Publications by authors named "Michael D Burkart"

169 Publications

Chemoenzymatic elaboration of the Raper-Mason pathway unravels the structural diversity within eumelanin pigments.

Chem Sci 2020 Jul 9;11(30):7836-7841. Epub 2020 Jul 9.

Department of Chemistry and Biochemistry, University of California San Diego, 9500 Gilman Drive La Jolla CA 92093-0358 USA

Melanin is a central polymer in living organisms, yet our understanding of its molecular structure remains unresolved. Here, we apply a biosynthetic approach to explore the composite structures accessible in one type of melanin, eumelanin. Using a combination of solid-state NMR, dynamic nuclear polarization, and electron microscopy, we reveal how a variety of monomers are enzymatically polymerized into their corresponding eumelanin pigments. We demonstrate how this approach can be used to unite structure with an understanding of enzymatic activity, substrate scope, and the regulation of nanostructural features. Overall, this data reveals how intermediate metabolites of the Raper-Mason metabolic pathway contribute to polymerization, allowing us to revisit the original proposal of how eumelanin is biosynthesized.
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http://dx.doi.org/10.1039/d0sc02262dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8163323PMC
July 2020

Unraveling the Role of Linker Design in Proteolysis Targeting Chimeras.

J Med Chem 2021 Jun 9;64(12):8042-8052. Epub 2021 Jun 9.

Department of Chemistry and Biochemistry, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093-0358, United States.

A current bottleneck in the development of proteolysis targeting chimeras (PROTACs) is the empirical nature of linker length structure-activity relationships (SARs). A multidisciplinary approach to alleviate the bottleneck is detailed here. First, we examine four published synthetic approaches that have been developed to increase synthetic throughput. We then discuss advances in structural biology and computational chemistry that have led to successful rational PROTAC design efforts and give promise to linker design . Lastly, we present a model generated from a curated list of linker SARs studies normalized to reflect how linear linker length affects the observed degradation potency (DC).
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http://dx.doi.org/10.1021/acs.jmedchem.1c00482DOI Listing
June 2021

Chemoenzymatic Generation of Phospholipid Membranes Mediated by Type I Fatty Acid Synthase.

J Am Chem Soc 2021 Jun 12;143(23):8533-8537. Epub 2021 May 12.

Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093, United States.

The formation of lipid membranes from minimal reactive precursors is a major goal in synthetic cell research. In nature, the synthesis of membrane phospholipids is orchestrated by numerous enzymes, including fatty acid synthases and membrane-bound acyltransferases. However, these enzymatic pathways are difficult to fully reproduce . As such, the reconstitution of phospholipid membrane synthesis from simple metabolic building blocks remains a challenge. Here, we describe a chemoenzymatic strategy for lipid membrane generation that utilizes a soluble bacterial fatty acid synthase (cgFAS I) to synthesize palmitoyl-CoA from acetyl-CoA and malonyl-CoA. The fatty acid derivative spontaneously reacts with a cysteine-modified lysophospholipid by native chemical ligation (NCL), affording a noncanonical amidophospholipid that self-assembles into micron-sized membrane-bound vesicles. To our knowledge, this is the first example of reconstituting phospholipid membrane formation directly from acetyl-CoA and malonyl-CoA precursors. Our results demonstrate that combining the specificity and efficiency of a type I fatty acid synthase with a highly selective bioconjugation reaction provides a biomimetic route for the formation of membrane-bound vesicles.
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http://dx.doi.org/10.1021/jacs.1c02121DOI Listing
June 2021

Synthase-Selective Exploration of a Tunicate Microbiome by Activity-Guided Single-Cell Genomics.

ACS Chem Biol 2021 05 6;16(5):813-819. Epub 2021 May 6.

Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0358, United States.

While thousands of environmental metagenomes have been mined for the presence of novel biosynthetic gene clusters, such computational predictions do not provide evidence of their biosynthetic functionality. Using fluorescent enzyme assay targeting carrier proteins common to polyketide (PKS) and nonribosomal peptide synthetases (NRPS), we applied fluorescence-activated cell sorting to tunicate microbiome to enrich for microbes with active secondary metabolic capabilities. Single-cell genomics uncovered the genetic basis for a wide biosynthetic diversity in the enzyme-active cells and revealed a member of marine harboring a novel NRPS gene cluster with high similarity to phylogenetically distant marine and terrestrial bacteria. Interestingly, this synthase belongs to a larger class of siderophore biosynthetic gene clusters commonly associated with pestilence and disease. This demonstrates activity-guided single-cell genomics as a tool to guide novel biosynthetic discovery.
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http://dx.doi.org/10.1021/acschembio.1c00157DOI Listing
May 2021

In silico identification and in vitro evaluation of a protein-protein interaction inhibitor of Escherichia coli fatty acid biosynthesis.

