Browsing by Author "Phillips, George N."

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    A discrete intermediate for the biosynthesis of both the enediyne core and the anthraquinone moiety of enediyne natural products
    (PNAS, 2023) Bhardwaj, Minakshi; Cui, Zheng; Daniel Hankore, Erome; Moonschi, Faruk H.; Saghaeiannejad Esfahani, Hoda; Kalkreuter, Edward; Gui, Chun; Yang, Dong; Phillips, George N.; Thorson, Jon S.; Shen, Ben; Van Lanen, Steven G.
    The enediynes are structurally characterized by a 1,5-diyne-3-ene motif within a 9- or 10-membered enediyne core. The anthraquinone-fused enediynes (AFEs) are a subclass of 10-membered enediynes that contain an anthraquinone moiety fused to the enediyne core as exemplified by dynemicins and tiancimycins. A conserved iterative type I polyketide synthase (PKSE) is known to initiate the biosynthesis of all enediyne cores, and evidence has recently been reported to suggest that the anthraquinone moiety also originates from the PKSE product. However, the identity of the PKSE product that is converted to the enediyne core or anthraquinone moiety has not been established. Here, we report the utilization of recombinant E. coli coexpressing various combinations of genes that encode a PKSE and a thioesterase (TE) from either 9- or 10-membered enediyne biosynthetic gene clusters to chemically complement ΔPKSE mutant strains of the producers of dynemicins and tiancimycins. Additionally, 13C-labeling experiments were performed to track the fate of the PKSE/TE product in the ΔPKSE mutants. These studies reveal that 1,3,5,7,9,11,13-pentadecaheptaene is the nascent, discrete product of the PKSE/TE that is converted to the enediyne core. Furthermore, a second molecule of 1,3,5,7,9,11,13-pentadecaheptaene is demonstrated to serve as the precursor of the anthraquinone moiety. The results establish a unified biosynthetic paradigm for AFEs, solidify an unprecedented biosynthetic logic for aromatic polyketides, and have implications for the biosynthesis of not only AFEs but all enediynes.
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    Evolution of substrate specificity in bacterial AA10 lytic polysaccharide monooxygenases
    (BioMed Central, 2014) Book, Adam J.; Yennamalli, Ragothaman M.; Takasuka, Taichi E.; Currie, Cameron R.; Phillips, George N.; Fox, Brian G.
    Background: Understanding the diversity of lignocellulose-degrading enzymes in nature will provide insights for the improvement of cellulolytic enzyme cocktails used in the biofuels industry. Two families of enzymes, fungal AA9 and bacterial AA10, have recently been characterized as crystalline cellulose or chitin-cleaving lytic polysaccharide monooxygenases (LPMOs). Here we analyze the sequences, structures, and evolution of LPMOs to understand the factors that may influence substrate specificity both within and between these enzyme families. Results: Comparative analysis of sequences, solved structures, and homology models from AA9 and AA10 LPMO families demonstrated that, although these two LPMO families are highly conserved, structurally they have minimal sequence similarity outside the active site residues. Phylogenetic analysis of the AA10 family identified clades with putative chitinolytic and cellulolytic activities. Estimation of the rate of synonymous versus non-synonymous substitutions (dN/dS) within two major AA10 subclades showed distinct selective pressures between putative cellulolytic genes (subclade A) and CBP21-like chitinolytic genes (subclade D). Estimation of site-specific selection demonstrated that changes in the active sites were strongly negatively selected in all subclades. Furthermore, all codons in the subclade D had dN/dS values of less than 0.7, whereas codons in the cellulolytic subclade had dN/dS values of greater than 1.5. Positively selected codons were enriched at sites localized on the surface of the protein adjacent to the active site. Conclusions: The structural similarity but absence of significant sequence similarity between AA9 and AA10 families suggests that these enzyme families share an ancient ancestral protein. Combined analysis of amino acid sites under Darwinian selection and structural homology modeling identified a subclade of AA10 with diversifying selection at different surfaces, potentially used for cellulose-binding and protein-protein interactions. Together, these data indicate that AA10 LPMOs are under selection to change their function, which may optimize cellulolytic activity. This work provides a phylogenetic basis for identifying and classifying additional cellulolytic or chitinolytic LPMOs.
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    Heterogeneity in M. tuberculosis β-lactamase inhibition by Sulbactam
    (Springer Nature, 2023) Malla, Tek Narsingh; Zielinski, Kara; Aldama, Luis; Bajt, Sasa; Feliz, Denisse; Hayes, Brendon; Hunter, Mark; Kupitz, Christopher; Lisova, Stella; Knoska, Juraj; Martin-Garcia, Jose Manuel; Mariani, Valerio; Pandey, Suraj; Poudyal, Ishwor; Sierra, Raymond G.; Tolstikova, Alexandra; Yefanov, Oleksandr; Yoon, Chung Hong; Ourmazd, Abbas; Fromme, Petra; Schwander, Peter; Barty, Anton; Chapman, Henry N.; Stojkovic, Emina A.; Batyuk, Alexander; Boutet, Sébastien; Phillips, George N.; Pollack, Lois; Schmidt, Marius
    For decades, researchers have elucidated essential enzymatic functions on the atomic length scale by tracing atomic positions in real-time. Our work builds on possibilities unleashed by mix-and-inject serial crystallography (MISC) at X-ray free electron laser facilities. In this approach, enzymatic reactions are triggered by mixing substrate or ligand solutions with enzyme microcrystals. Here, we report in atomic detail (between 2.2 and 2.7 Å resolution) by room-temperature, time-resolved crystallography with millisecond time-resolution (with timepoints between 3 ms and 700 ms) how the Mycobacterium tuberculosis enzyme BlaC is inhibited by sulbactam (SUB). Our results reveal ligand binding heterogeneity, ligand gating, cooperativity, induced fit, and conformational selection all from the same set of MISC data, detailing how SUB approaches the catalytic clefts and binds to the enzyme noncovalently before reacting to a trans-enamine. This was made possible in part by the application of singular value decomposition to the MISC data using a program that remains functional even if unit cell parameters change up to 3 Å during the reaction.
