ABSTRACT
Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and accounts for â¼84% of all lung cancer cases. NSCLC remains one of the leading causes of cancer-associated death, with a 5-year survival rate less than 25%. This type of cancer begins with healthy cells that change and start growing out of control, leading to the formation of lesions or tumors. Understanding the dynamics of how the tumor microenvironment promotes cancer initiation and progression that leads to cancer metastasis is crucial to help identify new molecular therapies. 3D primary cell tumor models have received renewed recognition due to their ability to better mimic the complexity of in vivo tumors and as a potential bridge between traditional 2D culture and in vivo studies. Vast improvements in 3D cell culture technologies make them much more cost effective and efficient largely because of the use of a cell-repellent surfaces and a novel angle plate adaptor technology. To exploit this technology, we accessed the Natural Products Library (NPL) at UF Scripps, which consists of crude extracts, partially purified fractions, and pure natural products (NPs). NPs generally are not very well represented in most drug discovery libraries and thus provide new insights to discover leads that could potentially emerge as novel molecular therapies. Herein we describe how we combined these technologies for 3D screening in 1536 well format using a panel of ten NSCLC cells lines (5 wild type and 5 mutant) against â¼1280 selected members of the NPL. After further evaluation, the selected active hits were prioritized to be screened against all 10 NSCLC cell lines as concentration response curves to determine the efficacy and selectivity of the compounds between wild type and mutant 3D cell models. Here, we demonstrate the methods needed for automated 3D screening using microbial NPs, exemplified by crude extracts, partially purified fractions, and pure NPs, that may lead to future use targeting human cancer.
Subject(s)
Antineoplastic Agents , Biological Products , Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Biological Products/pharmacology , Antineoplastic Agents/pharmacology , Drug Screening Assays, Antitumor , Spheroids, Cellular , Early Detection of Cancer , Tumor MicroenvironmentABSTRACT
Heme is an essential cofactor for many human proteins as well as the primary transporter of oxygen in blood. Recent studies have also established heme as a signaling molecule, imparting its effects through binding with protein partners rather than through reactivity of its metal center. However, the comprehensive annotation of such heme-binding proteins in the human proteome remains incomplete. Here, we describe a strategy which utilizes a heme-based photoaffinity probe integrated with quantitative proteomics to map heme-protein interactions across the proteome. In these studies, we identified 350+ unique heme-protein interactions, the vast majority of which were heretofore unknown and consist of targets from diverse functional classes, including transporters, receptors, enzymes, transcription factors, and chaperones. Among these proteins is the immune-related interleukin receptor-associated kinase 1 (IRAK1), where we provide preliminary evidence that heme agonizes its catalytic activity. Our findings should improve the current understanding of heme's regulation as well as its signaling functions and facilitate new insights of its roles in human disease.
Subject(s)
Heme , Proteomics , Carrier Proteins/metabolism , Heme/chemistry , Humans , Protein Binding , Proteome/metabolism , Signal Transduction , Transcription Factors/metabolismABSTRACT
The microbiome has a fundamental impact on the human host's physiology through the production of highly reactive compounds that can lead to disease development. One class of such compounds are carbonyl-containing metabolites, which are involved in diverse biochemical processes. Mass spectrometry is the method of choice for analysis of metabolites but carbonyls are analytically challenging. Herein, we have developed a new chemical biology tool using chemoselective modification to overcome analytical limitations. Two isotopic probes allow for the simultaneous and semi-quantitative analysis at the femtomole level as well as qualitative analysis at attomole quantities that allows for detection of more than 200 metabolites in human fecal, urine and plasma samples. This comprehensive mass spectrometric analysis enhances the scope of metabolomics-driven biomarker discovery. We anticipate that our chemical biology tool will be of general use in metabolomics analysis to obtain a better understanding of microbial interactions with the human host and disease development.
