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1.  Elevated Transglutaminase 2 Activity is Associated with Hypoxia-Induced Experimental Pulmonary Hypertension in Mice 
ACS chemical biology  2013;9(1):266-275.
Previous studies in human patients and animal models have suggested that transglutaminase 2 (TG2) is upregulated in pulmonary hypertension (PH), a phenomenon that appears to be associated with the effects of serotonin (5-hydroxytryptamine; 5-HT) in this disease. Using chemical tools to interrogate and inhibit TG2 activity in vivo, we have shown that pulmonary TG2 undergoes marked post-translational activation in a mouse model of hypoxia-induced PH. We have also identified irreversible fluorinated TG2 inhibitors that may find use as non-invasive positron emission tomography probes for diagnosis and management of this debilitating, lifelong disorder. Pharmacological inhibition of TG2 attenuated the elevated right ventricular pressure but had no effect on hypertrophy of the right ventricle of the heart. A longitudinal study of pulmonary TG2 activity in PH patients is warranted.
doi:10.1021/cb4006408
PMCID: PMC3947056  PMID: 24152195
2.  Metabolic Flux Between Unsaturated and Saturated Fatty Acids is Controlled by the FabA:FabB Ratio in the Fully Reconstituted Fatty Acid Biosynthetic Pathway of E. coli# 
Biochemistry  2013;52(46):10.1021/bi401116n.
The entire fatty acid biosynthetic pathway from Escherichia coli, starting from the acetyl-CoA carboxylase, has been reconstituted in vitro from fourteen purified protein components. Radiotracer analysis verified stoichiometric conversion of acetyl-CoA and NAD(P)H into the free fatty acid product, allowing implementation of a facile spectrophotometric assay for kinetic analysis of this multi-enzyme system. At steady state, a maximum turnover rate of 0.5 s−1 was achieved. Under optimal turnover conditions, the predominant products were C16 and C18 saturated as well as monounsaturated fatty acids. The reconstituted system allowed us to quantitatively interrogate the factors that influence metabolic flux toward unsaturated versus saturated fatty acids. In particular, the concentrations of the dehydratase FabA and the β-ketoacyl synthase FabB were found to be crucial for controlling this property. By altering these variables, the percentage of unsaturated fatty acid produced could be adjusted between 10 and 50% without significantly affecting the maximum turnover rate of the pathway. Our reconstituted system provides a powerful tool to understand and engineer rate-limiting and regulatory steps in this complex and practically significant metabolic pathway.
doi:10.1021/bi401116n
PMCID: PMC3858588  PMID: 24147979
3.  In vitro Reconstitution and Analysis of the 6-Deoxyerythronolide B Synthase 
Journal of the American Chemical Society  2013;135(45):10.1021/ja409048k.
Notwithstanding an extensive literature on assembly line polyketide synthases such as the 6-deoxyerythronolide B synthase (DEBS), a complete naturally occurring synthase has never been reconstituted in vitro from purified protein components. Here, we describe the fully reconstituted DEBS and quantitatively characterize some of the properties of the assembled system that have never been explored previously. The maximum turnover rate of the complete hexamodular system is 1.1 min−1, comparable to the turnover rate of a truncated trimodular derivative (2.5 min−1) but slower than a bimodular derivative (21 min−1). In the presence of similar concentrations of methylmalonyl- and ethylmalonyl-CoA substrates, DEBS synthesizes multiple regiospecifically modified analogs, one of which we have analyzed in detail. Our studies lay the foundation for biochemically interrogating and rationally engineering polyketide assembly lines in an unprecedented manner.
