Kewei Zhao

All Publications

• Zero-shot design of a de novo metalloenzyme. bioRxiv : the preprint server for biology Nesr, G. E., Dürr, S. L., Mathews, I. I., Wen, Q., Zhao, K., Sarangi, R., Röthlisberger, U., Sunden, F., Huang, P. S. 2026

  Abstract

  The de novo design of enzymes remains a central challenge, requiring consideration of catalytic mechanism and optimization across biochemical and biophysical criteria. To capture these criteria, we draw on principles from evolutionary biology. Here, we present dEVA (design by EVolutionary Algorithm), a multi-objective design framework for structure-based protein design. We apply dEVA to the zero-shot, de novo design of metalloenzymes by optimizing for the coordination sphere of catalytic metals. We fully characterize one of these designs: a bi-zinc metalloenzyme exhibiting promiscuous hydrolytic activity towards both phosphomonoesters and phosphodiesters. This design achieves a catalytic efficiency (kcat/KM) of up to 1500 M-1s-1 and a rate enhancement ((kcat/KM)/kw) of up to 3 × 1013, comparable to characterized natural phosphatases. dEVA offers a general and modular strategy for the programmable design of protein function without dependence on natural templates, predefined motif, or evolutionary information.

  View details for DOI 10.64898/2026.04.23.720277

  View details for PubMedID 42079180

  View details for PubMedCentralID PMC13131597

• Investigating weak axial ligation in corrinoids by X-ray absorption spectroscopy: Implications for corrinoid iron-sulfur protein. Journal of inorganic biochemistry Zhao, K., Abernathy, M. J., Griffith, C., Kountz, D. J., Esckilsen, D., Ragsdale, S. W., Sarangi, R. 2026; 281: 113323

  Abstract

  Corrinoid iron‑sulfur protein (CFeSP) is the methyltransferase in the Wood-Ljungdahl pathway, yet the geometric and electronic structure that enables its higher catalytic efficiency relative to small-molecule analogs remains a subject of debate. While axial ligation is known to tune the reactivity of B12-dependent enzymes, weak interactions in "base-off" species like methylated CFeSP (Me-CFeSP) have been difficult to define using standard structural probes, despite that solution-phase spectroscopy has suggested their presence. Here we utilize Co K-edge X-ray absorption spectroscopy (XAS) and theoretical analysis (DFT, TD-DFT, and QM/MM) to investigate a series of methylated corrinoids: methylcobalamin, methylcobinamide, and Me-CFeSP. Our results show that despite similar Co-CH3 bond distances across these species, the Co K-pre-edge feature undergoes a diagnostic increase in intensity and a shift to lower energy. Our analysis reveals that these spectral signatures are driven by modulations in the trans-axial Co-ligand distance, and because of the lower symmetry of the corrin ring, the spectral signature of the trans-axial ligand is decoupled from the methyl group. These results provide additional quantitative evidence that Me-CFeSP in solution possesses a weakly bound trans-axial water ligand rather than a 5-coordinate structure estimated by recent structural and XAS studies. This subtle ligation modulates frontier orbital energies to lower the CoC bond dissociation energy, explaining, in part, the enhanced methyl transfer rates of CFeSP. Collectively, this work establishes a spectroscopic and computational basis for diagnosing unresolved axial ligation in corrinoids, suggesting that such interactions may be a widespread, yet underappreciated, feature in "base-off" B12 enzymology.

  View details for DOI 10.1016/j.jinorgbio.2026.113323

  View details for PubMedID 41950862

• Cryo-EM captures the coordination of asymmetric electron transfer through a di-copper site in DPOR. Nature communications Kashyap, R., Walsh, N., Deveryshetty, J., Tokmina-Lukaszewska, M., Zhao, K., Gan, Y. J., Hoffman, B. M., Sarangi, R., Bothner, B., Bennett, B., Antony, E. 2025; 16 (1): 3866

  Abstract

  Enzymes that catalyze long-range electron transfer (ET) reactions often function as higher order complexes that possess two structurally symmetrical halves. The functional advantages for such an architecture remain a mystery. Using cryoelectron microscopy we capture snapshots of the nitrogenase-like dark-operative protochlorophyllide oxidoreductase (DPOR) during substrate binding and turnover. DPOR catalyzes reduction of the C17=C18 double bond in protochlorophyllide during the dark chlorophyll biosynthetic pathway. DPOR is composed of electron donor (L-protein) and acceptor (NB-protein) component proteins that transiently form a complex in the presence of ATP to facilitate ET. NB-protein is an alpha2beta2 heterotetramer with two structurally identical halves. However, our structures reveal that NB-protein becomes functionally asymmetric upon substrate binding. Asymmetry results in allosteric inhibition of L-protein engagement and ET in one half. Residues that form a conduit for ET are aligned in one half while misaligned in the other. An ATP hydrolysis-coupled conformational switch is triggered once ET is accomplished in one half. These structural changes are then relayed to the other half through a di-nuclear copper center at the tetrameric interface of the NB-protein and leads to activation of ET and substrate reduction. These findings provide a mechanistic blueprint for regulation of long-range electron transfer reactions.

  View details for DOI 10.1038/s41467-025-59158-7

  View details for PubMedID 40274796