Chem Biol Drug Des 2021 Jul 31;98(1):94-101. Epub 2021 May 31.

Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA.

To combat the rise in antibiotic resistance, new targets must be identified and probes against them developed. Protein-protein interactions (PPI) of bacterial type II fatty acid biosynthesis (FAS-II) represent an untapped, yet rich area for new antibiotic discovery. Here, we present a computational and in vitro workflow for the discovery of new inhibitors of PPI in Escherichia coli FAS-II. As part of this study, we identified suramin, an existing treatment for African sleeping sickness, to effectively block the interaction of E. coli dehydratase FabA and the acyl carrier protein EcACP, with an IC = 85 μΜ. This finding validates a workflow that combines in silico screening with in vitro PPI assays to identify probes appropriate for further optimization.
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http://dx.doi.org/10.1111/cbdd.13851DOI Listing
July 2021

Decoding allosteric regulation by the acyl carrier protein.

Proc Natl Acad Sci U S A 2021 Apr;118(16)

Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA 92093-0358;

Enzymes in multistep metabolic pathways utilize an array of regulatory mechanisms to maintain a delicate homeostasis [K. Magnuson, S. Jackowski, C. O. Rock, J. E. Cronan, Jr, 57, 522-542 (1993)]. Carrier proteins in particular play an essential role in shuttling substrates between appropriate enzymes in metabolic pathways. Although hypothesized [E. Płoskoń et al., 17, 776-785 (2010)], allosteric regulation of substrate delivery has never before been demonstrated for any acyl carrier protein (ACP)-dependent pathway. Studying these mechanisms has remained challenging due to the transient and dynamic nature of protein-protein interactions, the vast diversity of substrates, and substrate instability [K. Finzel, D. J. Lee, M. D. Burkart, 16, 528-547 (2015)]. Here we demonstrate a unique communication mechanism between the ACP and partner enzymes using solution NMR spectroscopy and molecular dynamics to elucidate allostery that is dependent on fatty acid chain length. We demonstrate that partner enzymes can allosterically distinguish between chain lengths via protein-protein interactions as structural features of substrate sequestration are translated from within the ACP four-helical bundle to the protein surface, without the need for stochastic chain flipping. These results illuminate details of cargo communication by the ACP that can serve as a foundation for engineering carrier protein-dependent pathways for specific, desired products.
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http://dx.doi.org/10.1073/pnas.2025597118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8072227PMC
April 2021

Renewable Polyurethanes from Sustainable Biological Precursors.

Biomacromolecules 2021 05 6;22(5):1770-1794. Epub 2021 Apr 6.

Department of Chemistry and Biochemistry, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093-0358, United States.

Due to the depletion of fossil fuels, higher oil prices, and greenhouse gas emissions, the scientific community has been conducting an ongoing search for viable renewable alternatives to petroleum-based products, with the anticipation of increased adaptation in the coming years. New academic and industrial developments have encouraged the utilization of renewable resources for the development of ecofriendly and sustainable materials, and here, we focus on those advances that impact polyurethane (PU) materials. Vegetable oils, algae oils, and polysaccharides are included among the major renewable resources that have supported the development of sustainable PU precursors to date. Renewable feedstocks such as algae have the benefit of requiring only sunshine, carbon dioxide, and trace minerals to generate a sustainable biomass source, offering an improved carbon footprint to lessen environmental impacts. Incorporation of renewable content into commercially viable polymer materials, particularly PUs, has increasing and realistic potential. Biobased polyols can currently be purchased, and the potential to expand into new monomers offers exciting possibilities for new product development. This Review highlights the latest developments in PU chemistry from renewable raw materials, as well as the various biological precursors being employed in the synthesis of thermoset and thermoplastic PUs. We also provide an overview of literature reports that focus on biobased polyols and isocyanates, the two major precursors to PUs.
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http://dx.doi.org/10.1021/acs.biomac.0c01610DOI Listing
May 2021

Elucidation of transient protein-protein interactions within carrier protein-dependent biosynthesis.

Commun Biol 2021 03 16;4(1):340. Epub 2021 Mar 16.

Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA.

Fatty acid biosynthesis (FAB) is an essential and highly conserved metabolic pathway. In bacteria, this process is mediated by an elaborate network of protein•protein interactions (PPIs) involving a small, dynamic acyl carrier protein that interacts with dozens of other partner proteins (PPs). These PPIs have remained poorly characterized due to their dynamic and transient nature. Using a combination of solution-phase NMR spectroscopy and protein-protein docking simulations, we report a comprehensive residue-by-residue comparison of the PPIs formed during FAB in Escherichia coli. This technique describes and compares the molecular basis of six discrete binding events responsible for E. coli FAB and offers insights into a method to characterize these events and those in related carrier protein-dependent pathways.
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http://dx.doi.org/10.1038/s42003-021-01838-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7966745PMC
March 2021

Unraveling the Structure and Function of Melanin through Synthesis.