doi:10.1021/ja409048k
PMCID: PMC3858836  PMID: 24161212
4.  Rationale for Using Social Media to Collect Patient-Reported Outcomes in Patients with Celiac Disease 
Patients with celiac disease (CD) are increasingly interconnected through social media, exchanging patient experiences and health-tracking information between individuals through various web-based platforms. Social media represents potentially unique communication interface between gastroenterologists and active social media users – especially young adults and adolescents with celiac disease-regarding adherence to the strict gluten-free diet, gastrointestinal symptoms, and meaningful discussion about disease management. Yet, various social media platforms may be underutilized for research purposes to collect patient-reported outcomes data. In this commentary, we summarize the scientific rationale and potential for future growth of social media in patient-reported outcomes research, focusing on college freshmen with celiac disease as a case study and provide overview of the methodological approach. Finally, we discuss how social media may impact patient care in the future through increasing mobile technology use.
doi:10.4172/2161-069X.1000166
PMCID: PMC4226063  PMID: 25392743
Social media; Social networking; Facebook; Patient-reported outcomes; Healthcare; Mobile technology; Quality-of-life
5.  Coupled Methyl-Group Epimerization and Reduction by Polyketide Synthase Ketoreductase Domains. Ketoreductase-Catalyzed Equilibrium Isotope Exchange 
Journal of the American Chemical Society  2013;135(44):10.1021/ja408944s.
Incubation of [2-2H]-(2S,3R)-2-methyl-3-hydroxypentanoyl-SACP ([2-2H]-1a) with the epimerizing ketoreductase domain EryKR1 in the presence of a catalytic amount NADP+ (0.05 equiv) resulted in time-and cofactor-dependent washout of deuterium from 1a, as a result of equilibrium isotope exchange of transiently generated [2-2H]-2-methyl-3-ketopentanoyl-ACP (2). Incubations of [2-2H]-(2S,3S)-2-methyl-3-hydroxypentanoyl-SACP ([2-2H]-1b) with RifKR7 and with NysKR1 also resulted in time-dependent loss of deuterium. By contrast, incubations of [2-2H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-SACP ([2-2H]-1c) and [2-2H]-(2R,3R)-2-methyl-3-hydroxypentanoyl-SACP ([2-2H]-1d) with the non-epimerizing ketoreductase domains EryKR6 and TylKR1, respectively, did not result in any significant washout of deuterium. The isotope exchange assay directly establishes that specific polyketide synthase ketoreductase domains also have an intrinsic epimerase activity, thus enabling mechanistic analysis of a key determinant of polyketide stereocomplexity.
doi:10.1021/ja408944s
PMCID: PMC3865775  PMID: 24161343
6.  Computational identification and analysis of orphan assembly-line polyketide synthases 
The Journal of antibiotics  2013;67(1):89-97.
The increasing availability of DNA sequence data offers an opportunity for identifying new assembly-line polyketide synthases (PKSs) that produce biologically active natural products. We developed an automated method to extract and consolidate all multimodular PKS sequences (including hybrid PKS/non-ribosomal peptide synthetases) in the National Center for Biotechnology Information (NCBI) database, generating a non-redundant catalog of 885 distinct assembly-line PKSs, the majority of which were orphans associated with no known polyketide product. Two in silico experiments highlight the value of this search method and resulting catalog. First, we identified an orphan that could be engineered to produce an analog of albocycline, an interesting antibiotic whose gene cluster has not yet been sequenced. Second, we identified and analyzed a hitherto overlooked family of metazoan multimodular PKSs, including one from Caenorhabditis elegans. We also developed a comparative analysis method that identified sequence relationships among known and orphan PKSs. As expected, PKS sequences clustered according to structural similarities between their polyketide products. The utility of this method was illustrated by highlighting an interesting orphan from the genus Burkholderia that has no close relatives. Our search method and catalog provide a community resource for the discovery of new families of assembly-line PKSs and their antibiotic products.
doi:10.1038/ja.2013.125
PMCID: PMC4028714  PMID: 24301183
assembly line; orphan antibiotics; polyketide synthase
7.  Expanding the fluorine chemistry of living systems using engineered polyketide synthase pathways 
Science (New York, N.Y.)  2013;341(6150):1089-1094.
