Jonathan Karty Research Collection

Permanent link for this collectionhttps://hdl.handle.net/2022/26904

Browse

Now showing 1 - 20 of 24

MpeV Is a Lyase Isomerase That Ligates a Doubly-Linked Phycourobilin on the β-Subunit of Phycoerythrin I and II in Marine Synechococcus
(Journal of Biological Chemistry, 2020-11) Carrigee, Lindsay; Frick, Jacob; Garczarek, Laurence; Karty, Jonathan; Partensky, Frederic; Schluchter, Wendy
Synechococcus cyanobacteria are widespread in the marine environment, as the extensive pigment diversity within their light-harvesting phycobilisomes enables them to utilize various wavelengths of light for photosynthesis. The phycobilisomes of Synechococcus sp. RS9916 contain two forms of the protein phycoerythrin (PEI and PEII), each binding two chromophores, green-light absorbing phycoerythrobilin and blue-light absorbing phycourobilin. These chromophores are ligated to specific cysteines via bilin lyases, and some of these enzymes, called lyase isomerases, attach phycoerythrobilin and simultaneously isomerize it to phycourobilin. MpeV is a putative lyase isomerase whose role in PEI and PEII biosynthesis is not clear. We examined MpeV in RS9916 using recombinant protein expression, absorbance spectroscopy, and tandem mass spectrometry. Our results show that MpeV is the lyase isomerase that covalently attaches a doubly linked phycourobilin to two cysteine residues (C50, C61) on the β-subunit of both PEI (CpeB) and PEII (MpeB). MpeV activity requires that CpeB or MpeB is first chromophorylated by the lyase CpeS (which adds phycoerythrobilin to C82). Its activity is further enhanced by CpeZ (a homolog of a chaperone-like protein first characterized in Fremyella diplosiphon). MpeV showed no detectable activity on the α-subunits of PEI or PEII. The mechanism by which MpeV links the A and D rings of phycourobilin to C50 and C61 of CpeB was also explored using site-directed mutants, revealing that linkage at the A ring to C50 is a critical step in chromophore attachment, isomerization, and stability. These data provide novel insights into β-PE biosynthesis and advance our understanding of the mechanisms guiding lyase isomerases.
Evolution of a Strategy for the Enantioselective Synthesis of (−)-Cajanusine
(Journal of the American Chemical Society, 2020) Brown, M. Kevin; Guo, Renyu; Witherspoon, Brittany
The first enantioselective synthesis of (−)-cajanusine is presented. Key features of the route include a rapid synthesis of the [4.2.0]bicyclooctane core by an enantioselective isomerization/stereoselective [2+2]-cycloaddition strategy as well as prominent use of catalytic methods for bond construction. The evolution of the approach is also presented that highlights unexpected roadblocks and how novel solutions were developed.
Lessons in Strain and Stability: Enantioselective Synthesis of (+)‐[5]‐Ladderanoic Acid
(Angewandte Chemie, 2020) Brown, M. Kevin; Hancock, Erin; Kuker, Erin; Tantillo, Dean
The synthesis of structurally complex and highly strained natural products provides unique challenges and unexpected opportunities for the development of new reactions and strategies. Herein, the synthesis of (+)-[5]-ladderanoic acid is reported. En route to the target, unusual and unexpected strain release driven transformations were uncovered. This occurrence required a drastic revision of the synthetic design that ultimately led to the development of a novel stepwise cyclobutane assembly by an allylboration/Zweifel olefination sequence.
N-Directed Fluorination of Unactivated Csp3–H Bonds
(Chemical Science, 2020) AbuSalim, Deyaa; Bingham, Jenna; Cook, Silas; Pinter, Emily
CpeF Is the Bilin Lyase That Ligates the Doubly Linked Phycoerythrobilin on β-Phycoerythrin in the Cyanobacterium Fremyella Diplosiphon.
