Deciphering the evolutionary origin of the stereoselectivity of short-chain dehydrogenases in the oxidation of the monoterpenol 1-borneol

This study elucidates how high enantioselectivity in borneol dehydrogenases evolved through a combination of a single active-site mutation (I111L) and peripheral mutations that modulate the hydrophobic pocket's solvent-accessible surface area, offering a blueprint for rational protein engineering.

The Gist

Imagine enzymes as highly specialized lock-and-key machines found in nature. In this story, the "key" is a molecule called 1-borneol (a type of plant oil), and the "lock" is an enzyme called borneol dehydrogenase (BDH). The goal of the enzyme is to change this key into something else, but it needs to be very picky: it should only work on one specific version of the key (like a left-handed glove) and ignore the mirror-image version (a right-handed glove). This pickiness is called enantioselectivity.

Scientists have long known that the "lock" has a deep, oily (hydrophobic) pocket where the key fits. However, understanding exactly how this oily pocket decides which key to accept has been like trying to solve a puzzle in the dark. It's hard to predict how to tweak these machines to make them more picky.

The Mystery of the Evolutionary Path
The researchers wanted to figure out how nature evolved these enzymes to become so picky over time. Did they change the shape of the main "lock" (the active site) directly? Or did they make small changes to the "frame" of the machine (the outer edges) that indirectly tightened the lock?

To solve this, they used a technique called ancestral sequence reconstruction. Think of this as a time machine for DNA. They looked at modern enzymes from plants like sage and rosemary (which are very picky) and worked backward to guess what their ancient, common ancestors looked like.

The Journey from "Messy" to "Precise"
They found a sequence of ancestors, starting with an ancient one (N30) that was very sloppy—it couldn't tell the difference between the left and right versions of the key. They then traced the path to a younger ancestor (N32) that was much more precise.

Here is the surprising part of the journey:

• Between the sloppy ancestor and the precise one, nature only made 19 small changes (swapping 19 amino acids, which are the building blocks of proteins).
• 18 of these changes happened on the outer rim of the enzyme, far away from the actual lock.
• Only 1 change happened right inside the oily pocket where the key fits.

Testing the Theory
To prove this was the cause, the scientists played "Frankenstein" with the proteins:

1. They took the sloppy ancient enzyme and made that single change inside the pocket. It became slightly more picky (twice as good), but not perfect.
2. They took the precise modern enzyme and reversed that single change. It became less picky.
3. Then, they added the 18 "outer rim" changes to the sloppy enzyme. Suddenly, it became truly specific, just like the modern plants.

The "Solvent" Secret
Using computer simulations, the team discovered a hidden rule: the pickiness of the enzyme is linked to how much water can reach the oily pocket. Imagine the pocket as a room. If the room is too open, water (solvent) can rush in and mess up the fit. If the room is tight and sealed, the key fits perfectly. The study found that as the enzyme evolved, the "doorway" to this pocket got smaller, keeping water out and forcing the enzyme to be precise about which key it accepts.

The Bottom Line
This paper tells the story of how nature built a super-picky enzyme. It wasn't just one big magic change; it was a combination of one small tweak in the center and many small tweaks on the outside working together. By understanding this specific evolutionary path, the authors believe we can learn how to design our own custom enzymes in the future, using nature's own blueprint as a guide.

Technical Summary

Technical Summary: Evolutionary Origins of Stereoselectivity in Borneol Dehydrogenases

Problem Statement
Hydrophobic pockets are widely recognized as critical determinants of enzyme selectivity, particularly for hydrophobic substrates. However, the specific interactions between these substrates and the highly dynamic hydrophobic surfaces of catalytic motifs remain poorly understood. This knowledge gap renders the rational design of enzymes with controlled selectivity exceedingly difficult. Specifically, the evolutionary mechanism by which borneol dehydrogenases (BDHs) acquired high enantioselectivity in the oxidation of the monoterpenol 1-borneol was unclear. It was unknown whether this selectivity arose from direct modifications within the active-site hydrophobic pocket or through indirect effects caused by peripheral mutations.

Methodology
The study employed a multi-faceted approach combining ancestral sequence reconstruction (ASR), structural biology, protein engineering, and computational simulation:

• Ancestral Sequence Reconstruction: A set of plant BDHs, comprising both selective and unselective enzymes, was analyzed to reconstruct a putative common ancestor (N6) and intermediate ancestors (N30 and N32).
• Structural Analysis: Crystal structures of two ancestral dehydrogenases were solved to compare their architecture with extant, highly selective enzymes from sage and rosemary.
• Site-Directed Mutagenesis: To probe the functional impact of specific mutations, researchers introduced substitutions into the reconstructed ancestors. This included testing the I111L mutation in the active site of the unselective ancestor N30 and the reverse L111I mutation in the selective ancestor N32. Additionally, three peripheral mutations (V136L, G169A, V183I) were introduced to N30 to assess their cumulative effect.
• Computational Simulation: Funnel-metadynamics simulations were utilized to analyze the relationship between the solvent-accessible surface area (SASA) of the active site and enzymatic selectivity.

Key Results

• Evolutionary Trajectory: The transition from the oldest unselective ancestor (N30) to the youngest selective ancestor (N32) involved only 19 amino acid substitutions. Notably, 18 of these mutations were located in the periphery of the enzyme, with only a single substitution (I111L) occurring directly within the active-site hydrophobic pocket.
• Structural Conservation: Crystal structures revealed very high structural similarity among the reconstructed ancestors and the extant selective enzymes, suggesting that the overall fold remained stable during the evolution of selectivity.
• Functional Impact of Mutations:
  • The single active-site substitution (I111L) in the unselective ancestor N30 increased enantioselectivity by approximately two-fold.
  • Conversely, the back-mutation (L111I) in the selective ancestor N32 reduced its selectivity.
  • Crucially, the introduction of three additional peripheral mutations (V136L, G169A, V183I) to the N30 background (specifically the N30 L111I/V136L/G169A/V183I variant) restored high specificity. This demonstrates that high enantioselectivity in extant dehydrogenases results from a combination of active-site and peripheral mutations.
• Mechanistic Insight: Molecular simulations established a correlation between the solvent-accessible surface area (SASA) of the active site and the enzyme's selectivity, offering a potential molecular explanation for the observed stereoselectivity.

Significance and Claims
The paper posits that the reconstitution of a plausible natural evolutionary pathway for the formation of stereoselectivity in biosynthetic enzymes provides a valuable framework for future protein engineering. By elucidating how a combination of direct active-site modifications and indirect peripheral effects can drive the evolution of high enantioselectivity, the study offers guidance for the rational design of enzymes, addressing the current difficulty in controlling selectivity for hydrophobic substrates. The authors modestly claim that this work helps decipher the evolutionary origin of stereoselectivity, suggesting that understanding these natural pathways is essential for advancing the field of enzyme engineering.