Organofluorines represent a rapidly expanding proportion of molecules used in pharmaceuticals, diagnostics, agrochemicals, and materials. Despite the prevalence of fluorine in synthetic compounds, the known biological scope is limited to a single pathway that produces fluoroacetate. Here, we demonstrate that this pathway can be exploited as a source of fluorinated building blocks for introduction of fluorine into natural product scaffolds. Specifically, we have constructed pathways involving two polyketide synthase systems and show that fluoroacetate can be used to incorporate fluorine into the polyketide backbone in vitro. We further show that fluorine can be introduced site-selectively and introduced into polyketide products in vivo. These results highlight the prospects for the production of complex fluorinated natural products using synthetic biology.
doi:10.1126/science.1242345
PMCID: PMC4057101  PMID: 24009388
8.  Dihydroisoxazole inhibitors of Anopheles gambiae seminal transglutaminase AgTG3 
Malaria Journal  2014;13:210.
Background
Current vector-based malaria control strategies are threatened by the rise of biochemical and behavioural resistance in mosquitoes. Researching mosquito traits of immunity and fertility is required to find potential targets for new vector control strategies. The seminal transglutaminase AgTG3 coagulates male Anopheles gambiae seminal fluids, forming a ‘mating plug’ that is required for male reproductive success. Inhibitors of AgTG3 can be useful both as chemical probes of A. gambiae reproductive biology and may further the development of new chemosterilants for mosquito population control.
Methods
A targeted library of 3-bromo-4,5-dihydroxoisoxazole inhibitors were synthesized and screened for inhibition of AgTG3 in a fluorescent, plate-based assay. Positive hits were tested for in vitro activity using cross-linking and mass spectrometry, and in vivo efficacy in laboratory mating assays.
Results
A targeted chemical library was screened for inhibition of AgTG3 in a fluorescent plate-based assay using its native substrate, plugin. Several inhibitors were identified with IC50 < 10 μM. Preliminary structure-activity relationships within the library support the stereo-specificity and preference for aromatic substituents in the chemical scaffold. Both inhibition of plugin cross-linking and covalent modification of the active site cysteine of AgTG3 were verified. Administration of an AgTG3 inhibitor to A. gambiae males by intrathoracic injection led to a 15% reduction in mating plug transfer in laboratory mating assays.
Conclusions
A targeted screen has identified chemical inhibitors of A. gambiae transglutaminase 3 (AgTG3). The most potent inhibitors are known inhibitors of human transglutaminase 2, suggesting a common binding pose may exist within the active site of both enzymes. Future efforts to develop additional inhibitors will provide chemical tools to address important biological questions regarding the role of the A. gambiae mating plug. A second use for transglutaminase inhibitors exists for the study of haemolymph coagulation and immune responses to wound healing in insects.
doi:10.1186/1475-2875-13-210
PMCID: PMC4113009  PMID: 24888439
9.  Stereochemistry of Reductions Catalyzed by Methyl-Epimerizing Ketoreductase Domains of Polyketide Synthases 
Ketoreductase (KR) domains from modular polyketide synthases catalyze reduction of 2-methyl-3-ketoacyl-ACP substrates and in certain cases epimerization of the 2-methyl group as well. The structural and mechanistic basis of epimerization is poorly understood, and only a small number of such KRs been studied. We have studied three recombinant KR domains with putative epimerase activity – NysKR1 from module 1 of the nystatin PKS whose stereospecificity can be predicted from both protein sequence and product structure, RifKR7 from module 7 of the rifamycin PKS whose stereospecificity cannot be predicted from protein sequence, and RifKR10 from module 10 of the rifamycin PKS whose specificity is unclear from both sequence and structure. Each KR was individually incubated with NADPH and (2R)- or (2RS)-2-methyl-3-ketopentanoyl-ACP generated enzymatically in situ or via chemo-enzymatic synthesis, respectively. Chiral GC-MS analysis revealed that each KR stereospecifically produced the corresponding (2S,3S)-2-methyl-3-hydroxypentanoyl-ACP in which the 2-methyl substituent had undergone KR-catalyzed epimerization. Thus, our results have led to the identification of a prototypical set of KR domains that generate (2S,3S)-2-methyl-3-hydroxyacyl products in the course of polyketide biosynthesis.
