approaches are desired but require a deep understanding of P450 substrate scope.

2. Methodology

The authors employed a multi-faceted approach combining bioinformatics, heterologous expression, and biotransformation:

• Genome Mining: Used the conserved Diels-Alderase (DA) sequence (PyiF) as a query to identify cryptic cytochalasan BGCs in six fungal species (Aspergillus heteromorphus, A. sclerotioniger, Colletotrichum spinosum, C. sidae, Metarhizium brunneum, and M. robertsii).
• Bioinformatic Analysis:
  • Annotated BGCs to identify core enzymes (PKS-NRPS, trans-ER, HYD, DA) and tailoring enzymes.
  • Identified a conserved thioredoxin-like (TRX) gene co-occurring with Baeyer-Villiger monooxygenases (BVMOs).
  • Constructed phylogenetic trees of P450s to correlate sequence with regioselectivity (macrocycle vs. cyclohexene oxidation).
  • Used AlphaFold to model P450 structures, focusing on N-terminal $\alpha$-helices (ER anchoring).
• Combinatorial Biosynthesis (Heterologous Expression):
  • Host: Magnaporthe grisea mutant strains ($\Delta$pyiD and $\Delta$pyiG) lacking specific P450s, producing pyrichalasin H analogues.
  • Strategy: Expressed cryptic and characterized P450s (from Chaetomium, Aspergillus, Xylaria, Metarhizium, Colletotrichum) in the M. grisea host to test if they could restore wild-type pyrichalasin H production or generate new metabolites.
  • Verification: Used RT-PCR to confirm transcription and splicing of introns in the heterologous host.
• Biotransformation: Purified the intermediate 7-deshydroxypyrichalasin H and fed it exogenously to M. grisea strains to test if native enzymes could modify the external substrate.
• Analytical Techniques: HR-LCMS/MS, MZmine, MS-DIAL, and NMR for metabolite identification and structural elucidation.

3. Key Contributions

A. Discovery of Cryptic BGCs and a Putative New Metabolite

• Identified cryptic BGCs in six fungal species.
• Discovered a highly conserved TRX-like gene co-occurring with BVMOs across diverse cytochalasan BGCs, hypothesizing its role in scavenging peroxide radicals released during BVMO catalysis (uncoupling).
• Cultivated A. heteromorphus CBS 117.55 and detected a putative new cytochalasan (m/z 496.2291, C$_{28}$H$_{34}$NO$_7^+$) via HR-LCMS/MS. Based on BGC homology and MS fragmentation, it is proposed to be a C18-derived cytochalasan lacking methyl groups at C-16 and C-18.

B. Phylogenetic and Structural Insights

• Regioselectivity Clustering: Phylogenetic analysis confirmed that P450s cluster based on their target site: cyclohexene oxidation (epoxidation) vs. macrocycle oxidation (hydroxylation).
• Structural Correlation: Macrocycle-associated P450s sub-clades reflect specific structural features of the final molecule, including polyketide chain length (C16 vs. C18) and the type of amino acid incorporated.
• N-terminal Helix: Identified that macrocycle P450s typically possess an extended N-terminal $\alpha$-helix (20–60 aa) for ER anchoring, which is often truncated in cryptic sequences but can be bioinformatically recovered.

C. Functional Characterization of P450s

The study systematically tested the activity of seven P450s in M. grisea:

• Active:
  • CHGG_01243 (from C. globosum): Restored pyrichalasin H production (hydroxylated C-18), though it did not act iteratively on the non-native substrate.
  • AhCYP2 (from A. heteromorphus): Successfully restored pyrichalasin H production, confirming it is a functional macrocycle P450.
  • CspCYP2 (from C. spinosum): Restored pyrichalasin H in a $\Delta$pyiG host, confirming its role as a cyclohexene epoxidase.
• Inactive:
  • CcsD (A. clavatus), XsCYP2 (Xylaria sp.), MrCYP1 (M. robertsii), and CspCYP1 (C. spinosum) failed to produce new metabolites or restore pyrichalasin H, despite correct transcription.
  • Note: Inactivity of CcsD was partly attributed to intron splicing issues in the host.

D. Identification of Stereochemical Restrictions

The most significant finding is the stereochemical restriction of P450 activity:

• Macrocycle Size vs. Stereochemistry: The size of the macrocycle (e.g., C16 vs. C18) was not the primary barrier to activity.
• Stereochemistry is Critical: The inactivity of XsCYP2 and MrCYP1 on pyrichalasin H intermediates was attributed to the stereochemistry of methyl groups at C-16 and C-18.
  • Pyrichalasin H has cis-methyl groups at these positions.
  • The native substrates for XsCYP2 and MrCYP1 (e.g., 19,20-epoxycytochalasin D) have trans-methyl groups.
  • The P450 active sites appear to be highly sensitive to the 3D orientation of these methyl groups, preventing hydrogen abstraction on non-native substrates with mismatched stereochemistry.

4. Results

• Biotransformation Success: Exogenous feeding of 7-deshydroxypyrichalasin H to M. grisea $\Delta$pyiD successfully restored pyrichalasin H production, proving that cytochalasans can penetrate the fungal cell wall and be modified by native enzymes. This offers a faster alternative to combinatorial biosynthesis for generating analogues.
• Iterative vs. Non-Iterative: The known iterative P450 (CHGG_01243) acted as a single hydroxylase on the non-native substrate, suggesting iterative behavior is substrate-dependent.
• Gene Knockout Failure: Attempts to knock out the TRX-like gene in A. clavatus failed, likely due to the host's reliance on non-homologous end-joining (NHEJ) rather than homologous recombination.

5. Significance

• Biocatalyst Selection: The study provides a framework for selecting P450s for chemoenzymatic synthesis. It demonstrates that while P450s have intrinsic promiscuity, their utility is strictly limited by the stereochemistry of the substrate backbone, not just the macrocycle size.
• Natural Product Discovery: The identification of cryptic BGCs and the detection of a new putative cytochalasan in A. heteromorphus expands the known chemical space of this family.
• Methodological Advancement: The successful use of biotransformation (exogenous feeding) offers a streamlined route to functionalize cytochalasan skeletons without the laborious steps of gene cloning, transformation, and screening required for combinatorial biosynthesis.
• Evolutionary Insight: The correlation between P450 phylogeny and the specific stereochemical features of the polyketide chain suggests that the PKS-NRPS machinery and P450 tailoring enzymes co-evolved to maintain specific stereochemical configurations essential for enzyme recognition.

In conclusion, this paper moves beyond simple substrate promiscuity to define the stereochemical boundaries of cytochalasan P450s, offering critical insights for the rational design of biocatalytic pathways to produce novel, bioactive cytochalasan derivatives.