(Journal of Biological Chemistry, 2019-01) Boutaghou, M. Nazim; Cole, Richard; Frick, Jacob; Gutu, Andrian; Hernandez, Carla; Hernandez, Leanora; Karty, Jonathan; Kehoe, David; Kronfel, Christina; Schluchter, Wendy
Phycoerythrin (PE) is a green light–absorbing protein present in the light-harvesting complex of cyanobacteria and red algae. The spectral characteristics of PE are due to its prosthetic groups, or phycoerythrobilins (PEBs), that are covalently attached to the protein chain by specific bilin lyases. Only two PE lyases have been identified and characterized so far, and the other bilin lyases are unknown. Here, using in silico analyses, markerless deletion, biochemical assays with purified and recombinant proteins, and site-directed mutagenesis, we examined the role of a putative lyase-encoding gene, cpeF, in the cyanobacterium Fremyella diplosiphon. Analyzing the phenotype of the cpeF deletion, we found that cpeF is required for proper PE biogenesis, specifically for ligation of the doubly linked PEB to Cys-48/Cys-59 residues of the CpeB subunit of PE.We also show that in a heterologous host, CpeF can attach PEB to Cys-48/Cys-59 of CpeB, but only in the presence of the chaperone-like protein CpeZ. Additionally, we report that CpeF likely ligates the A ring of PEB to Cys-48 prior to the attachment of the D ring to Cys-59. We conclude that CpeF is the bilin lyase responsible for attachment of the doubly ligated PEB to Cys-48/Cys-59 of CpeB and together with other specific bilin lyases contributes to the post-translational modification and assembly of PE into mature light-harvesting complexes.
Lactate dehydrogenase and glycerol-3-phosphate dehydrogenase cooperatively regulate growth and carbohydrate metabolism during Drosophila melanogaster larval development
(Development, 2019) Buddika, Kasun; Burton, Anna; Chawla, Geetanjali; Gosney, Chelsea; Julick, Cole; Karty, Jonathan; Li, Hongde; Luhur, Arthur; Mahmoudzadeh, Nader; Montooth, Kristi; Pletcher, Rose; Rai, Madhulika; Sokol, Nicholas; Tennessen, Jason
The dramatic growth that occurs during Drosophila larval development requires rapid conversion of nutrients into biomass. Many larval tissues respond to these biosynthetic demands by increasing carbohydrate metabolism and lactate dehydrogenase (LDH) activity. The resulting metabolic program is ideally suited for synthesis of macromolecules and mimics the manner by which cancer cells rely on aerobic glycolysis. To explore the potential role of Drosophila LDH in promoting biosynthesis, we examined how Ldh mutations influence larval development. Our studies unexpectedly found that Ldh mutants grow at a normal rate, indicating that LDH is dispensable for larval biomass production. However, subsequent metabolomic analyses suggested that Ldh mutants compensate for the inability to produce lactate by generating excess glycerol-3-phosphate (G3P), the production of which also influences larval redox balance. Consistent with this possibility, larvae lacking both LDH and G3P dehydrogenase (GPDH1) exhibit growth defects, synthetic lethality and decreased glycolytic flux. Considering that human cells also generate G3P upon inhibition of lactate dehydrogenase A (LDHA), our findings hint at a conserved mechanism in which the coordinate regulation of lactate and G3P synthesis imparts metabolic robustness to growing animal tissues.
Multi-metal Restriction by Calprotectin Impacts De Novo Flavin Biosynthesis in Acinetobacter baumannii
(Cell Chemical Biology, 2019) Andreini, Claudia; Chazin, Walter; Giedroc, David; Gonzalez-Gutierrez, Giovanni; Karty, Jonathan; Lonergan, Zachery; Maxwell, Christina; Nairn, Brittany; Skaar, Eric; Trinidad, Jonathan; Wang, Jiefei; Zhang, Yixiang
Phosphate–phosphate oligomerization drives higher order co-assemblies with stacks of cyanostar macrocycles
(Chemical Science, 2018) Fatila, Elisabeth; Flood, Amar; Karty, Jonathan; Pink, Maren; Twum, Eric
Modular Self-Assembly of Protein Cage Lattices for Multistep Catalysis
(ACS Nano, 2017-11) Douglas, Trevor; Fukuto, Masafumi; Karty, Jonathan; LaFrance, Ben; McCoy, Kimberly; Miettinen, Heini; Patterson, Dustin; Prevelige, Peter Jr.; Schwarz, Benjamin; Uchida, Masaki; Yang, Lin; Yoshimura, Hideyuki
The assembly of individual molecules into hierarchical structures is a promising strategy for developing three-dimensional materials with properties arising from interaction between the individual building blocks. Virus capsids are elegant examples of biomolecular nanostructures, which are themselves hierarchically assembled from a limited number of protein subunits. Here, we demonstrate the bio-inspired modular construction of materials with two levels of hierarchy: the formation of catalytically active individual virus-like particles (VLPs) through directed self-assembly of capsid subunits with enzyme encapsulation, and the assembly of these VLP building blocks into three-dimensional arrays. The structure of the assembled arrays was successfully altered from an amorphous aggregate to an ordered structure, with a face-centered cubic lattice, by modifying the exterior surface of the VLP without changing its overall morphology, to modulate interparticle interactions. The assembly behavior and resultant lattice structure was a consequence of interparticle interaction between exterior surfaces of individual particles and thus independent of the enzyme cargos encapsulated within the VLPs. These superlattice materials, composed of two populations of enzyme-packaged VLP modules, retained the coupled catalytic activity in a two-step reaction for isobutanol synthesis. This study demonstrates a significant step toward the bottom-up fabrication of functional superlattice materials using a self-assembly process across multiple length scales and exhibits properties and function that arise from the interaction between individual building blocks.
