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Overall Assessment

The manuscript by Cui, Balskus, and coworkers describes the identification and characterization of a new class of metal-dependent bile salt hydrolases (BSHs) from the human gut bacterium Bilophila wadsworthia. Gut microbial BSHs modify the bile acid pool in the human gut by deconjugating glycine- or taurine-conjugated bile acids, thereby influencing host digestion and nutrient absorption. Prior to this work, all known BSHs were classified within the N-terminal nucleophile (Ntn) hydrolase superfamily, which uses a catalytic, N-terminal cysteine residue to hydrolyze amide bonds. Using ICP-MS analysis, activity assays, mutagenesis, and docking studies, the authors provide strong evidence that BwBSHs are distinct from Ntn-BSHs due to their metal dependency, specificity toward taurine-conjugated bile salts, and reliance on additional domains for secretion and enhanced activity. Although further experimentation is needed to confirm the identity of the metallocofactor, the authors suggest the possibility of a dinuclear Zn²⁺ metal center based on conserved ligands identified in an AlphaFold model of the enzyme. Importantly, the discovery of this enzyme class impactfully contributes to gut microbial enzymology, eliminating the notion that a single enzyme class is responsible for BSH activity in the gut, and building the foundation for metalloBSH research focused on human gut health. The following suggestions are made to improve data presentation and increase reader confidence in the conclusions drawn.

Major Points

1. In Figure 2d, chelators PHN and HQS appear to substantially decrease BwBSH activity relative to the control, providing convincing evidence of the enzyme’s metal dependence. However, in Extended Data Figure 2c, chelator treatment appears to have a much lesser impact on initial activity compared to the control, potentially weakening the conclusion derived from Fig. 2d. If this possible discrepancy is due to inconsistencies in experimental design, the authors should consider recollecting the data to maintain uniformity, thereby strengthening the credibility of the metal-dependency claim. If data recollection yields similar results, the authors might offer an explanation in the main text to aid reader interpretation of these findings.

2. Lines 209-210 state that following reconstitution with ZnCl₂ or CuCl₂, unbound metal ions were removed via overnight dialysis. However, in the methods section, lines 840-841 state that excess metal ions were separated from the reconstituted protein using a PD-10 column. Considering that ICP-MS analysis showed 4 equivalents of Zn per monomer of protein when BwBSH is predicted to contain a dinuclear Zn²⁺ cofactor, the method of excess metal ion removal seems pertinent. Ensuring clear and consistent methods may assist readers in confidently assessing and criticizing this key result.

3. The authors might consider including statistical analysis throughout all data presented in bar graph format, particularly the data in Figure 2. This addition would strengthen the paper’s main claims about the enzyme’s metal-dependency.

Minor Points

1. Throughout the main-text and extended data figures, activity assay data is presented in bar graphs with relative activity normalized to the control on the y-axis (e.g. Fig. 2, panels a, d, e, and f). This method of data presentation makes it easy for the reader to compare the experimental group to the control within a single experiment, but comparing activity across different experiments is challenging. Authors might consider displaying the concentration of taurine produced on the y-axis of activity assay graphs to minimize this challenge.

2. In Figure 2a, incubating BwBSH with an Ntn-BSH inhibitor resulted in a concentration-dependent enhancement of BwBSH activity. While this data supports the claim that BwBSH behaves differently than canonical BSHs, the authors should consider offering a potential explanation for the observed increase in activity, as the reason for this result is not immediately apparent. Providing reasoning could prevent reader speculation from diminishing the main purpose of this experiment.

3. In Figure 4a, the active site of the predicted BwBSH with TCA docked is shown. Since the alpha and beta Zn²⁺ ions are referenced in the main text, it might be useful to label the two ions in the figure or indicate the location of each in the figure caption (e.g. alpha on the left and beta on the right). This addition might make it easier for the reader to connect the description in lines 301-324 with the image in Figure 4a.

4. In the introduction, the authors emphasize the importance of the work by detailing the roles of bile acids and BSHs in human health. While the information and sources provided are useful, it would be even more compelling if there were studies referenced that detail the health implications of taurine and taurine-conjugated bile acids specifically. BwBSHs exhibiting activity solely on taurine-conjugated bile acids is an interesting attribute, and emphasizing the health implications of this attribute may increase reader investment in the work.

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.