J Am Chem Soc 2021 02 9;143(7):2622-2637. Epub 2021 Feb 9.

Department of Chemistry & Biochemistry, University of California, San Diego, La Jolla, California 92093, United States.

Melanin is ubiquitous in living organisms across different biological kingdoms of life, making it an important, natural biomaterial. Its presence in nature from microorganisms to higher animals and plants is attributed to the many functions of melanin, including pigmentation, radical scavenging, radiation protection, and thermal regulation. Generally, melanin is classified into five types-eumelanin, pheomelanin, neuromelanin, allomelanin, and pyomelanin-based on the various chemical precursors used in their biosynthesis. Despite its long history of study, the exact chemical makeup of melanin remains unclear, and it moreover has an inherent diversity and complexity of chemical structure, likely including many functions and properties that remain to be identified. Synthetic mimics have begun to play a broader role in unraveling structure and function relationships of natural melanins. In the past decade, polydopamine, which has served as the conventional form of synthetic eumelanin, has dominated the literature on melanin-based materials, while the synthetic analogues of other melanins have received far less attention. In this perspective, we will discuss the synthesis of melanin materials with a special focus beyond polydopamine. We will emphasize efforts to elucidate biosynthetic pathways and structural characterization approaches that can be harnessed to interrogate specific structure-function relationships, including electron paramagnetic resonance (EPR) and solid-state nuclear magnetic resonance (ssNMR) spectroscopy. We believe that this timely Perspective will introduce this class of biopolymer to the broader chemistry community, where we hope to stimulate new opportunities in novel, melanin-based poly-functional synthetic materials.
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http://dx.doi.org/10.1021/jacs.0c12322DOI Listing
February 2021

Traceless Staudinger ligation enabled parallel synthesis of proteolysis targeting chimera linker variants.

Chem Commun (Camb) 2021 Jan 6;57(8):1026-1029. Epub 2021 Jan 6.

Department of Chemistry and Biochemistry, UC San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0358, USA.

A parallel, one-pot assembly approach to proteolysis targeting chimeras (PROTACs) is demonstrated utilizing activated esters generated in situ, and traceless Staudinger ligation chemistry. The method described allows for rapid structure-activity relationship studies of PROTAC linker variants. Two previously studied systems, cereblon and BRD4 degraders, are examined as test cases for the synthetic method. The two related strategies to assemble PROTAC linker variants discussed can accommodate the chromotographic separations capabilities of labs of many sizes and incorporates commercially available degrader building blocks, thereby easing synthetic entry into PROTAC chemical space.
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http://dx.doi.org/10.1039/d0cc05395cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7962863PMC
January 2021

Screening and characterization of polyhydroxyalkanoate granules, and phylogenetic analysis of polyhydroxyalkanoate synthase gene PhaC in cyanobacteria.

J Phycol 2021 06 5;57(3):754-765. Epub 2021 Apr 5.

Scripps Institution of Oceanography, University of California, San Diego, La Jolla, California, 92093-0202, USA.

Using Nile Red and BODIPY 493/503 dye-staining and fluorescence microscopy, twenty cyanobacterial strains, including ten commercially available strains and ten environmental isolates from estuaries, freshwater ponds, and lagoons, were screened for the accumulation of ecologically important and potentially biotechnologically significant carbon storage granules such as polyhydroxyalkanoates (PHA). Dye-staining granules were observed in six strains. Three Synechocystis, spp. strains WHSYN, LSNM, and CGF-1, and a Phormidium-like sp. CGFILA were isolated from environmental sources and found to produce granules of polyhydroxyalkanoate (PHA) according to PHA synthase gene (phaC) PCR screening and H NMR analyses. The environmental isolate, Nodularia sp. Las Olas and commercially available Phormidium cf. iriguum CCALA 759 displayed granules but screened negative for PHA according to phaC PCR and H NMR analyses. Partial polyhydroxyalkanoate synthase subunit C (phaC) and 16S rRNA gene sequences obtained from the PHA-accumulating strains and analyzed alongside publicly available phaC, phaE, 16S rRNA, and 23S rRNA data help in understanding the distribution and evolutionary history of PHA biosynthesis within the phylum Cyanobacteria. The data show that the presence of phaC is highly conserved within the genus Synechocystis, and present in at least one isolate of Phormidium. Maximum likelihood analyses and cophylogenetic modeling of PHA synthase gene sequences provide evidence of a recent horizontal gene transfer event between distant genera of cyanobacteria related to Pleurocapsa sp. PCC 7327 and Phormidium-like sp. CGFILA. These findings will help guide additional screening for PHA producers, and may explain why some Phormidium species produce PHAs, while others do not.
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http://dx.doi.org/10.1111/jpy.13123DOI Listing
June 2021

Dynamic visualization of type II peptidyl carrier protein recognition in pyoluteorin biosynthesis.

