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Summary:

This study introduces a novel approach called Riff-Diff (rotamer inverted fragment finder – diffusion) for designing de novo enzymes based on  optimized catalytic centers. Using Riff-Diff, the authors designed 36 retro-aldolase sequences based on a highly evolved catalytic tetrad. Biochemical and structural characterization combined with molecular dynamics simulations showed that many of the designed enzymes exhibited remarkable catalytic activities, with rate accelerations exceeding 5 million-fold over the uncatalyzed reaction. Remarkably, this study demonstrates that precise positioning of the catalytic center residues and enzymatic activity are not correlated.  Their study reveals potential limitations of static structure-based analysis of de novo enzyme designs and underscores the importance of incorporating ensemble-function relationships in motif scaffolding to develop more efficient enzymatic designs. Overall,  it suggests that either artificial motifs must be designed with even sub-Angstrom accuracy to productively sample catalytically competent conformations or that directionality/modes of displacement from the mean position should be considered in an ensemble-function relationships in motif scaffolding.

Major comments:

1. With reference to Fig 2F in particular, can mutagenesis better discover factors directly contribute to the >100 fold difference between the k_cat of the evolved retro aldolase RA95.5-8f (10 s^-1) and that of the most catalytically proficient variants obtained through the Riff-Diff approach (k_cat values of 0.036 s^-1 and 0.031 s^-1)?

2. We found it particularly intriguing that certain designed variants maintain measurable catalytic activity, albeit to different extents, even after the catalytic lysine is mutated to alanine. A discussion on the mechanistic and evolutionary implications of catalytic performance of these variants and the variability therein would be valuable.

Minor points:

1. The authors claim in the results (second paragraph) that “Success rates for motif-scaffolding with RFdiffusion have been shown to correlate with increasing size of the selected motifs.” Is there a reference or specific data to support this statement?

2. Under the section ‘Scaffolding of a catalytic tetrad yielded highly proficient enzymes’, the authors mention “Sequence-structure pairs designed by the Riff-Diff pipeline generally passed structural quality filters.” This statement seems a little vague. It will be good to list those filters.

Competing interests

The authors declare that they have no competing interests.