Browsing by Author "Dong, Liao-Bin"

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    Characterization and Crystal Structure of a Nonheme Diiron Monooxygenase Involved in Platensimycin and Platencin Biosynthesis
    (American Chemical Society, 2019) Dong, Liao-Bin; Liu, Yu-Chen; Cepeda, Alexis J.; Kalkreuter, Edward; Deng, Ming-Rong; Rudolf, Jeffrey D.; Chang, Changsoo; Joachimiak, Andrzej; Phillips, George N.Jr.; Shen, Ben
    Nonheme diiron monooxygenases make up a rapidly growing family of oxygenases that are rarely identified in secondary metabolism. Herein, we report the in vivo, in vitro, and structural characterizations of a nonheme diiron monooxygenase, PtmU3, that installs a C-5 β-hydroxyl group in the unified biosynthesis of platensimycin and platencin, two highly functionalized diterpenoids that act as potent and selective inhibitors of bacterial and mammalian fatty acid synthases. This hydroxylation sets the stage for the subsequent A-ring cleavage step key to the unique diterpene-derived scaffolds of platensimycin and platencin. PtmU3 adopts an unprecedented triosephosphate isomerase (TIM) barrel structural fold for this class of enzymes and possesses a noncanonical diiron active site architecture with a saturated six-coordinate iron center lacking a μ-oxo bridge. This study reveals the first member of a previously unidentified superfamily of TIM-barrel-fold enzymes for metal-dependent dioxygen activation, with the majority predicted to act on CoA-linked substrates, thus expanding our knowledge of nature’s repertoire of nonheme diiron monooxygenases and TIM-barrel-fold enzymes.
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    Natural separation of the acyl-CoA ligase reaction results in a non-adenylating enzyme
    (Springer Nature, 2018) Wang, Nan L.; Rudolf, Jeffrey D.; Dong, Liao-Bin; Osipiuk, Jerzy; Hatzos-Skintges, Catherine; Endres, Michael; Chang, Chin-Yuan; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N.Jr.; Shen, Ben
    Acyl-coenzyme A (CoA) ligases catalyze the activation of carboxylic acids via a two-step reaction of adenylation followed by thioesterification. Here, we report the discovery of a non-adenylating acyl-CoA ligase PtmA2 and the functional separation of an acyl-CoA ligase reaction. Both PtmA1 and PtmA2, two acyl-CoA ligases from the biosynthetic pathway of platensimycin and platencin, are necessary for the two steps of CoA activation. Gene inactivation of ptmA1 and ptmA2 resulted in the accumulation of free acid and adenylate intermediates, respectively. Enzymatic and structural characterization of PtmA2 confirmed its ability to only catalyze thioesterification. Structural characterization of PtmA2 revealed it binds both free acid and adenylate substrates and undergoes the established mechanism of domain alternation. Finally, site-directed mutagenesis restored both the adenylation and complete CoA activation reactions. This study challenges the currently accepted paradigm of adenylating enzymes and inspires future investigations on functionally separated acyl-CoA ligases and their ramifications in biology.