RSC Chem Biol 2020 Apr 24;1(1):8-12. Epub 2020 Mar 24.

Department of Chemistry and Biochemistry, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0358, USA.

Using a covalent chemical probe and X-ray crystallography coupled to nuclear magnetic resonance data, we elucidated the dynamic molecular basis of protein recognition between the carrier protein and adenylation domain in pyoluteorin biosynthesis. These findings reveal a unique binding mode, which contrasts previously solved carrier protein and partner protein interfaces.
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http://dx.doi.org/10.1039/c9cb00015aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7723355PMC
April 2020

Interfacial plasticity facilitates high reaction rate of FAS malonyl-CoA:ACP transacylase, FabD.

Proc Natl Acad Sci U S A 2020 09 14;117(39):24224-24233. Epub 2020 Sep 14.

Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA 92093-0358;

Fatty acid synthases (FASs) and polyketide synthases (PKSs) iteratively elongate and often reduce two-carbon ketide units in de novo fatty acid and polyketide biosynthesis. Cycles of chain extensions in FAS and PKS are initiated by an acyltransferase (AT), which loads monomer units onto acyl carrier proteins (ACPs), small, flexible proteins that shuttle covalently linked intermediates between catalytic partners. Formation of productive ACP-AT interactions is required for catalysis and specificity within primary and secondary FAS and PKS pathways. Here, we use the FAS AT, FabD, and its cognate ACP, AcpP, to interrogate type II FAS ACP-AT interactions. We utilize a covalent crosslinking probe to trap transient interactions between AcpP and FabD to elucidate the X-ray crystal structure of a type II ACP-AT complex. Our structural data are supported using a combination of mutational, crosslinking, and kinetic analyses, and long-timescale molecular dynamics (MD) simulations. Together, these complementary approaches reveal key catalytic features of FAS ACP-AT interactions. These mechanistic inferences suggest that AcpP adopts multiple, productive conformations at the AT binding interface, allowing the complex to sustain high transacylation rates. Furthermore, MD simulations support rigid body subdomain motions within the FabD structure that may play a key role in AT activity and substrate selectivity.
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http://dx.doi.org/10.1073/pnas.2009805117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7533678PMC
September 2020

Activity Mapping the Acyl Carrier Protein: Elongating Ketosynthase Interaction in Fatty Acid Biosynthesis.

Biochemistry 2020 09 11;59(38):3626-3638. Epub 2020 Sep 11.

Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0358, United States.

Elongating ketosynthases (KSs) catalyze carbon-carbon bond-forming reactions during the committed step for each round of chain extension in both fatty acid synthases (FASs) and polyketide synthases (PKSs). A small α-helical acyl carrier protein (ACP) shuttles fatty acyl intermediates between enzyme active sites. To accomplish this task, the ACP relies on a series of dynamic interactions with multiple partner enzymes of FAS and associated FAS-dependent pathways. Recent structures of the FAS ACP, AcpP, in covalent complexes with its two cognate elongating KSs, FabF and FabB, provide high-resolution details of these interfaces, but a systematic analysis of specific interfacial interactions responsible for stabilizing these complexes has not yet been undertaken. Here, we use site-directed mutagenesis with both and activity analyses to quantitatively evaluate these contacting surfaces between AcpP and FabF. We delineate the FabF interface into three interacting regions and demonstrate the effects of point mutants, double mutants, and region deletion variants. Results from these analyses reveal a robust and modular FabF interface capable of tolerating seemingly critical interface mutations with only the deletion of an entire region significantly compromising activity. Structure and sequence analyses of FabF orthologs from related type II FAS pathways indicate significant conservation of type II FAS KS interface residues and, overall, support its delineation into interaction regions. These findings strengthen our mechanistic understanding of molecular recognition events between ACPs and FAS enzymes and provide a blueprint for engineering ACP-dependent biosynthetic pathways.
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http://dx.doi.org/10.1021/acs.biochem.0c00605DOI Listing
September 2020

Enzyme-Directed Functionalization of Designed, Two-Dimensional Protein Lattices.

Biochemistry 2021 Apr 3;60(13):1050-1062. Epub 2020 Aug 3.

Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093, United States.

