Biology subjects

Mai, B. K.

Publications and source records attributed to Mai, B. K..

3 recordsLinked to original sources

bioRxiv2026

A binuclear copper enzyme platform for enantioconvergent radical (pseudo)halogenation

Despite their intriguing native metalloenzymology, naturally occurring copper enzymes remain largely underexploited for new-to-nature biocatalytic reactions. Herein, we report the systematic investigation and reprogramming of natural copper enzymes to catalyze unnatural free radical (pseudo)halogenation reaction in a highly enantioselective fashion. Evaluating Cu enzymes in the decarboxylative azidation of redox-active esters revealed activity across multiple Cu enzyme families, with type III binuclear Cu enzymes, particularly the Bacillus megaterium tyrosinase (BmTyr), exhibiting superior activity and enantioselectivity across both stabilized and unstabilized alkyl radicals upon further engineering. The strong halide binding affinity of the binuclear Cu system also enabled challenging enantioconvergent bromination, chlorination and isothiocyanation reactions, which remained inaccessible to repurposed nonheme Fe enzymes. Further EPR and UV-visible spectroscopic analyses confirmed the coupled binuclear nature of wild-type and engineered bacterial tyrosinases and provided insights into the origin of their enhanced activity. Collectively, this study establishes binuclear copper enzymes as a powerful platform for new-to-nature stereoselective radical reactions, expanding the scope of metalloenzyme catalysis beyond mononuclear systems.

biochemistry↗

bioRxiv2026

Photometallobiocatalytic Asymmetric Radical-Mediated Cross-Coupling of Organotrifluoroborate Salts and Pyridotriazoles

The cooperative integration of photoredox catalysis and metalloenzyme catalysis has emerged as a powerful strategy for enabling stereoselective radical transformations beyond the capabilities of either catalytic mode alone. Herein, we report a photometallobiocatalytic enantioselective intermolecular C-C cross-coupling of pyridotriazoles and secondary alkyltrifluoroborate salts through cooperative catalysis between an organic photosensitizer and an engineered protoglobin. By combining visible-light-mediated radical generation with enzymatic activation of pyridotriazoles to form reactive Fe carbenoid intermediates, this transformation enabled highly enantioselective radical C-C bond formation through a proposed outer-sphere coupling mechanism. Through biocatalyst mining and directed evolution, engineered Aeropyrum pernix protoglobin catalysts were developed that catalyzed this radical C-C coupling with excellent efficiency and stereocontrol. The photobiocatalytic platform exhibited a broad substrate scope with respect to both secondary alkyltrifluoroborate salts and pyridotriazoles, affording a range of valuable N-heterocyclic products in excellent yields and enantioselectivities. Mechanistic studies supported the involvement of radical intermediates and revealed spontaneous binding between the photocatalyst eosin B and the engineered metalloenzyme. By leveraging cooperative photometallobiocatalysis, this work established an underexplored strategy for asymmetric intermolecular radical cross-coupling via an outer-sphere mechanism, further expanding the catalytic repertoire of transition-metal carbenoid chemistry. Entry for the Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/744224v1_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@39f55borg.highwire.dtl.DTLVardef@11b3dfeorg.highwire.dtl.DTLVardef@1832ac2org.highwire.dtl.DTLVardef@6c4f56_HPS_FORMAT_FIGEXP M_FIG An enantioselective photometallobiocatalytic cross-coupling of pyridotriazoles and secondary alkyltrifluoroborate salts is developed. Cooperative catalysis using eosin B and an engineered protoglobin combines visible-light-mediated radical generation with enzymatic metal carbenoid activation, affording valuable N-heterocyclic products in excellent yield and enantioselectivity through an outer-sphere radical coupling pathway. C_FIG

biochemistry↗

bioRxiv2026

Influence of Primary Coordination Sphere on Anion Rebound Selectivity in Nonheme Fe Enzyme-Catalyzed C(sp3)-H Functionalization: A Comparative Experimental and Computational Study of EgtB and ACCO

Developing enzymatic mechanisms for C-F bond formation remains a long-standing challenge. Here, we repurposed the biosynthetic nonheme Fe enzyme EgtB, which features a three-histidine facial triad, to catalyze C(sp3)-H fluorination reactions. Directed evolution of EgtB afforded two new-to-nature fluorine atom transferases with opposite enantiopreference, EgtBCHF1 and EgtBCHF2, with up to 28-fold improved total activity. In contrast to our previously evolved nonheme Fe fluorine atom transfer biocatalyst ACCOCHF, which contains a two-histidine-one-carboxylate facial triad, the evolved EgtBCHF variants displayed unexpected hydroxylation activity. 18O-labeling experiments showed that the hydroxy group originated from water rather than residual O2. Computational studies suggested that the three-histidine-supported Fe(III) center exhibits enhanced Lewis acidity compared to the two-histidine-one-carboxylate system, allowing deprotonation of Fe(III)-bound water to form a Fe(III)-OH species to catalyze radical hydroxylation. Primary coordination-sphere mutagenesis in EgtB and ACCO further supported the critical role of Fe coordination chemistry in controlling radical rebound reactivity and selectivity. Computational studies revealed that Fe coordination chemistry strongly influences both fluorine atom abstraction and radical rebound, with the intrinsic C-X (X = F, OH, and N3) bond forming radical rebound preference following the order N3 > OH > F. Furthermore, multivariate linear regression analysis revealed that fluorine atom abstraction is primarily governed by the intrinsic Fe-F bond strength, whereas fluorine rebound is predominantly controlled by the electronic structure of the Fe(III) intermediate. Together, these findings provide mechanistic insights into nonheme Fe enzymology and reprogramming toward selective radical rebound reactions, including challenging C-H fluorination. Table of Contents (TOC) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/737789v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@143b503org.highwire.dtl.DTLVardef@2102aaorg.highwire.dtl.DTLVardef@135e7fdorg.highwire.dtl.DTLVardef@11b68a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