Significance Iodine-induced cleavage is widely used for detecting bacterial DNA phosphorothioation in gel electrophoresis, deep sequencing, and single-molecule optical mapping. However, we lack quantitative understanding of the phosphorothioate DNA (PT-DNA) cleavage efficiency and the chemoselectivity of this method for determination of phosphorothioate vs. phosphate. Computational explorations now reveal why iodine selectively attacks at sulfur in phosphorothioate links but not at normal phosphates. The active role of Tris buffer in the PT-DNA cleavage, and the factors controlling cleavage efficiency, were also revealed. Cleavage efficiency is limited by competition between the desired DNA backbone cleavage and unwanted P-S/P-O conversion. These mechanistic studies will guide the development of new methods for iodine-induced specific PT-DNA cleavage.
Enzymes have long been characterized for their specificity and efficiency in catalyzing one substrate to one product. However, a SnoaL-like cyclase (XimE) that was originally discovered in xiamenmycin biosynthesis not only catalyzes the pyran-forming cyclization of the natural epoxide metabolites generated by XimD, but also enhances the furan-forming cyclization of the unnatural epoxide isomer. We have investigated and elucidated the reaction mechanism for this potentially unique substrate control of enzyme function. We explored the plausible pathways occurring at the hydrophobic active sites with a combination of theozyme (a small cluster model) calculations, pre- and post-reaction molecular dynamics (MD) simulations, ONIOM(ωB97X-D/6-31G(d):AMBER) transition state searching, and QM/SCRF(VS) dielectric constant scanning. Both the pyran and furan pathways share similar general acid–base catalytic mechanisms in which E136 and H102 act as proton donor and acceptor, respectively; pyran is generated by a fused-TS that proceeds via a general-acid-catalyzed mode, while furan is generated via a spiro-TS catalyzed by a general-base-catalyzed mode. The relative energies of the four possible transition states were found by ONIOM calculations to be Fused-S ≲ Spiro-S < Spiro-R < Fused-R. The regiochemical preference of the XimE-catalyzed pyran formation from S-epoxide and furan formation from R-epoxide stems from the induced-fit interaction between the enzyme and its transition states, which carries over to products. XimE apparently evolved along with the natural S-epoxide substrate generated by the upstream XimD epoxidase, and accidentally is also able to catalyze a different reaction of the enantiomeric R-epoxide via a similar catalytic mechanism.
Fluostatins, benzofluorene-containing aromatic polyketides in the atypical angucycline family, conjugate into dimeric and even trimeric compounds in the post-biosynthesis. The formation of the C–C bond involves a non-enzymatic stereospecific coupling reaction. In this work, the unusual regio- and enantioselectivities were rationalized by density functional theory calculations with the M06-2X (SMD, water)/6–311 + G(d,p)//6–31G(d) method. These DFT calculations reproduce the lowest energy C1-(R)-C10′-(S) coupling pathway observed in a nonenzymatic reaction. Bonding of the reactive carbon atoms (C1 and C10′) of the two reactant molecules maximizes the HOMO–LUMO interactions and Fukui function involving the highest occupied molecular orbital (HOMO) of nucleophile p-QM and lowest unoccupied molecular orbital (LUMO) of electrophile FST2− anion. In particular, the significant π–π stacking interactions of the low-energy pre-reaction state are retained in the lowest energy pathway for C–C coupling. The distortion/interaction–activation strain analysis indicates that the transition state (TScp-I) of the lowest energy pathway involves the highest stabilizing interactions and small distortion among all possible C–C coupling reactions. One of the two chiral centers generated in this step is lost upon aromatization of the phenol ring in the final difluostatin products. Thus, the π–π stacking interactions between the fluostatin 6-5-6 aromatic ring system play a critical role in the stereoselectivity of the nonenzymatic fluostatin conjugation.
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