Subject(s)
Acetaldehyde/analysis , Acetone/analysis , Aldehydes/analysis , Butanones/analysis , Dihydroxyacetone/analysis , Metabolomics/methods , Acetaldehyde/blood , Acetaldehyde/chemistry , Acetaldehyde/urine , Acetamides/chemistry , Acetone/blood , Acetone/chemistry , Acetone/urine , Aldehydes/blood , Aldehydes/chemistry , Aldehydes/urine , Butanones/blood , Butanones/chemistry , Butanones/urine , Carbon/chemistry , Carbon Isotopes/chemistry , Dihydroxyacetone/blood , Dihydroxyacetone/chemistry , Dihydroxyacetone/urine , Feces/chemistry , Gastrointestinal Microbiome , Humans , Indicators and Reagents/chemistry , Limit of Detection , Urine/chemistryABSTRACT
BACKGROUND: Genes in the Ras pathway have somatic mutations in at least 60 % of colorectal cancers. Despite activating the same pathway, the BRAF V600E mutation and the prevalent mutations in codon 12 and 13 of KRAS have all been linked to different clinical outcomes, but the molecular mechanisms behind these differences largely remain to be clarified. METHODS: To characterize the similarities and differences between common activating KRAS mutations and between KRAS and BRAF mutations, we used genome editing to engineer KRAS G12C/D/V and G13D mutations in colorectal cancer cells that had their mutant BRAF V600E allele removed and subjected them to transcriptome sequencing, global proteomics and metabolomics analyses. RESULTS: By intersecting differentially expressed genes, proteins and metabolites, we uncovered (i) two-fold more regulated genes and proteins when comparing KRAS to BRAF mutant cells to those lacking Ras pathway mutation, (ii) five differentially expressed proteins in KRAS mutants compared to cells lacking Ras pathway mutation (IFI16, S100A10, CD44, GLRX and AHNAK2) and 6 (CRABP2, FLNA, NXN, LCP1, S100A10 and S100A2) compared to BRAF mutant cells, (iii) 19 proteins expressed differentially in a KRAS mutation specific manner versus BRAF V600E cells, (iv) regulation of the Integrin Linked Kinase pathway by KRAS but not BRAF mutation, (v) regulation of amino acid metabolism, particularly of the tyrosine, histidine, arginine and proline pathways, the urea cycle and purine metabolism by Ras pathway mutations, (vi) increased free carnitine in KRAS and BRAF mutant RKO cells. CONCLUSIONS: This comprehensive integrative -omics analysis confirms known and adds novel genes, proteins and metabolic pathways regulated by mutant KRAS and BRAF signaling in colorectal cancer. The results from the new model systems presented here can inform future development of diagnostic and therapeutic approaches targeting tumors with KRAS and BRAF mutations.
Subject(s)
Colorectal Neoplasms/genetics , Mutation , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Humans , Phenotype , Proto-Oncogene Proteins B-raf/metabolism , Proto-Oncogene Proteins p21(ras)/metabolismABSTRACT
The use of photo-affinity reagents for the mapping of noncovalent small molecule-protein interactions has become widespread. Recently, several 'fully-functionalized' (FF) chemical tags have been developed wherein a photoactivatable capture group, an enrichment handle, and a functional group for synthetic conjugation to a molecule of interest are integrated into a single modular tag. Diazirine-based FF tags in particular are increasingly employed in chemical proteomic investigations; however, despite routine usage, their relative utility has not been established. Here, we systematically evaluate several diazirine-containing FF tags, including a terminal diazirine analog developed herein, for chemical proteomic investigations. Specifically, we compared the general reactivity of five diazirine tags and assessed their impact on the profiles of various small molecules, including fragments and known inhibitors revealing that such tags can have profound effects on the proteomic profiles of chemical probes. Our findings should be informative for chemical probe design, photo-affinity reagent development, and chemical proteomic investigations.
ABSTRACT
Aberrant Ras signaling is linked to a wide spectrum of hyperproliferative diseases, and components of the signaling pathway, including Ras, have been the subject of intense and ongoing drug discovery efforts. The cellular activity of Ras is modulated by its association with the guanine nucleotide exchange factor Son of sevenless (Sos), and the high-resolution crystal structure of the Ras-Sos complex provides a basis for the rational design of orthosteric Ras ligands. We constructed a synthetic Sos protein mimic that engages the wild-type and oncogenic forms of nucleotide-bound Ras and modulates downstream kinase signaling. The Sos mimic was designed to capture the conformation of the Sos helix-loop-helix motif that makes critical contacts with Ras in its switch region. Chemoproteomic studies illustrate that the proteomimetic engages Ras and other cellular GTPases. The synthetic proteomimetic resists proteolytic degradation and enters cells through macropinocytosis. As such, it is selectively toxic to cancer cells with up-regulated macropinocytosis, including those that feature oncogenic Ras mutations.