Capillary electrophoresis–mass spectrometry for direct structural identification of serum N-glycans
(Journal of Chromatography A, 2017) Fonslow, Bryan; Jacobson, Stephen; Karty, Jonathan; Novotny, Milos; Snyder, Christa; Zhou, Xiaomei
Metabolomic Analysis Reveals That the Drosophila melanogaster Gene lysine Influences Diverse Aspects of Metabolism
(Genetics, 2017-10) Ashraf, Usman; Karty, Jonathan; Li, Hongde; St. Clair, Samantha; Tennessen, Jason
The fruit fly Drosophila melanogaster has emerged as a powerful model for investigating the molecular mechanisms that regulate animal metabolism. However, a major limitation of these studies is that many metabolic assays are tedious, dedicated to analyzing a single molecule, and rely on indirect measurements. As a result, Drosophila geneticists commonly use candidate gene approaches, which, while important, bias studies toward known metabolic regulators. In an effort to expand the scope of Drosophila metabolic studies, we used the classic mutant lysine (lys) to demonstrate how a modern metabolomics approach can be used to conduct forward genetic studies. Using an inexpensive and well-established gas chromatography-mass spectrometry-based method, we genetically mapped and molecularly characterized lys by using free lysine levels as a phenotypic readout. Our efforts revealed that lys encodes the Drosophila homolog of Lysine Ketoglutarate Reductase/Saccharopine Dehydrogenase, which is required for the enzymatic degradation of lysine. Furthermore, this approach also allowed us to simultaneously survey a large swathe of intermediate metabolism, thus demonstrating that Drosophila lysine catabolism is complex and capable of influencing seemingly unrelated metabolic pathways. Overall, our study highlights how a combination of Drosophila forward genetics and metabolomics can be used for unbiased studies of animal metabolism, and demonstrates that a single enzymatic step is intricately connected to diverse aspects of metabolism.
Synergism between genome sequencing, tandem mass spectrometry and bio-inspired synthesis reveals insights into nocardioazine B biogenesis
(Organic & Biomolecular Chemistry, 2015) Alqahtani, Norah; Bis, Dana; James, Elle; Karty, Jonathan; Lane, Amy; Porwal, Suheel; Viswanathan, Rajesh
Marine actinomycete-derived natural products continue to inspire chemical and biological investigations. Nocardioazines A and B (3 and 4), from Nocardiopsis sp. CMB-M0232, are structurally unique alkaloids featuring a 2,5-diketopiperazine (DKP) core functionalized with indole C3-prenyl as well as indole C3 and N-methyl groups. The logic of their assembly remains cryptic. Bioinformatics analyses of the Nocardiopsis sp. CMB-M0232 draft genome afforded the noz cluster, split across two regions of the genome, and encoding putative open reading frames with roles in nocardioazine biosynthesis, including cyclodipeptide synthase (CDPS), prenyltransferase, methyltransferase, and cytochrome P450 homologs. Heterologous expression of a twelve gene contig from the noz cluster in Streptomyces coelicolor resulted in accumulation of cyclo-L-Trp-L-Trp DKP (5). This experimentally connected the noz cluster to indole alkaloid natural product biosynthesis. Results from bioinformatics analyses of the noz pathway along with challenges in actinomycete genetics prompted us to use asymmetric synthesis and mass spectrometry to determine biosynthetic intermediates in the noz pathway. The structures of hypothesized biosynthetic intermediates 5 and 12–17 were firmly established through chemical synthesis. LC-MS and MS-MS comparison of these synthetic compounds with metabolites present in chemical extracts from Nocardiopsis sp. CMB-M0232 revealed which of these hypothesized intermediates were relevant in the nocardioazine biosynthetic pathway. This established the early and mid-stages of the biosynthetic pathway, demonstrating that Nocardiopsis performs indole C3-methylation prior to indole C3-normal prenylation and indole N1’-methylation in nocardioazine B assembly. These results highlight the utility of merging bioinformatics analyses, asymmetric synthetic approaches, and mass spectrometric metabolite profiling in probing natural product biosynthesis.
