Browsing by Subject "Enzyme mechanism"

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Investigation of the post-polyketide synthase (PKS) modifications during spinosyn A biosynthesis in Saccharopolyspora spinosa
(2010-08) Kim, Hak Joong, 1974-; Liu, Hung-wen, 1952-
Diverse biological activities of polyketide natural products are often associated with specific structural motifs, biosynthetically introduced after construction of the polyketide core. Therefore, investigation of such "post-polykektide synthase (PKS)" modifications is important, and the accumulated knowledge on these processes can be applied for combinatorial biosynthesis to generate new polyketide derivatives with enhanced biological activities. In addition to the practical value, a lot of unprecedented chemical mechanisms can be found in the enzymes involved therein, which will significantly advance our understanding of enzyme catalysis. The works described in this dissertation focus on elucidating a number of post-PKS modifications involved in the biosynthesis of an insecticidal polyketide, spinosyn A, in Saccharopolyspora spinosa. First, three methyltransferases, SpnH, SpnI, and SpnK, responsible for the modification of the rhamnose moiety, have been investigated to verify their functions and to study how they are coordinated to achieve the desired level of methylation of rhamnose. In vitro assays using purified enzymes not only established that SpnH, SpnI, and SpnK are the respective rhamnose 4ʹ-, 2ʹ-, and 3ʹ-O-methyltransferase, but also validated their roles in the permethylation process of spinosyn A. Investigation of the order of the methylation events revealed that only one route catalyzed by SpnI, SpnK, and SpnH in sequence is productive for the permethylation of the rhamnose moiety, which is likely achieved by the proper control of the expression levels of the methyltransferase genes involved in vivo. The key structural feature of spinosyn A is the presence of the unique tetracyclic architecture likely derived from the monocyclic PKS product. To elucidate this "cross-bridging" process, which had been hypothesized to involve four enzymes, SpnF, SpnJ, SpnL, and SpnM, the presumed polyketide substrate was chemically synthesized using Julia-Kocienski olefination, Stille cross-coupling, and Yamaguchi macrolactonization as key reactions. Incubation of the synthesized substrate with SpnJ produced a new product where the 15-OH group of the substrate is oxidized to the ketone. Next, it was demonstrated that incubation of this ketone intermediate with SpnM produces a tricyclic compound, via a transient monocyclic intermediate with high degree of unsaturation. Whereas it was initially thought that SpnM catalyzes both dehydration and [4+2] cycloaddition in sequence, detailed kinetic analysis revealed that SpnM is only responsible for the dehydration step, and the [4+2] cycloaddition step is indeed catalyzed by SpnF. Finally, successful conversion of the tricyclic intermediate to the tetracyclic core was demonstrated using SpnL. Proposed chemical mechanisms of SpnF and SpnL, Diels-Alder and Rauhut-Currier reactions, respectively, are interesting because enzymes capable of catalyzing these reactions have yet to be characterized in vitro. This work not only establishes the biosynthetic pathway for constructing the spinosyn tetracyclic core, but also epitomizes the significance of the post-PKS modification as a rich source of new enzyme catalysis.
