Archives

  • 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • br PFL AE as a model for the GRE

    2020-04-03


    PFL-AE as a model for the GRE–AEs Most of the GRE–AEs have proven difficult to study due to instability, difficulty in overexpression, lability of the iron-sulfur cluster, or other reasons. PFL-AE is the exception, and after the initial discovery of the iron–sulfur cluster in this enzyme [60], considerable understanding of radical SAM TNF-alpha, recombinant rat protein in general, and GRE–AEs in particular, has come about via detailed studies of this enzyme.
    Conclusions The GRE–AE enzymes are a unique class of radical SAM enzymes, activating a much larger protein. One of the most prominent GRE–AEs, PFL-AE, was one of the first radical SAM enzymes characterized and has provided considerable insights into the SAM-cluster interactions for this superfamily. PFL-AE has also shown unusual active site electronic structure in vivo that can be replicated in the presence of small molecules in vitro, and may provide insights into control of reactivity in the GRE–AEs. Recent studies have provided insight into how PFL-AE catalyzes direct H-atom abstraction on a buried glycine residue of PFL, and similar complex protein–protein interactions are also likely for the structurally related GRE–AE/GRE pairs described herein. It is likely that additional GREs and their cognate activating enzymes will continue to be discovered, and the understanding of PFL and PFL-AE will provide an important foundation for elucidating their structures, mechanisms, and interactions.
    Acknowledgments Research on radical SAM activating enzymes in the Broderick laboratory is supported by the National Institutes of Health (GM54608).
    6-Tuliposides (Pos), the major secondary metabolites in tulip (), are glucose esters of 4-hydroxy-2-methylenebutanoate and (3)-3,4-dihydroxy-2-methylenebutanoate; the former and the latter are referred to as PosA and PosB, respectively ()., , PosA and PosB serve as precursors of the antimicrobial α-methylene-γ-butyrolactones, tulipalins A (PaA) and B (PaB), respectively, which are formed from the hydroxyl acids at the C-6 position of -glucose ()., , We previously discovered a unique Pos-converting enzymes (tuliposide-converting enzymes, TgTCEs) that specifically catalyze the conversion reactions of Pos to Pa ()., , , , , , , , Two distinct types of TgTCE, PosA-converting enzyme (TgTCEA) and PosB-converting enzyme (TgTCEB), are present in tulip tissues, and several isozymes with distinct expression profiles in tulip tissues have been identified for each of TgTCEA and TgTCEB (): TgTCEA from bulbs, and petals, and TgTCEB from pollen grains, roots, and leaves. Both types of TgTCE belong to the plant class I carboxylesterase family in the α/β-hydrolase fold superfamily. Canonical carboxylesterase catalyzes the hydrolysis of a carboxylic ester to form a carboxylic acid and an alcohol, but TgTCEs catalyze only intramolecular transesterification of Pos to form Pa and -glucose in a stoichiometric manner, and never catalyze hydrolysis of Pos to form hydroxy acids (). Hence, TgTCEs were identified as unique “non-ester-hydrolyzing carboxylesterases” and classified as lyases (EC 4.2.99.22 for TgTCEA and EC 4.2.99.23 for TgTCEB), but not as hydrolases. The enzyme reaction by TgTCEs begins with a nucleophilic attack by the catalytic Ser, whose hydroxyl group is activated by the charge relay of the catalytic triad, on the carbonyl carbon of Pos. This is followed by the formation of a tetrahedral intermediate, which is stabilized by the oxyanion hole structure formed by the two Gly residues of the HGG motif. Then, following the elimination of glucose, the acyl-enzyme complex is formed, and an intramolecular nucleophilic attack by a terminal hydroxyl group of Pos, but not by water, occurs, and this nucleophilic attack results in the formation of the five-membered ring structure of Pa. Although we proposed this reaction mechanism based on that of the canonical ester-hydrolyzing carboxylesterase and the site-directed mutagenesis analysis of the enzyme, it has not yet been verified experimentally by the crystallographic analysis. Moreover, there has so far been no information regarding the requisite substrate structures to be recognized by TgTCEs. This prompted us to examine the TNF-alpha, recombinant rat protein structure-activity relationship using structural analogues of Pos. Considering that natural Pos are chemically labile under neutral to basic conditions, such information is also useful to design more simple, stable substrate mimics that can be applied to co-crystallization experiments with TgTCEs, which lead to the verification of the reaction mechanism, including the formation of tetrahedral intermediate and the intramolecular nucleophilic attack. We hereby focused on the structural requirements of the alcohol moiety of Pos for recognition by TgTCEs. We synthesized many analogues of PosA and PosB by combinations of acyl units (for PosA, racemic ()-PosB, and PosB) with various alcohol units (), and assessed their effects on the enzyme activities of TgTCEA and TgTCEB. Moreover, stabilities of the synthetic analogues in aqueous solution were examined and compared with those of natural substrates, PosA and PosB.