Subject(s)
Multiprotein Complexes/ultrastructure , Protein Conformation , Son of Sevenless Protein, Drosophila/ultrastructure , ras Proteins/ultrastructure , Animals , Biomimetics , Crystallography, X-Ray , Drug Discovery , GTP Phosphohydrolases/chemistry , GTP Phosphohydrolases/ultrastructure , HCT116 Cells , Helix-Loop-Helix Motifs/genetics , Humans , Models, Molecular , Multiprotein Complexes/chemistry , Multiprotein Complexes/genetics , Proteome/genetics , Signal Transduction/genetics , Son of Sevenless Protein, Drosophila/chemistry , Son of Sevenless Protein, Drosophila/genetics , ras Proteins/chemistry , ras Proteins/geneticsABSTRACT
The ID of disease-modifying, chemically accessible targets remains a central priority of modern therapeutic discovery. The phenotypic screening of small-molecule libraries not only represents an attractive approach to identify compounds that may serve as drug leads but also serves as an opportunity to uncover compounds with novel mechanisms of action (MoAs). However, a major bottleneck of phenotypic screens continues to be the ID of pharmacologically relevant target(s) for compounds of interest. The field of chemoproteomics aims to map proteome-wide small-molecule interactions in complex, native systems, and has proved a key technology to unravel the protein targets of pharmacological modulators. In this review, we discuss the application of modern chemoproteomic methods to identify protein targets of phenotypic screening hits and investigate MoAs, with a specific focus on the development of chemoproteomic-enabled compound libraries to streamline target discovery.
Subject(s)
Pharmaceutical Preparations/chemistry , Proteomics , Small Molecule Libraries/chemistry , Drug Discovery , Drug Evaluation, Preclinical , Humans , PhenotypeABSTRACT
A new, dynamic diastereomeric crystallization method has been developed, in which the mother liquors are continuously separated, racemized over a fixed-bed catalyst, and recirculated to the crystallizer in a resolution-racemization-recycle (R3) process. Separating the racemization from crystallization overcomes problems of using catalysts in situ, that suffer conflicting sets of conditions, inhibition, and separation. Continuous racemization has been achieved through the covalent attachment of [IrCp*I2]2 SCRAM catalyst to Wang resin solid support to give a fixed-bed catalyst. One tertiary and a variety of secondary optically enriched amines have been racemized efficiently, with residence times compatible with the crystallization (2.25-30 min). The catalyst demonstrates lower turnover (TOF) than the homogeneous analogue but with reuse shows a long lifetime (e.g., 40 recycles, 190 h) giving acceptable turnover number (TON) (up to 4907). The slow release of methylamine during racemization of N-methyl amines was found to inactivate the catalyst, which could be partially reactivated using hydroiodic acid. Dynamic crystallization is achieved in the R3 process through the continual removal of the more soluble diastereomer and supply of the less soluble one. The solubility of the diastereomers was determined, and the difference correlates to the rate of resolution but is also affected by the rates of racemization, crystal growth, and dissolution. A variety of cyclic and acyclic amine salts were resolved using mandelic acid (MA) and ditoluoyl tartaric acid (DTTA) with higher resolvability (S = yield × d.e.) than the simple diastereomeric crystallization alone. Comparing resolvabilities, resolutions were 1.6-44 times more effective with the R3 process than batch, though one case was worse. Further investigation of this revealed an unusual thermodynamic switching behavior: rac-N-methylphenethylamine was initially resolved as an (S,S)-bis-alkylammonium tartrate crystal but over time became the equivalent (R,S) salt. Thermal, mixing, concentration, stoichiometry, and seeding conditions were all found to affect the onset of the switching behavior which is only associated with difunctional resolving reagents.
ABSTRACT
N-Acetyltransferases play critical roles in the deactivation and clearance of xenobiotics, including clinical drugs. NAT2 has been classified as an arylamine N-acetyltransferase that mainly converts aromatic amines, hydroxylamines, and hydrazines. Herein, we demonstrate that the human arylamine N-acetyltransferase NAT2 also acetylates aliphatic endogenous amines. Metabolomic analysis and chemical synthesis revealed increased intracellular concentrations of mono- and diacetylated spermidine in human cell lines expressing the rapid compared to the slow acetylator NAT2 phenotype. The regioselective N8 -acetylation of monoacetylated spermidine by NAT2 answers the long-standing question of the source of diacetylspermidine. We also identified selective acetylation of structurally diverse alkylamine-containing drugs by NAT2, which may contribute to variations in patient responses. The results demonstrate a previously unknown functionality and potential regulatory role for NAT2, and we suggest that this enzyme should be considered for re-classification.