doi:10.1021/ja4014776
PMCID: PMC3699853  PMID: 23659177
10.  Gluten-sensitive enteropathy coincides with decreased capability of intestinal T cells to secrete IL-17 and IL-22 in a macaque model for celiac disease 
Celiac disease (CD) is an autoimmune disorder caused by intolerance to dietary gluten. The interleukin (IL)-17 and IL-22 function as innate regulators of mucosal integrity. Impaired but not well-understood kinetics of the IL-17/22 secretion was described in celiac patients. Here, the IL-17 and IL-22-producing intestinal cells were studied upon their in vitro stimulation with mitogens in class II major histocompatibility complex-defined, gluten-sensitive rhesus macaques. Pediatric biopsies were collected from distal duodenum during the stages of disease remission and relapse. Regardless of dietary gluten content, IL-17 and IL-22-producing cells consisted of CD4+ and CD8+ T lymphocytes as well as of lineage-negative (Lin−) cells. Upon introduction of dietary gluten, capability of intestinal T cells to secrete IL-17/22 started to decline (p < 0.05), which was paralleled with gradual disruption of epithelial integrity. These data indicate that IL-17/22-producing cells play an important role in maintenance of intestinal mucosa in gluten-sensitive primates.
doi:10.1016/j.clim.2013.02.012
PMCID: PMC3732447  PMID: 23518597
Celiac disease; Th17; Rhesus; Tissue transglutaminase; Autoimmunity
11.  Mechanism and Specificity of an Acyltransferase Domain from a Modular Polyketide Synthase 
Biochemistry  2013;52(11):1839-1841.
Acyltransferase (AT) domains of modular polyketide synthases exercise tight control over the choice of α-carboxyacyl-CoA substrates, but the mechanistic basis for this specificity is unknown. We show that whereas the specificity for the electrophilic malonyl or methylmalonyl component is primarily expressed in the first half-reaction (formation of the acyl enzyme intermediate), the second half-reaction shows comparable specificity for the acyl carrier protein that carries the nucleophilic pantetheine arm. We also show that currently used approaches for engineering AT domain specificity work mainly by degrading specificity for the natural substrate rather than by enhancing specificity for alternative substrates.
doi:10.1021/bi400185v
PMCID: PMC3612939  PMID: 23452124
12.  Analysis and Refactoring of the A-74528 Biosynthetic Pathway 
A-74528 is a C-30 polyketide natural product that functions as an inhibitor of 2′,5′-oligoadenylate phosphodiesterase (2′-PDE), a key regulatory enzyme of the interferon pathway. Modulation of 2′-PDE represents a unique therapeutic approach to regulate viral infections. The gene cluster responsible for biosynthesis of A-74528 yields minute amounts of this natural product together with considerably larger quantities of a structurally dissimilar C-30 cytotoxic agent, fredericamycin. Through construction and analysis of a series of knockout mutants, we identified the necessary genes for A-74528 biosynthesis. Remarkably, the formation of six stereocenters and the regiospecific formation of six rings in A- 74528 appears to be catalyzed by only two tailoring enzymes, a cyclase and an oxygenase, in addition to the core polyketide synthase. The inferred pathway was genetically refactored in a heterologous host, Streptomyces coelicolor CH999, to produce 3 mg/L A-74528 in the absence of fredericamycin.
doi:10.1021/ja311579s
PMCID: PMC3601660  PMID: 23442197
13.  Selective Inhibition of Extracellular Thioredoxin by Asymmetric Disulfides 
Journal of medicinal chemistry  2013;56(3):1301-1310.