Chelation-induced diradical formation as an approach to modulation of the amyloid-b aggregation pathway
(Chemical Science, 2015) Karty, Jonathan; Kochi, Akiko; Lim, Mi Hee; Porter, Meghan; Zaleski, Jeffrey
Nanopipettes: probes for local sample analysis
(Chemical Science, 2015) Baker, Lane; Hieftje, Gary; Karty, Jonathan; Ray, Steven; Saha-Shah, Anumita; Weber, Anna
Nanopipettes (pipettes with diameters <1 mm) were explored as pressure-driven fluid manipulation tools for sampling nanoliter volumes of fluids. The fundamental behavior of fluids confined in the narrow channels of the nanopipette shank was studied to optimize sampling volume and probe geometry. This method was utilized to collect nanoliter volumes (<10 nL) of sample from single Allium cepa cells and live Drosophila melanogaster first instar larvae. Matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) was utilized to characterize the collected sample. The use of nanopipettes for surface sampling of mouse brain tissue sections was also explored. Lipid analyses were performed on mouse brain tissues with spatial resolution of sampling as small as 50 mm. Nanopipettes were shown to be a versatile tool that will find further application in studies of sample heterogeneity and population analysis for a wide range of samples.
Vitamin B12 regulates photosystem gene expression via the CrtJ antirepressor AerR in Rhodobacter capsulatus
(Molecular Microbiology, 2014-01) Bauer, Carl; Cheng, Zhuo; Hammad, Loubna; Karty, Jonathan; Keran, Li
The tetrapyrroles haem, bacteriochlorophyll and cobalamin (B12) exhibit a complex interrelationship regarding their synthesis. In this study, we demonstrate that AerR functions as an antirepressor of the tetrapyrrole regulator CrtJ. We show that purified AerR contains B12 that is bound to a conserved histidine (His145) in AerR. The interaction of AerR to CrtJ was further demonstrated in vitro by pull down experiments using AerR as bait and quantified using microscale thermophoresis. DNase I DNA footprint assays show that AerR containing B12 inhibits CrtJ binding to the bchC promoter. We further show that bchC expression is greatly repressed in a B12 auxotroph of Rhodobacter capsulatus and that B12 regulation of gene expression is mediated by AerR’s ability to function as an antirepressor of CrtJ. This study thus provides a mechanism for how the essential tetrapyrrole, cobalamin controls the synthesis of bacteriochlorophyll, an essential component of the photosystem.
Integrated Metabolomics Approach Facilitates Discovery of an Unpredicted Natural Product Suite from Streptomyces coelicolor M145
(ACS Chemical Biology, 2013-06) Carlson, Erin; Johnson, Andrew; Karty, Jonathan; Sidebottom, Ashley; Trader, Darci
Natural products exhibit a broad range of biological properties and have been a crucial source of therapeutic agents and novel scaffolds. Although bacterial secondary metabolomes are widely explored, they remain incompletely cataloged by current isolation and characterization strategies. To identify metabolites residing in unexplored chemical space, we have developed an integrated discovery approach that combines bacterial growth perturbation, accurate mass spectrometry, comparative mass spectra data analysis, and fragmentation spectra clustering for the identification of low-abundant, novel compounds from complex biological matrices. In this investigation, we analyzed the secreted metabolome of the extensively studied Actinomycete, Streptomyces coelicolor M145, and discovered a low-abundant suite of 15 trihydroxamate, amphiphilic siderophores. Compounds in this class have primarily been observed in marine microorganisms making their detection in the soil-dwelling S. coelicolor M145 significant. At least 10 of these ferrioxamine-based molecules are not known to be produced by any organism, and none have previously been detected from S. coelicolor M145. In addition, we confirmed the production of ferrioxamine D1, a relatively hydrophilic family member that has not been shown to be biosynthesized by this organism. The identified molecules are part of only a small list of secondary metabolites that have been discovered since sequencing of S. coelicolor M145 revealed that it possessed numerous putative secondary metabolite-producing gene clusters with no known metabolites. Thus, the identified siderophores represent the unexplored metabolic potential of both well-studied and new organisms that could be uncovered with our sensitive and robust approach.