Subject(s)
Amines/metabolism , Arylamine N-Acetyltransferase/metabolism , Acetylation , Arylamine N-Acetyltransferase/genetics , Cell Line, Tumor , Chromatography, Liquid/methods , Genotype , Humans , Kinetics , Mass Spectrometry/methodsABSTRACT
Metabolites with ketone or aldehyde functionalities comprise a large proportion of the human metabolome, most notably in the form of sugars. However, these reactive molecules are also generated through oxidative stress or gut microbiota metabolism and have been linked to disease development. The discovery and structural validation of this class of metabolites over the large concentration range found in human samples is crucial to identify their links to pathogenesis. Herein, we have utilized an advanced chemoselective probe methodology alongside bioinformatic analysis to identify carbonyl-metabolites in urine and fecal samples. In total, 99 metabolites were identified in urine samples and the chemical structure for 40 metabolites were unambiguously validated using a co-injection procedure. We also describe the preparation of a metabolite-conjugate library of 94 compounds utilized to efficiently validate these ketones and aldehydes. This method was used to validate 33 metabolites in a pooled fecal sample extract to demonstrate the potential for rapid and efficient metabolite detection over a wide metabolite concentration range. This analysis revealed the presence of six metabolites that have not previously been detected in either sample type. The constructed library can be utilized for straightforward, large-scale, and expeditious analysis of carbonyls in any sample type.
Subject(s)
Aldehydes/urine , Feces/chemistry , Ketones/urine , Aldehydes/chemistry , Computational Biology , Humans , Ketones/chemistry , Mass Spectrometry/methods , Metabolome , Metabolomics/methods , Pancreatic Neoplasms/urine , Small Molecule Libraries/analysis , Small Molecule Libraries/chemistryABSTRACT
New strategies are required for the discovery of unknown bioactive molecules produced by gut microbiota in the human host. Herein, we utilize a chemoselective probe immobilized to magnetic beads for analysis of carbonyls in human fecal samples. We identified 112 metabolites due to femtomole analysis and an increased mass spectrometric sensitivity by up to six orders of magnitude.
Subject(s)
Aldehydes/analysis , Feces/chemistry , Gastrointestinal Microbiome/physiology , Ketones/analysis , Molecular Probes/analysis , Molecular Probes/chemistry , Aldehydes/metabolism , Feces/microbiology , Female , Humans , Ketones/metabolism , Male , Molecular Probes/chemical synthesis , Molecular Structure , Particle Size , Surface PropertiesABSTRACT
Sialic acids are a family of acidic monosaccharides often found on the termini of cell surface proteins or lipid glycoconjugates of higher animals. Herein we describe the enzymatic synthesis of the two isotopically labeled sialic acid derivatives d3-X-Gal-α-2,3-Neu5Ac and d3-X-Gal-α-2,3-Neu5Gc. Using deuterium oxide as the reaction solvent, deuterium atoms could be successfully introduced during the enzymatic epimerization and aldol addition reactions when the sialosides were generated. NMR and mass spectrometric analyses confirmed that the resulting sialosides were indeed tri-deuterated. These compounds may be of interest as internal standards in liquid chromatography/mass spectrometric assays for biochemical or clinical studies of sialic acids. This was further exemplified by the use of this tri-deuterated sialosides as internal standards for the quantification of sialic acids in meat and egg samples.
Subject(s)
Deuterium Oxide/chemistry , Enzymes/metabolism , Sialic Acids/biosynthesis , Magnetic Resonance Spectroscopy , Mass Spectrometry , Oxo-Acid-Lyases/metabolism , Racemases and Epimerases/metabolism , Sialic Acids/chemistryABSTRACT
Sulfatases hydrolyze sulfated metabolites to their corresponding alcohols and are present in all domains of life. These enzymes have found major application in metabolic investigation of drugs, doping control analysis and recently in metabolomics. Interest in sulfatases has increased due to a link between metabolic processes involving sulfated metabolites and pathophysiological conditions in humans. Herein, we present the first comprehensive substrate specificity and kinetic analysis of the most commonly used arylsulfatase extracted from the snail Helix pomatia. In the past, this enzyme has been used in the form of a crude mixture of enzymes, however, recently we have purified this sulfatase for a new application in metabolomics-driven discovery of sulfated metabolites. To evaluate the substrate specificity of this promiscuous sulfatase, we have synthesized a series of new sulfated metabolites of diverse structure and employed a mass spectrometric assay for kinetic substrate hydrolysis evaluation. Our analysis of the purified enzyme revealed that the sulfatase has a strong preference for metabolites with a bi- or tricyclic aromatic scaffold and to a lesser extent for monocyclic aromatic phenols. This metabolite library and mass spectrometric method can be applied for the characterization of other sulfatases from humans and gut microbiota to investigate their involvement in disease development.