Whereas the role of mammalian thioredoxin (Trx) as an intracellular protein cofactor is widely appreciated, its function in the extracellular environment is not well understood. Only few extracellular targets of Trx-mediated thiol-disulfide exchange are known. For example, Trx activates extracellular transglutaminase 2 (TG2) via reduction of an intramolecular disulfide bond. Because hyperactive TG2 is thought to play a role in various diseases, understanding the biological role of extracellular Trx may provide critical insight into the pathogenesis of these disorders. Starting from a clinical-stage asymmetric disulfide lead, we have identified analogs with >100-fold specificity for Trx. Structure-activity relationship and computational docking model analyses have provided insights into the features important for enhancing potency and specificity. The most active compound identified had an IC50 below 0.1µM in cell culture, and may be appropriate for in vivo use to interrogate the role of extracellular Trx in health and disease.
doi:10.1021/jm301775s
PMCID: PMC3574193  PMID: 23327656
14.  Molecular Insights into the Biosynthesis of Guadinomine, a Type III Secretion System Inhibitor 
Journal of the American Chemical Society  2012;134(42):17797-17806.
Guadinomines are a recently discovered family of anti-infective compounds produced by Streptomyces sp. K01-0509 with a novel mode of action. With an IC50 of 14 nM, guadinomine B is the most potent known inhibitor of the Type III Secretion System (TTSS) of Gram-negative bacteria. TTSS activity is required for the virulence of many pathogenic Gram-negative bacteria including Escherichia coli, Salmonella spp., Yersinia spp., Chlamydia spp., Vibrio spp., and Pseudomonas spp. The guadinomine (gdn) biosynthetic gene cluster has been cloned and sequenced, and includes 26 open reading frames spanning 51.2 kb. It encodes a chimeric multimodular polyketide synthase – nonribosomal peptide synthetase, along with enzymes responsible for the biosynthesis of the unusual aminomalonyl-ACP extender unit and the signature carbamoylated cyclic guanidine. Its identity was established by targeted disruption of the gene cluster, as well as by heterologous expression and analysis of key enzymes in the biosynthetic pathway. Identifying the guadinomine gene cluster provides critical insight into the biosynthesis of these scarce but potentially important natural products.
doi:10.1021/ja308622d
PMCID: PMC3483642  PMID: 23030602
15.  Engineering the acyltransferase substrate specificity of assembly line polyketide synthases 
Polyketide natural products act as a broad range of therapeutics, including antibiotics, immunosuppressants and anti-cancer agents. This therapeutic diversity stems from the structural diversity of these small molecules, many of which are produced in an assembly line manner by modular polyketide synthases. The acyltransferase (AT) domains of these megasynthases are responsible for selection and incorporation of simple monomeric building blocks, and are thus responsible for a large amount of the resulting polyketide structural diversity. The substrate specificity of these domains is often targeted for engineering in the generation of novel, therapeutically active natural products. This review outlines recent developments that can be used in the successful engineering of these domains, including AT sequence and structural data, mechanistic insights and the production of a diverse pool of extender units. It also provides an overview of previous AT domain engineering attempts, and concludes with proposed engineering approaches that take advantage of current knowledge. These approaches may lead to successful production of biologically active ‘unnatural’ natural products.
doi:10.1098/rsif.2013.0297
PMCID: PMC3971720  PMID: 23720536
polyketide; acyltransferase; antibiotics; enzyme engineering
16.  Novel chemo-sensitizing agent, ERW1227B, impairs cellular motility and enhances cell death in glioblastomas 
Journal of neuro-oncology  2010;103(2):207-219.
Glioblastomas display variable phenotypes that include increased drug-resistance associated with enhanced migratory and anti-apoptotic characteristics. These shared characteristics contribute to failure of clinical treatment regimens. Identification of novel compounds that promote cell death and impair cellular motility is a logical strategy to develop more effective clinical protocols. We recently described the ability of the small molecule, KCC009, a tissue transglutaminase (TG2) inhibitor, to sensitize glioblastoma cells to chemotherapy. In the current study, we synthesized a series of related compounds that show variable ability to promote cell death and impair motility in glioblastomas, irrespective of their ability to inhibit TG2. Each compound has a 3-bromo-4,5-dihydroisoxazole component that presumably reacts with nucleophilic cysteine thiol residues in the active sites of proteins that have an affinity to the small molecule.