The design and construction of crystalline protein arrays to selectively assemble ordered nanoscale materials have potential applications in sensing, catalysis, and medicine. Whereas numerous designs have been implemented for the bottom-up construction of protein assemblies, the generation of artificial functional materials has been relatively unexplored. Enzyme-directed post-translational modifications are responsible for the functional diversity of the proteome and, thus, could be harnessed to selectively modify artificial protein assemblies. In this study, we describe the use of phosphopantetheinyl transferases (PPTases), a class of enzymes that covalently modify proteins using coenzyme A (CoA), to site-selectively tailor the surface of designed, two-dimensional (2D) protein crystals. We demonstrate that a short peptide (ybbR) or a molecular tag (CoA) can be covalently tethered to 2D arrays to enable enzymatic functionalization using Sfp PPTase. The site-specific modification of two different protein array platforms is facilitated by PPTases to afford both small molecule- and protein-functionalized surfaces with no loss of crystalline order. This work highlights the potential for chemoenzymatic modification of large protein surfaces toward the generation of sophisticated protein platforms reminiscent of the complex landscape of cell surfaces.
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http://dx.doi.org/10.1021/acs.biochem.0c00363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7855359PMC
April 2021

Selenomelanin: An Abiotic Selenium Analogue of Pheomelanin.

J Am Chem Soc 2020 07 8;142(29):12802-12810. Epub 2020 Jul 8.

Department of Chemistry, Northwestern University, Evanston, Illinois 60208, United States of America.

Melanins are a family of heterogeneous biopolymers found ubiquitously across plant, animal, bacterial, and fungal kingdoms where they act variously as pigments and as radiation protection agents. There exist five multifunctional yet structurally and biosynthetically incompletely understood varieties of melanin: eumelanin, neuromelanin, pyomelanin, allomelanin, and pheomelanin. Although eumelanin and allomelanin have been the focus of most radiation protection studies to date, some research suggests that pheomelanin has a better absorption coefficient for X-rays than eumelanin. We reasoned that if a selenium enriched melanin existed, it would be a better X-ray protector than the sulfur-containing pheomelanin because the X-ray absorption coefficient is proportional to the fourth power of the atomic number (Z). Notably, selenium is an essential micronutrient, with the amino acid selenocysteine being genetically encoded in 25 natural human proteins. Therefore, we hypothesize that selenomelanin exists in nature, where it provides superior ionizing radiation protection to organisms compared to known melanins. Here we introduce this novel selenium analogue of pheomelanin through chemical and biosynthetic routes using selenocystine as a feedstock. The resulting selenomelanin is a structural mimic of pheomelanin. We found selenomelanin effectively prevented neonatal human epidermal keratinocytes (NHEK) from G2/M phase arrest under high-dose X-ray irradiation. Provocatively, this beneficial role of selenomelanin points to it as a sixth variety of yet to be discovered natural melanin.
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http://dx.doi.org/10.1021/jacs.0c05573DOI Listing
July 2020

Quantifying protein-protein interactions of the acyl carrier protein with solvatochromic probes.

Methods Enzymol 2020 24;638:321-340. Epub 2020 Apr 24.

Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, United States. Electronic address:

Protein-protein interactions (PPIs) are universal to life and their study and understanding is critical to drug discovery and bioengineering efforts. Historically, X-ray crystallography, isothermal titration calorimetry and other biophysical methods have been used to study PPIs, but can be costly and are low throughput, hindering progress towards rapid evaluation of these interactions. Recent interest in targeting PPIs and in engineering biosynthetic pathways in which PPIs play a critical role has driven innovation in their evaluation but a universal screen is still needed. One of the best characterized systems relying upon PPIs is Escherichia coli type II fatty acid biosynthesis in which the central acyl carrier protein (EcACP) shuttles substrates to a series of partner enzymes. Here we present a method by which EcACP is labeled with a solvatochromic dye, 4-DMN, and then allowed to interact with its various partner enzymes. Upon interaction, there is a large increase in fluorescence intensity which is easily monitored via fluorometer or plate reader. This method is useful in the study of known PPI, hypothetical PPI and in evaluation of inhibitors of both partner enzyme active site and of the PPI itself.
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http://dx.doi.org/10.1016/bs.mie.2020.03.016DOI Listing
June 2021

Structural basis for selectivity in a highly reducing type II polyketide synthase.

Nat Chem Biol 2020 07 4;16(7):776-782. Epub 2020 May 4.

Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.

In type II polyketide synthases (PKSs), the ketosynthase-chain length factor (KS-CLF) complex catalyzes polyketide chain elongation with the acyl carrier protein (ACP). Highly reducing type II PKSs, represented by IgaPKS, produce polyene structures instead of the well-known aromatic skeletons. Here, we report the crystal structures of the Iga11-Iga12 (KS-CLF) heterodimer and the covalently cross-linked Iga10=Iga11-Iga12 (ACP=KS-CLF) tripartite complex. The latter structure revealed the molecular basis of the interaction between Iga10 and Iga11-Iga12, which differs from that between the ACP and KS of Escherichia coli fatty acid synthase. Furthermore, the reaction pocket structure and site-directed mutagenesis revealed that the negative charge of Asp 113 of Iga11 prevents further condensation using a β-ketoacyl product as a substrate, which distinguishes IgaPKS from typical type II PKSs. This work will facilitate the future rational design of PKSs.
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http://dx.doi.org/10.1038/s41589-020-0530-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556716PMC
July 2020

Gating mechanism of elongating β-ketoacyl-ACP synthases.