Subject(s)
Arylsulfatases/metabolism , Helix, Snails/enzymology , Animals , Helix, Snails/metabolism , Hydrolysis , Kinetics , Mass Spectrometry , Substrate SpecificityABSTRACT
While metabolites derived from gut microbiota metabolism have been linked to disease development in the human host, the chemical tools required for their detailed analysis and the discovery of biomarkers are limited. A unique and multifunctional chemical probe for mass spectrometric analysis, which contains p-nitrocinnamyloxycarbonyl as a new bioorthogonal cleavage site has been designed and synthesized. Coupled to magnetic beads, this chemical probe allows for straightforward extraction of metabolites from human samples and release under mild conditions. This isolation from the sample matrix results in significantly reduced ion suppression, an increased mass spectrometric sensitivity, and facilitates the detection of metabolites in femtomole quantities. The chemoselective probe was applied to the analysis of human fecal samples, resulting in the discovery of four metabolites previously unreported in this sample type and confirmation of the presence of medically relevant gut microbiota-derived metabolites.
Subject(s)
Gastrointestinal Microbiome , Molecular Probes/chemistry , Chromatography, Liquid/methods , Humans , Magnetics , Mass Spectrometry/methodsABSTRACT
Gut microbiota significantly impact human physiology through metabolic interaction. Selective investigation of the co-metabolism of bacteria and their human host is a challenging task and methods for their analysis are limited. One class of metabolites associated with this co-metabolism are O-sulfated compounds. Herein, we describe the development of a new enzymatic assay for the selective mass spectrometric investigation of this phase II modification class. Analysis of human urine and fecal samples resulted in the detection of 206 sulfated metabolites, which is three times more than reported in the Human Metabolome Database. We confirmed the chemical structure of 36 sulfated metabolites including unknown and commonly reported microbiota-derived sulfated metabolites using synthesized internal standards and mass spectrometric fragmentation experiments. Our findings demonstrate that enzymatic sample pre-treatment combined with state-of-the-art metabolomics analysis represents a new and efficient strategy for the discovery of unknown microbiota-derived metabolites in human samples. Our described approach can be adapted for the targeted investigation of other metabolite classes as well as the discovery of biomarkers for diseases affected by microbiota.
ABSTRACT
Within human biology, combinations of regioisomeric motifs of α2,6- or α2,3-sialic acids linked to galactose are frequently observed attached to glycoconjugates. These include glycoproteins and glycolipids, with each linkage carrying distinct biological information and function. Microbial linkage-specific sialidases have become important tools for studying the role of these sialosides in complex biological settings, as well as being used as biocatalysts for glycoengineering. However, currently, there is no α2,6-specific sialidase available. This gap has been addressed herein by exploiting the ability of a Photobacterium sp. α2,6-sialyltransferase to catalyze trans-sialidation reversibly and in a highly linkage-specific manner, acting as a pseudosialidase in the presence of cytidine monophosphate. Selective, near quantitative removal of α2,6-linked sialic acids was achieved from a wide range of sialosides including small molecules conjugates, simple glycan, glycopeptide and finally complex glycoprotein including both linkages.
Subject(s)
Neuraminidase/metabolism , Sialic Acids/metabolism , Galactose/chemistry , Galactose/metabolism , Glycoconjugates/chemistry , Glycoconjugates/metabolism , Glycolipids/chemistry , Glycolipids/metabolism , Glycoproteins/chemistry , Glycoproteins/metabolism , Kinetics , Salmonella typhimurium/enzymology , Sialic Acids/chemistryABSTRACT
Galactokinases are a class of enzymes which belong to the GHMP (galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase) superfamily and catalyse the phosphorylation of galactose in the first step of the Leloir pathway. Here we report the discovery of three enzymes from Shewanella woodyi which have been classified as galactokinases based on sequence similarity. However, each of these enzymes show little to no significant activity towards galactose and instead exhibit a strong preference for glucose. Furthermore, in contrast to the usual galactose-1-phosphate product of the galactokinase-catalysed reaction, these enzymes produce glucose-6-phosphate. This radical change in enzyme functionality is postulated to be linked to the mutation of a glycine residue which is conserved in all other sequenced galactokinases.