Our studies focused on the effects of the compound, ERW1227B. Treatment of glioblastoma cells with ERW1227B was associated with both down-regulation of the PI-3 kinase/Akt pathway, which enhanced cell death; as well as disruption of focal adhesive complexes and intracellular actin fibers, which impaired cellular mobility. Bioassays as well as time-lapse photography of glioblastoma cells treated with ERW1227B showed cell death and rapid loss of cellular motility. Mice studies with in vivo glioblastoma models demonstrated the ability of ERW1227B to sensitize tumor cells to cell death after treatment with either chemotherapy or radiation. The above findings identify ERW1227B as a potential novel therapeutic agent in the treatment of glioblastomas.
doi:10.1007/s11060-010-0379-2
PMCID: PMC3732656  PMID: 20824305
Focal adhesive complexes; Glioblastomas; ERW1227B; Cell death; Drug-resistance; Cellular motility
17.  Tissue transgluaminase 2 expression in meningiomas 
Journal of neuro-oncology  2008;90(2):125-132.
Meningiomas are common intracranial tumors that occur in extra-axial locations, most often over the cerebral convexities or along the skull-base. Although often histologically benign these tumors frequently present challenging clinical problems. Primary clinical management of patients with symptomatic tumors is surgical resection. Radiation treatment may arrest growth or delay recurrence of these tumors, however, meningioma cells are generally resistant to apoptosis after treatment with radiation. Tumor cells are known to alter their expression of proteins that interact in the ECM to provide signals important in tumor progression. One such protein, fibronectin, is expressed in elevated levels in the ECM in a number of tumors including meningiomas. We recently reported that levels of both extracellular fibronectin and tissue transglutaminase 2 (TG2) were increased in glioblastomas. We examined the expression of fibronectin and its association TG2 in meningiomas. Both fibronectin and TG2 were strongly expressed in all meningiomas studied. TG2 activity was markedly elevated in meningiomas, and TG2 was found to co-localize with fibronectin. Treatment of meningiomas with the small molecule TG2 inhibitor, KCC009, inhibited the binding of TG2 to fibronectin and blocked disposition of linear strands of fibronectin in the ECM. KCC009 treatment promoted apoptosis and enhanced radiation sensitivity both in cultured IOMM-Lee meningioma cells and in meningioma tumor explants. These findings support a potential protective role for TG2 in meningiomas.
doi:10.1007/s11060-008-9642-1
PMCID: PMC3732188  PMID: 18587533
Brain tumor; Cell death; tissue transglutaminase inhibitor; Radiation
18.  Precursor Directed Biosynthesis of an Orthogonally Functional Erythromycin Analogue: Selectivity in the Ribosome Macrolide Binding Pocket 
Journal of the American Chemical Society  2012;134(29):12259-12265.
The macrolide antibiotic erythromycin A and its semisynthetic analogues have been among the most useful antibacterial agents for the treatment of infectious diseases. Using a recently developed chemical genetic strategy for precursor-directed biosynthesis and colony bioassay of 6-deoxyerythromycin D analogues, we identified a new class of alkynyl- and alkenyl-substituted macrolides with activity comparable to that of the natural product. Further analysis revealed a marked and unexpected dependence of antibiotic activity on the size and degree of unsaturation of the precursor. Based on these leads, we also report the precursor-directed biosynthesis of 15-propargyl erythromycin A, a novel antibiotic that is not only as potent as erythromycin A with respect to its ability to inhibit bacterial growth and cell-free ribosomal protein biosynthesis, but also harbors an orthogonal functional group that is capable of facile chemical modification.
doi:10.1021/ja304682q
PMCID: PMC3405186  PMID: 22741553
19.  Noninflammatory Gluten Peptide Analogs as Biomarkers for Celiac Sprue 
Chemistry & biology  2009;16(8):868-881.
