Similarly, enzyme function can be designed de novo by creating a new active site within a natural protein scaffold that lacks the target activity, even if that catalytic function exists in nature.

See below for an early example:

www.pnas.org/doi/full/10....

No, I don’t think that’s necessarily implied. For example, a TIM barrel can be designed from scratch without referencing any specific natural sequence or structure, even if this fold exists in nature. I consider this de novo design. See example below:

www.nature.com/articles/nch...

The design and creation of a protein sequence, structure or function from scratch, rather than modifying a pre-existing sequence, structure or function.

An early pioneer of this field is Bill DeGrado, see below.

www.science.org/doi/10.1126/...

Overall, our study:
✅ Introduces a new strategy to transform minimal protein scaffolds into biocatalysts
✅ Provides mechanistic insights from crystallography & molecular dynamics
✅ Opens the door to designing custom lids for more complex reactions, which we’re now exploring

Thanks for reading! 🧵🧬

Our crystal structure validated the designed fold, confirming that the lid was correctly folded.

However, a subtle 1.8 Å lid shift disrupted a key catalytic contact, likely contributing to the modest activity. But structural analysis reveals paths to improve activity in the next round of design!

One of our designs, KempTIM4, showed catalytic efficiency comparable to many first-round de novo Kemp eliminases generated by traditional methods.

Using CANVAS, we designed a structural lid onto a minimal, de novo TIM barrel to anchor catalytic residues and create an active site for the Kemp elimination reaction.

TIM barrels are among nature’s most powerful enzyme scaffolds but making them from scratch with catalytic function has been a challenge.

Enter CANVAS: a computational pipeline combining Triad, RFdiffusion & ProteinMPNN to customize minimal TIM barrels into functional enzymes.

Congratulations! Looking forward to seeing all the exciting science that will come out of your lab! 🧪

The take-home message? Distal residues actively shape enzyme catalysis. Optimizing them can remove bottlenecks in substrate binding & product release—boosting activity. Want to dive deeper? Read our full study here: www.biorxiv.org/content/10.1...
(6/6)

Molecular dynamics simulations showed that distal mutations enhance active-site accessibility—either by loosening loops covering the active site or widening bottlenecks for substrate entry & product exit. The enzyme breathes more efficiently! 🌬️ (5/6)

Kinetic solvent viscosity effects & stopped-flow experiments showed that distal mutations don’t just tweak structure—they accelerate substrate binding & product release. (4/6)

Crystal structures showed that active-site mutations pre-organize the catalytic machinery. But distal mutations? They subtly tune conformational dynamics—enhancing productive substates & reshaping the energy landscape of the catalytic cycle. (3/6)

We engineered "Core" and "Shell" variants of three evolved Kemp eliminases to dissect the effects of active-site vs. distal mutations. Core mutations dramatically boosted catalysis. Shell mutations alone? Not much—until they worked together in evolved enzymes. 🔍 (2/6)

Thank you for the feedback!

Together, our findings reveal how distal mutations sculpt enzyme function by reshaping the catalytic cycle for more efficient catalysis.

What does this mean for enzyme design? Find out in our preprint: www.biorxiv.org/content/10.1...
#Enzymology #Biophysics #Catalysis #EnzymeDesign