Nat Commun 2020 04 7;11(1):1727. Epub 2020 Apr 7.

Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0358, USA.

Carbon-carbon bond forming reactions are essential transformations in natural product biosynthesis. During de novo fatty acid and polyketide biosynthesis, β-ketoacyl-acyl carrier protein (ACP) synthases (KS), catalyze this process via a decarboxylative Claisen-like condensation reaction. KSs must recognize multiple chemically distinct ACPs and choreograph a ping-pong mechanism, often in an iterative fashion. Here, we report crystal structures of substrate mimetic bearing ACPs in complex with the elongating KSs from Escherichia coli, FabF and FabB, in order to better understand the stereochemical features governing substrate discrimination by KSs. Complemented by molecular dynamics (MD) simulations and mutagenesis studies, these structures reveal conformational states accessed during KS catalysis. These data taken together support a gating mechanism that regulates acyl-ACP binding and substrate delivery to the KS active site. Two active site loops undergo large conformational excursions during this dynamic gating mechanism and are likely evolutionarily conserved features in elongating KSs.
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http://dx.doi.org/10.1038/s41467-020-15455-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7138838PMC
April 2020

Splice Modulation Synergizes Cell Cycle Inhibition.

ACS Chem Biol 2020 03 17;15(3):669-674. Epub 2020 Feb 17.

Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0358, United States.

While recognized as a therapeutic target, the spliceosome may offer a robust vector to improve established therapeutics against other protein targets. Here, we describe how modulating the spliceosome using small molecule splice modulators (SPLMs) can prime a cell for sensitivity to a target-specific drug. Using the cell cycle regulators aurora kinase and polo-like kinase as models, this study demonstrates how the combination of SPLM treatment in conjunction with kinase inhibition offers synergy for antitumor activity using reduced, sublethal levels of SPLM and kinase inhibitors. This concept of splice-modulated drug attenuation suggests a possible approach to enhance therapeutic agents that have shown limited applicability due to high toxicity or low efficacy.
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http://dx.doi.org/10.1021/acschembio.9b00833DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7570451PMC
March 2020

Charting the Complexity of the Marine Microbiome through Single-Cell Genomics.

Cell 2019 12;179(7):1623-1635.e11

Bigelow Laboratory for Ocean Sciences, East Boothbay, Maine, 04544, USA. Electronic address:

Marine bacteria and archaea play key roles in global biogeochemistry. To improve our understanding of this complex microbiome, we employed single-cell genomics and a randomized, hypothesis-agnostic cell selection strategy to recover 12,715 partial genomes from the tropical and subtropical euphotic ocean. A substantial fraction of known prokaryoplankton coding potential was recovered from a single, 0.4 mL ocean sample, which indicates that genomic information disperses effectively across the globe. Yet, we found each genome to be unique, implying limited clonality within prokaryoplankton populations. Light harvesting and secondary metabolite biosynthetic pathways were numerous across lineages, highlighting the value of single-cell genomics to advance the identification of ecological roles and biotechnology potential of uncultured microbial groups. This genome collection enabled functional annotation and genus-level taxonomic assignments for >80% of individual metagenome reads from the tropical and subtropical surface ocean, thus offering a model to improve reference genome databases for complex microbiomes.
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http://dx.doi.org/10.1016/j.cell.2019.11.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6919566PMC
December 2019

Role of MyD88 in IL-1β and Ethanol Modulation of GABAergic Transmission in the Central Amygdala.

Brain Sci 2019 Dec 7;9(12). Epub 2019 Dec 7.

Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA.

Myeloid differentiation primary response protein (MyD88) is a critical neuroimmune adaptor protein in TLR (Toll-like receptor) and IL-1R (Interleukin-1 receptor) signaling complexes. These two pro-inflammatory families play an important role in the neurobiology of alcohol use disorder, specifically MyD88 regulates ethanol drinking, ethanol-induced sedation, and ethanol-induced deficits in motor coordination. In this study, we examined the role of MyD88 in mediating the effects of IL-1β and ethanol on GABAergic transmission in the central amygdala (CeA) of male mice using whole-cell patch-clamp recordings in combination with pharmacological (AS-1, a mimetic that prevents MyD88 recruitment by IL-1R) and genetic ( knockout mice) approaches. We demonstrate through both approaches that IL-1β and ethanol's modulatory effects at CeA GABA synapses are not dependent on MyD88. knockout potentiated IL-1β's actions in reducing postsynaptic GABA receptor function. Pharmacological inhibition of MyD88 modulates IL-1β's action at CeA GABA synapses similar to knockout mice. Additionally, ethanol-induced CeA GABA release was greater in knockout mice compared to wildtype controls. Thus, MyD88 is not essential to IL-1β or ethanol regulation of CeA GABA synapses but plays a role in modulating the magnitude of their effects, which may be a potential mechanism by which it regulates ethanol-related behaviors.
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http://dx.doi.org/10.3390/brainsci9120361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6956324PMC
December 2019

Dissecting modular synthases through inhibition: A complementary chemical and genetic approach.