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    Structural Studies in Natural Product Biosynthesis and Structure Determination
    (2016-08-17) Han, Lu; Phillips, George N.
    Natural living organisms produce many natural compounds with diverse structures. They are one of the most productive sources of drug/bio-probe discovery and development, biosynthesis and enzymology, and organic synthesis. So far, more than 40% of current commercial drugs are natural products or natural product derivatives. However, given the highly selective modification site of natural products, de novo chemical synthesis and modification of natural products can be hard to design and sometimes problematic. To better exploit nature’s tool, I studied the structures of biosynthesizing enzymes for several potential drug leads by X-ray crystallography. Specifically, I studied the loop dynamics of TDP-rhamnose 3’-O-methyltransferase (Cals11), an enzyme in calicheamicin biosynthesis. I also characterized the structure and mechanism of decarboxylase (TtnD) as well as a non-heme FeII/α-ketoglutarate dependent hydroxylase (TtnM), both of which are involved in Tautomycetin biosynthesis. These results would provide us with guidance to engineering more efficient enzymes with different substrate specificity. Also, the impact of natural products in bioactive probe or drug lead discovery/development has faltered in the last decade due to an inability of natural products discovery technologies to keep pace with monumental advances in both screening and library synthesis platforms. Importantly, this de-emphasis in natural products discovery programs correlates with an overall reduction in new chemical entities/leads in the development pipeline. Thus, technological innovation is needed to realign natural product discovery with next generation technologies. The two most significant challenges in the discovery of new microbial natural products are: i) how to rapidly identify strains capable of novel chemistries (strain dereplication); and ii) how to expedite the subsequent structure determination of new natural products (structure elucidation). I propose a new paradigm for rapid unambiguous natural product structure elucidation, which will require very small amounts (g) of target natural product and, when merged with the rapid metabolomics-based dereplication process described herein, offers the potential to transform the natural products discovery process.
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    The Structure of the Arabidopsis PEX4-PEX22 Peroxin Complex—Insights Into Ubiquitination at the Peroxisomal Membrane
    (Frontiers Media S.A., 2022) Traver, Melissa S.; Bradford, Sarah E.; Olmos, Jose Luis; Wright, Zachary J.; Miller, Mitchell D.; Xu, Weijun; Phillips, George N.; Bartel, Bonnie
    Peroxisomes are eukaryotic organelles that sequester critical oxidative reactions and process the resulting reactive oxygen species into less toxic byproducts. Peroxisome function and formation are coordinated by peroxins (PEX proteins) that guide peroxisome biogenesis and division and shuttle proteins into the lumen and membrane of the organelle. Despite the importance of peroxins in plant metabolism and development, no plant peroxin structures have been reported. Here we report the X-ray crystal structure of the PEX4-PEX22 peroxin complex from the reference plant Arabidopsis thaliana. PEX4 is a ubiquitin-conjugating enzyme (UBC) that ubiquitinates proteins associated with the peroxisomal membrane, and PEX22 is a peroxisomal membrane protein that anchors PEX4 to the peroxisome and facilitates PEX4 activity. We co-expressed Arabidopsis PEX4 as a translational fusion with the soluble PEX4-interacting domain of PEX22 in E. coli. The fusion was linked via a protease recognition site, allowing us to separate PEX4 and PEX22 following purification and solve the structure of the complex. We compared the structure of the PEX4-PEX22 complex to the previously published structures of yeast orthologs. Arabidopsis PEX4 displays the typical UBC structure expected from its sequence. Although Arabidopsis PEX22 lacks notable sequence identity to yeast PEX22, it maintains a similar Rossmann fold-like structure. Several salt bridges are positioned to contribute to the specificity of PEX22 for PEX4 versus other Arabidopsis UBCs, and the long unstructured PEX22 tether would allow PEX4-mediated ubiquitination of distant peroxisomal membrane targets without dissociation from PEX22. The Arabidopsis PEX4-PEX22 structure also revealed that the residue altered in pex4-1 (P123L), a mutant previously isolated via a forward-genetic screen for peroxisomal dysfunction, is near the active site cysteine of PEX4. We demonstrated in vitro UBC activity for the PEX4-PEX22 complex and found that the pex4-1 enzyme has reduced in vitro ubiquitin-conjugating activity and altered specificity compared to PEX4. Our findings illuminate the role of PEX4 and PEX22 in peroxisome structure and function and provide tools for future exploration of ubiquitination at the peroxisome surface.