SUMMARY
New tools are needed for managing celiac sprue, a lifelong immune disease of the small intestine. Ongoing drug trials are also prompting a search for noninvasive biomarkers of gluten-induced intestinal change. We have synthesized and characterized noninflammatory gluten peptide analogs in which key Gln residues are replaced by Asn or His. Like their proinflammatory counterparts, these biomarkers are resistant to gastrointestinal proteases, susceptible to glutenases, and permeable across enterocyte barriers. Unlike gluten peptides, however, they are not appreciably recognized by transglutaminase, HLA-DQ2, or disease-specific T cells. In vitro and animal studies show that the biomarkers can detect intestinal permeability changes as well as glutenase-catalyzed gastric detoxification of gluten. Accordingly, controlled clinical studies are warranted to evaluate the use of these peptides as probes for abnormal intestinal permeability in celiac patients and for glutenase efficacy in clinical trials and practice.
doi:10.1016/j.chembiol.2009.07.009
PMCID: PMC3721637  PMID: 19716477
20.  Role of Transglutaminase 2 in Celiac Disease Pathogenesis 
Seminars in immunopathology  2012;34(4):513-522.
doi:10.1007/s00281-012-0305-0
PMCID: PMC3712867  PMID: 22437759
21.  The Biochemical Basis for Stereochemical Control in Polyketide Biosynthesis 
Journal of the American Chemical Society  2009;131(51):18501-18511.
One of the most striking features of complex polyketides is the presence of numerous methyl- and hydroxyl-bearing stereogenic centers. In order to investigate the biochemical basis for the control of polyketide stereochemistry and to establish the timing and mechanism of the epimerization at methyl-bearing centers, a series of incubations was carried out using reconstituted components from a variety of modular polyketide synthases. In all cases the stereochemistry of the product was directly correlated with the intrinsic stereospecificity of the ketoreductase domain, independent of the particular chain elongation domains that were used, thereby establishing that methyl group epimerization, when it does occur, takes place after ketosynthase-catalyzed chain elongation. The finding that there were only minor differences in the rates of product formation observed for parallel incubations using an epimerizing ketoreductase domain and the non-epimerizing ketoreductase domain supports the proposal that the epimerization is catalyzed by the ketoreductase domain itself.
doi:10.1021/ja908296m
PMCID: PMC3699857  PMID: 19928853
22.  Resolving Multiple Protein–peptide Binding Events: Implication for HLA-DQ2 Mediated Antigen Presentation in Celiac Disease 
Chemistry, an Asian journal  2012;7(5):992-999.
Techniques that can effectively separate protein–peptide complexes from free peptides have shown great value in MHC–peptide binding studies. However, most of the available techniques are limited to measuring the binding of a single peptide to MHC molecule. As antigen presentation in vivo involves both endogenous ligands and exogenous antigens, the deconvolution of multiple binding events necessitates the implementation of a more powerful technique. Here we show that capillary electrophoresis coupled to fluorescence detection (CE-FL) can resolve multiple MHC peptide binding events owing to its superior resolution and the ability to simultaneously monitor multiple emission channels. We utilized CE-FL to investigate competition and displacement of endogenous peptides by an immunogenic gluten peptide for binding to HLA-DQ2. Remarkably, this immunogenic peptide could displace CLIP peptides from the DQ2 binding site at neutral but not acidic pH. This unusual ability of the gluten peptide supports a direct loading mechanism of antigen presentation in extracellular environment, a property that could explain the antigenicity of dietary gluten in celiac disease.
doi:10.1002/asia.201101041
PMCID: PMC3382112  PMID: 22411856
protein-peptide interactions; multiple ligands; antigen presentation; electrophoresis; fluorescence
23.  Combinatorial Biosynthesis of Polyketides – A Perspective 
Current Opinion in Chemical Biology  2012;16(1-2):117-123.