RegB Kinase Activity Is Repressed by Oxidative Formation of Cysteine Sulfenic Acid
(Journal of Biological Chemistry, 2013-02) Bauer, Carl; Carroll, Kate; Cheng, Zhuo; Hammad, Loubna; Karty, Jonathan; Reddie, Khalilah; Wu, Jiang
RegB/RegA comprise a global redox-sensing signal transduction system utilized by a wide range of proteobacteria to sense environmental changes in oxygen tension. The conserved cysteine 265 in the sensor kinase RegB was previously reported to form an intermolecular disulfide bond under oxidizing conditions that converts RegB from an active dimer into an inactive tetramer. In this study, we demonstrate that a stable sulfenic acid (-SOH) derivative also forms at Cys-265 in vitro and in vivo when RegB is exposed to oxygen. This sulfenic acid modification is reversible and stable in the air. Autophosphorylation assay shows that reduction of the SOH at Cys-265 to a free thiol (SH) can increase RegB kinase activity in vitro. Our results suggest that a sulfenic acid modification at Cys-265 performs a regulatory role in vivo and that it may be the major oxidation state of Cys-265 under aerobic conditions. Cys-265 thus functions as a complex redox switch that can form multiple thiol modifications in response to different redox signals to control the kinase activity of RegB.
Redox and Light Control the Heme-Sensing Activity of AppA
(mBio, 2013-08) Bauer, Carl; Dan III, Charles; Dragnea, Vladimira; Feldman, George; Hammad, Loubna; Karty, Jonathan; Yin, Liang
The DNA binding activity of the photosystem-specific repressor PpsR is known to be repressed by the antirepressor AppA. AppA contains a blue-light-absorbing BLUF domain and a heme-binding SCHIC domain that controls the interaction of AppA with PpsR in response to light and heme availability. In this study, we have solved the structure of the SCHIC domain and identified the histidine residue that is critical for heme binding. We also demonstrate that dark-adapted AppA binds heme better than light-excited AppA does and that heme bound to the SCHIC domain significantly reduces the length of the BLUF photocycle. We further show that heme binding to the SCHIC domain is affected by the redox state of a disulfide bridge located in the Cys-rich carboxyl-terminal region. These results demonstrate that light, redox, and heme are integrated inputs that control AppA’s ability to disrupt the DNA binding activity of PpsR.
In a Variable Thermal Environment Selection Favors Greater Plasticity of Cell Membranes in Drosophila Melanogaster
(Evolution, 2012) Cooper, B.S.; Hammad, L.A.; Fisher, N.P.; Karty, Jonathan; Montooth, K.L.
Theory predicts that developmental plasticity, the capacity to change phenotypic trajectory during development, should evolve when the environment varies sufficiently among generations, owing to temporal (e.g., seasonal) variation or to migration among environments. We characterized the levels of cellular plasticity during development in populations of Drosophila melanogaster experimentally evolved for over three years in either constant or temporally variable thermal environments. We used two measures of the lipid composition of cell membranes as indices of physiological plasticity (a.k.a. acclimation): (1) change in the ratio of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) and (2) change in lipid saturation (number of double bonds) in cool (16°C) relative to warm (25°C) developmental conditions. Flies evolved under variable environments had a greater capacity to acclimate the PE/PC ratio compared to flies evolved in constant environments, supporting the prediction that environments with high among-generation variance favor greater developmental plasticity. Our results are consistent with the selective advantage of a more environmentally sensitive allele that may have associated costs in constant environments.
Activity of the tetrapyrrole regulator CrtJ is controlled by oxidation of a redox active cysteine located in the DNA binding domain
(Molecular Microbiology, 2012) Cheng, Z.; Wu, J.; Setterdahl, A.; Reddie, K.; Carroll, K.; Hammad, L.A.; Karty, Jonathan; Bauer, C.E.
CrtJ from Rhodobacter capsulatus is a regulator of genes involved in the biosynthesis of haem, bacteriochlorophyll, carotenoids as well as structural proteins of the light harvesting-II complex. Fluorescence anisotropy-based DNA-binding analysis demonstrates that oxidized CrtJ exhibits ??20-fold increase in binding affinity over that of reduced CrtJ. Liquid chromatography electrospray tandem ionization mass spectrometric analysis using DAz-2, a sulfenic acid (-SOH)-specific probe, demonstrates that exposure of CrtJ to oxygen or to hydrogen peroxide leads to significant accumulation of a sulfenic acid derivative of Cys420 which is located in the helix-turn-helix (HTH) motif. In vivo labelling with 4-(3-azidopropyl)cyclohexane-1,3-dione (DAz-2) shows that Cys420 also forms a sulfenic acid modification in vivo when cells are exposed to oxygen. Moreover, a Cys420 to Ala mutation leads to a ∼ 60-fold reduction of DNA binding activity while a Cys to Ser substitution at position 420 that mimics a cysteine sulfenic acid results in a ∼ 4-fold increase in DNA binding activity. These results provide the first example where sulfenic acid oxidation of a cysteine in a HTH-motif leads to differential effects on gene expression.

Browse

Recent Submissions