Bioorg Med Chem Lett 2020 01 25;30(2):126820. Epub 2019 Nov 25.

Department of Chemistry and Biochemistry, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0358, USA. Electronic address:

Modular synthases, such as fatty acid, polyketide, and non-ribosomal peptide synthases (NRPSs), are sophisticated machineries essential in both primary and secondary metabolism. Various techniques have been developed to understand their genetic background and enzymatic abilities. However, uncovering the actual biosynthetic pathways remains challenging. Herein, we demonstrate a pipeline to study an assembly line synthase by interrogating the enzymatic function of each individual enzymatic domain of BpsA, a NRPS that produces the blue 3,3'-bipyridyl pigment indigoidine. Specific inhibitors for each biosynthetic domain of BpsA were obtained or synthesized, and the enzymatic performance of BpsA upon addition of each inhibitor was monitored by pigment development in vitro and in living bacteria. The results were verified using genetic mutants to inactivate each domain. Finally, the results complemented the currently proposed biosynthetic pathway of BpsA.
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http://dx.doi.org/10.1016/j.bmcl.2019.126820DOI Listing
January 2020

Tailoring chemoenzymatic oxidation via in situ peracids.

Org Biomol Chem 2019 11;17(43):9418-9424

Department of Chemistry and Biochemistry, UC San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0358, USA.

Epoxidation chemistry often suffers from the challenging handling of peracids and thus requires in situ preparation. Here, we describe a two-phase enzymatic system that allows the effective generation of peracids and directly translate their activity to the epoxidation of olefins. We demonstrate the approach by application to lipid and olefin epoxidation as well as sulfide oxidation. These methods offer useful applications to synthetic modifications and scalable green processes.
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http://dx.doi.org/10.1039/c9ob01814jDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7751277PMC
November 2019

Type II non-ribosomal peptide synthetase proteins: structure, mechanism, and protein-protein interactions.

Nat Prod Rep 2020 03;37(3):355-379

Department of Chemistry and Biochemistry, University of California, 9500 Gilman Drive, La Jolla, San Diego, California 92093-0358, USA.

Covering: 1990 to 2019 Many medicinally-relevant compounds are derived from non-ribosomal peptide synthetase (NRPS) products. Type I NRPSs are organized into large modular complexes, while type II NRPS systems contain standalone or minimal domains that often encompass specialized tailoring enzymes that produce bioactive metabolites. Protein-protein interactions and communication between the type II biosynthetic machinery and various downstream pathways are critical for efficient metabolite production. Importantly, the architecture of type II NRPS proteins makes them ideal targets for combinatorial biosynthesis and metabolic engineering. Future investigations exploring the molecular basis or protein-protein recognition in type II NRPS pathways will guide these engineering efforts. In this review, we consolidate the broad range of NRPS systems containing type II proteins and focus on structural investigations, enzymatic mechanisms, and protein-protein interactions important to unraveling pathways that produce unique metabolites, including dehydrogenated prolines, substituted benzoic acids, substituted amino acids, and cyclopropanes.
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http://dx.doi.org/10.1039/c9np00047jDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7101270PMC
March 2020

Shifting the Hydrolysis Equilibrium of Substrate Loaded Acyl Carrier Proteins.

Biochemistry 2019 08 14;58(34):3557-3560. Epub 2019 Aug 14.

Department of Chemistry and Biochemistry , University of California, San Diego , 9500 Gilman Drive , La Jolla , California 92093-0358 , United States.

Acyl carrier proteins (ACP)s transport intermediates through many primary and secondary metabolic pathways. Studying the effect of substrate identity on ACP structure has been hindered by the lability of the thioester bond that attaches acyl substrates to the 4'-phosphopantetheine cofactor of ACP. Here we show that an acyl acyl-carrier protein synthetase (AasS) can be used in real time to shift the hydrolysis equilibrium toward favoring acyl-ACP during solution NMR spectroscopy. Only 0.005 molar equivalents of AasS enables 1 week of stability to palmitoyl-AcpP from . 2D NMR spectra enabled with this method revealed that the tethered palmitic acid perturbs nearly every secondary structural region of AcpP. This technique will allow previously unachievable structural studies of unstable acyl-ACP species, contributing to the understanding of these complex biosynthetic pathways.
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http://dx.doi.org/10.1021/acs.biochem.9b00612DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7605104PMC
August 2019

Tuning the ultrasonic and photoacoustic response of polydopamine-stabilized perfluorocarbon contrast agents.