Since their discovery, polyketide synthases have been attractive targets of biosynthetic engineering to make “unnatural” natural products. Although combinatorial biosynthesis has made encouraging advances over the past two decades, the field remains in its infancy. In this enzyme-centric perspective, we discuss the scientific and technological challenges that could accelerate the adoption of combinatorial biosynthesis as a method of choice for the preparation of encoded libraries of bioactive small molecules. Borrowing a page from the protein structure prediction community, we propose a periodic challenge program to vet the most promising methods in the field, and to foster the collective development of useful tools and algorithms.
doi:10.1016/j.cbpa.2012.01.018
PMCID: PMC3328608  PMID: 22342766
24.  Structural and Biochemical Studies of the Hedamycin Type II Polyketide Ketoreductase (HedKR): Molecular Basis of Stereo- and Regio-Specificities 
Biochemistry  2011;50(34):7426-7439.
Bacterial aromatic polyketides that include many antibiotic and antitumor therapeutics are biosynthesized by the type II polyketide synthase (PKS), which consists of 5 – 10 stand-alone enzymatic domains. Hedamycin, an antitumor antibiotic polyketide, is uniquely primed with a hexadienyl group generated by a type I PKS followed by coupling to a downstream type II PKS to biosynthesize a 24-carbon polyketide, whose C9 position is reduced by hedamycin type II ketoreductase (hedKR). HedKR is homologous to the actinorhodin KR (actKR), for which we have conducted extensive structural studies previously. How hedKR can accommodate a longer polyketide substrate than the actKR, and the molecular basis of its regio- and stereospecificities, is not well understood. Here we present a detailed study of hedKR that sheds light on its specificity. Sequence alignment of KRs predicts that hedKR is less active than actKR, with significant differences in substrate/inhibitor recognition. In vitro and in vivo assays of hedKR confirmed this hypothesis. The hedKR crystal structure further provides the molecular basis for the observed differences between hedKR and actKR in the recognition of substrates and inhibitors. Instead of the 94-PGG-96 motif observed in actKR, hedKR has the 92-NGG-94 motif, leading to S-dominant stereospecificity, whose molecular basis can be explained by the crystal structure. Together with mutations, assay results, docking simulations, and the hedKR crystal structure, a model for the observed regio- and stereospecificities is presented herein that elucidates how different type II KRs recognize substrates with different chain lengths, yet precisely reduce only the C9-carbonyl group. The molecular features of hedKR important for regio- and stereospecificities can potentially be applied to biosynthesize new polyketides via protein engineering that rationally controls polyketide ketoreduction.
doi:10.1021/bi2006866
PMCID: PMC3175028  PMID: 21776967
25.  Analysis of the Ketosynthase-Chain Length Factor Heterodimer from the Fredericamycin Polyketide Synthase 
Chemistry & biology  2011;18(8):1021-1031.
SUMMARY
The pentadecaketide fredericamycin has the longest carbon chain backbone among polycyclic aromatic polyketide antibiotics whose biosynthetic genes have been sequenced. This backbone is synthesized by the bimodular fdm polyketide synthase (PKS). The initiation module is thought to synthesize a C6 intermediate that is then transferred onto the elongation PKS module, which extends it into a C30 poly-β-ketoacyl product. Here we demonstrate that the bimodular fdm PKS as well as its elongation module alone synthesize undecaketides and dodecaketides. Thus, unlike other homologues, the fdm ketosynthase – chain length factor (KS-CLF) heterodimer does not exclusively control the backbone length of its natural product. Using sequence- and structure-based approaches, 48 multiple mutants of the CLF were engineered and analyzed. Unexpectedly, the I134F mutant was unable to turn over, but could initiate and at least partially elongate the polyketide chain. This unprecedented mutant suggests that the KS-CLF heterodimer harbors an as yet uncharacterized chain termination mechanism. Together, our findings reveal fundamental mechanistic differences between the fdm PKS and its well-studied homologues.
doi:10.1016/j.chembiol.2011.07.015
PMCID: PMC3172819  PMID: 21867917

Results 1-25 (73)