Subject(s)
Galactokinase/metabolism , Glucose/metabolism , Shewanella/enzymology , Glucose-6-Phosphate/metabolism , Hexokinase/metabolism , Phosphorylation , Substrate SpecificityABSTRACT
CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.
Subject(s)
Chromatography, High Pressure Liquid , Liver/chemistry , Milk/chemistry , Mixed Function Oxygenases/genetics , Sialic Acids/analysis , Animals , CRISPR-Cas Systems/genetics , Liver/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Mixed Function Oxygenases/deficiency , Sialic Acids/isolation & purification , Video RecordingABSTRACT
BACKGROUND: The biocatalytic oxidation of UDP-glucose in the presence of NAD+ is catalyzed by UDP-glucose dehydrogenases. OBJECTIVES: The main objective of this study was the characterization of a UDP-glucose dehydrogenase (AmUGD) from Akkermansia muciniphila, a bacterium originally isolated from human faeces in an anaerobic medium containing gastric mucin as the sole carbon source. METHODS: The biochemical analysis of AmUGD was performed using a plate reader-based assay measuring the reaction by-product NADH. Furthermore, HPLC- and MALDI-ToF-MS- based methods were used for the enzyme characterization. RESULTS: The recombinant form of the protein was expressed in E. coli and the purified enzyme exhibited optimum levels of activity at 37°C and pH 9.0. While the enzyme is active in the absence of metal ions, the presence of Zn2+ ions results in markedly enhanced levels of catalysis. CONCLUSION: This study describes the first characterization of a nucleotide-processing enzyme from A. muciniphila. The ease of expression and purification of this enzyme make it ideal for biotechnological applications such as the enzymatic synthesis of nucleotide sugars, which may in turn be used for the synthesis of complex carbohydrates or glycoconjugates.
Subject(s)
Bacterial Proteins/metabolism , NAD/metabolism , Uridine Diphosphate Glucose Dehydrogenase/metabolism , Uridine Diphosphate Glucose/metabolism , Verrucomicrobia/chemistry , Bacterial Proteins/genetics , Cations, Divalent , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Hot Temperature , Hydrogen-Ion Concentration , Kinetics , NAD/chemistry , Plasmids/chemistry , Plasmids/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate Specificity , Uridine Diphosphate Glucose/chemistry , Uridine Diphosphate Glucose Dehydrogenase/genetics , Verrucomicrobia/enzymology , Zinc/chemistry , Zinc/metabolismABSTRACT
BACKGROUND: The biosynthesis of NDP-glucoses is based on the nucleotide transfer from NTP donor substrates to glucose-1-phosphates catalyzed by glucose-1-phosphate nucleotidyltransferases. OBJECTIVES: The cloning and biochemical characterization of a glucose-1-phosphate nucleotidyltransferase (TiGPNT) from the deep sea bacterium Thermodesulfatator indicus. METHODS: The biochemical parameters of recombinant TiGPNT were determined using a plate reader-based coupled enzymatic assay, in which the reaction product UDP-glucose is oxidized in the presence of NAD+ forming UDP-Glucuronic acid and NADH. The substrate promiscuity of the enzyme was determined using thin-layer chromatography and MALDI-ToF mass spectrometry. RESULTS: TiGPNT was recombinantly expressed under the control of the T7 promoter in Escherichia coli and could be successfully enriched by heat treatment at 80°C for 30 min. The obtained enzyme worked best at pH 7.5 and the optimum reaction temperature was determined to be 50°C. Interestingly, TiGPNT could fully retain its activity even after extended incubation periods at temperatures of up to 80°C. The enzyme was strongly inhibited in the presence of Cu2+ and Fe2+ ions and EDTA. Among the tested glycosyl donor substrates, TiGPNT showed strict specificity towards glucose-1-phosphate. At the same time, TiGPNT was highly promiscuous towards all tested nucleotide donor substrates. CONCLUSION: TiGPNT shows comparable biochemical features in regards to pH optima, temperature optima and the substrate specificity to characterized glucose-1-phosphate nucleotidyltransferase from other species. The enzyme was capable of utilizing glucose-1-phosphate and all tested nucleoside triphosphate donors as substrates. The high activity of the enzyme and the simple purification protocol make TiGPNT an interesting new biocatalyst for the synthesis of glucose-diphospho nucleosides.