Molecular mechanism of poly(ADP-ribosyl)ation catalyzed by human poly(ADP-ribose) polymerase-1
(2015-08-21) Lin, Ke-Yi; Liu, Hung-wen, 1952-; Whitman, Christian P; Fast, Walter; Russell, Rick; Lee, Seongmin
Human poly(ADP-ribose) polymerase-1 (PARP-1) is an abundant nuclear enzyme which catalyzes protein poly(ADP-ribosyl)ation upon binding to DNA. NAD+ is used as a co-substrate in the reaction via iterative transfer of its ADP-ribose moiety to acceptor proteins including PARP-1 itself, yielding elongated and branched poly(ADP-ribose) (PAR) polymers. This type of protein posttranslational modification has been demonstrated in the regulation of diverse biological processes including DNA repair, gene expression, cell cycle, etc. Therefore, elucidating the catalytic mechanism of PARP-1 would not only advance our understanding of how its enzymatic activity is regulated under physiological and pathophysiological conditions, but also greatly benefit the development of novel therapeutics involving pharmacological manipulation of PARP-1. In this dissertation, the molecular mechanism of DNA-dependent poly(ADP-ribosyl)ation by human PARP-1 was addressed from an enzymological perspective in terms of the allosteric ligand DNA, the substrate NAD+, and the PARP-1 protein–DNA complex as a whole. By site-specific labeling of the DNA-binding domain AB of PARP-1 and DNA ligands with fluorophores, quantitative binding kinetics of AB with DNA was investigated by single-molecule fluorescence spectroscopy. Two binding modes, one involving a strongly-associated protein–DNA complex and the other being transient, were suggested by the experimental data. To probe the catalytic mechanism of the initiation, elongation, and branching step of poly(ADP-ribosyl)ation with regard to NAD+ substrate scope, analogues of NAD+ with fluoro-substituted ribose ring were synthesized chemoenzymatically and employed as substrates. The results are consistent with the proposed mechanism that the ADP-ribosyl transfer reaction proceeds through an oxocarbenium-like transition state. Mass spectrometry and biochemical approaches were utilized to decipher the poly(ADP-ribosyl)ation sites on PARP-1 and the chemical nature of PAR–protein linkages. The data confirm the existence of automodification sites beyond domain D, and lysine could be the targeted residue for poly(ADP-ribosyl)ation, either enzymatically or nonenzymatically. The macromolecular mechanism of DNA-dependent PARP-1 automodification was established by an in vitro radioactivity-based poly(ADP-ribosyl)ation assay using structurally distinguishable PARP-1 mutants. The data support the model of an intermolecular process. Top-down MS analysis and crosslinking assay bolster the monomeric structure of domain C in solution and its participation in interdomain contacts during PARP-1 catalysis. Taken together, these mechanistic studies provide further insight into the catalytic strategies exploited by human PARP-1 complementary to recent reports of structural characterization, and may help discover better therapeutic agents modulating poly(ADP-ribosyl)ation.
Probing chemical mechanism of two enzyme-catalyzed reactions by chiral substrate analogues
(2014-02-04) Liu, Cheng-Hao; Liu, Hung-wen, 1952-; Anslyn, Eric V; Bielawski, Christopher W; Whitman, Christian P; Fast, Walter L
Enzymes are biological catalysts which greatly accelerate the rate of chemical reactions with remarkable substrate specificity and stereoselectivity. To optimize their catalytic abilities, many enzymes have also evolved to cooperate with coenzymes. The variety of coenzymes largely enhances the scope of enzyme-catalyzed reactions that are not accessible to the twenty canonical amino acids. In general, the chiral enzyme active sites allow enzymes to selectively bind substrates with a specific conformation which can lower the activation energy of the reactions by stabilizing high energy intermediates. Therefore, if the substrate of an enzyme contains chirality, in many cases, enzymes preferably accept one of the stereoisomers for catalysis. Inspired by this phenomenon, the work described in this dissertation focuses on mechanistic studies of two enzyme-catalyzed unusual chemical reactions by using chemically synthesized chiral substrate analogues. The first section of this dissertation focuses on 1-aminocyclopropane-1-carboxylic acid deaminase (ACCD), an enzyme that plays a role in regulating the production of the potent plant hormone, ethylene. ACCD is a pyridoxal-5ʹ-phosphate (PLP)-dependent enzyme that catalyzes a C-C bond cleavage event that is unique among the PLP-dependent enzymes. (R)- and (S)-2,2-difluoro-1-aminocyclopropane-1-carboxylic acid are synthesized as mechanism based inhibitors. Our studies suggest that the previously proposed acid-catalyzed mechanism for the ACCD-catalyzed reaction is less likely, and instead, a nucleophilic attack mechanism is consistent with the accumulated experimental results from the mechanistic studies of the ACCD reaction. The second section of this dissertation focuses on (E)-4-hydroxyl-3-methylbutenyl-1-diphosphate reductase (IspH), an essential enzyme in the non-mevalonate pathway (MEP pathway) for isoprenoid biosynthesis that employs a [4Fe-4S] cluster as cofactor for catalysis. Stereospecific labeling of tritium on the substrate and substrate analogue of IspH are used to generate chiral methyl groups in the products of the IspH reaction via catalysis in D2O-based buffer condition. Our results suggest that a configuration inversion at C4 of the substrate during the catalysis of IspH reaction is a universal step among all currently proposed mechanisms for the IspH reaction. Furthermore, we also provide the first experimental evidence for the direction of protonation of the allyl anion intermediate during IspH catalysis.