J Mater Chem B 2019 08;7(31):4833-4842

Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093, USA.

Contrast-enhanced ultrasound (CEUS) offers the exciting prospect of retaining the ease of ultrasound imaging while enhancing imaging clarity, diagnostic specificity, and theranostic capability. To advance the capabilities of CEUS, the synthesis and understanding of new ultrasound contrast agents (UCAs) is a necessity. Many UCAs are nano- or micro-scale materials composed of a perfluorocarbon (PFC) and stabilizer that synergistically induce an ultrasound response that is both information-rich and easily differentiated from natural tissue. In this work, we probe the extent to which CEUS is modulated through variation in a PFC stabilized with fluorine-modified polydopamine nanoparticles (PDA NPs). The high level of synthetic tunability in this system allows us to study signal as a function of particle aggregation and PFC volatility in a systematic manner. Separation of aggregated and non-aggregated nanoparticles lead to a fundamentally different signal response, and for this system, PFC volatility has little effect on CEUS intensity despite a range of over 50 °C in boiling point. To further explore the imaging tunability and multimodality, Fe3+-chelation was employed to generate an enhanced photoacoustic (PA) signal in addition to the US signal. In vitro and in vivo results demonstrate that PFC-loaded PDA NPs show stronger PA signal than the non-PFC ones, indicating that the PA signal can be used for in situ differentiation between PFC-loading levels. In sum, these data evince the rich role synthetic chemistry can play in guiding new directions of development for UCAs.
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http://dx.doi.org/10.1039/c9tb00928kDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6690494PMC
August 2019

Antitumor Activity of 1,18-Octadecanedioic Acid-Paclitaxel Complexed with Human Serum Albumin.

J Am Chem Soc 2019 07 18;141(30):11765-11769. Epub 2019 Jul 18.

Department of Chemistry & Biochemistry , University of California San Diego , La Jolla , California 92093 , United States.

We describe the design, synthesis, and antitumor activity of an 18 carbon α,ω-dicarboxylic acid monoconjugated via an ester linkage to paclitaxel (PTX). This 1,18-octadecanedioic acid-PTX () prodrug readily forms a noncovalent complex with human serum albumin (HSA). Preservation of the terminal carboxylic acid moiety on enables binding to HSA in the same manner as native long-chain fatty acids (LCFAs), within hydrophobic pockets, maintaining favorable electrostatic contacts between the ω-carboxylate of and positively charged amino acid residues of the protein. This carrier strategy for small molecule drugs is based on naturally evolved interactions between LCFAs and HSA, demonstrated here for PTX. shows differentiated pharmacokinetics, higher maximum tolerated doses and increased efficacy in multiple subcutaneous murine xenograft models of human cancer, as compared to two FDA-approved clinical formulations, Cremophor EL-formulated paclitaxel (crPTX) and Abraxane (nanoparticle albumin-bound (nab)-paclitaxel).
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http://dx.doi.org/10.1021/jacs.9b04272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6676409PMC
July 2019

A Single Tool to Monitor Multiple Protein-Protein Interactions of the Acyl Carrier Protein.

ACS Infect Dis 2019 09 30;5(9):1518-1523. Epub 2019 Jul 30.

Department of Chemistry and Biochemistry , University of California, San Diego , 9500 Gilman Drive , La Jolla , California 92093-0358 , United States.

Protein-protein interactions are ubiquitous to all domains of life and have gained recent interest as drug targets. However, many current methods to study protein-protein interactions can be costly and are low-throughput. Here, we demonstrate a solvatochromic tool based on the natural post-translational modification of the acyl carrier protein (EcACP) used to visualize protein-protein interactions between EcACP and 13 different partner enzymes from several biosynthetic pathways. We use this tool to confirm proposed interactions between EcACP and both catalytic and regulatory proteins. We also show the utility of this method toward detecting allosteric changes to partner enzyme structure and the validation of active site inhibitors. We anticipate the future adaptation of this assay into a high-throughput screen for antibiotic discovery.
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http://dx.doi.org/10.1021/acsinfecdis.9b00150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7607843PMC
September 2019

Molecular basis for interactions between an acyl carrier protein and a ketosynthase.

Nat Chem Biol 2019 07 17;15(7):669-671. Epub 2019 Jun 17.

Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA, USA.

Fatty acid synthases are dynamic ensembles of enzymes that can biosynthesize long hydrocarbon chains efficiently. Here we visualize the interaction between the Escherichia coli acyl carrier protein (AcpP) and β-ketoacyl-ACP-synthase I (FabB) using X-ray crystallography, NMR, and molecular dynamics simulations. We leveraged this structural information to alter lipid profiles in vivo and provide a molecular basis for how protein-protein interactions can regulate the fatty acid profile in E. coli.
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http://dx.doi.org/10.1038/s41589-019-0301-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7323458PMC
July 2019