FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2284865753> ?p ?o ?g. }

• W2284865753 endingPage "28976" @default.
• W2284865753 startingPage "28963" @default.
• W2284865753 abstract "Innovations in the discovery of the functions of uncharacterized proteins/enzymes have become increasingly important as advances in sequencing technology flood protein databases with an exponentially growing number of open reading frames. This study documents one such innovation developed by the Enzyme Function Initiative (EFI; U54GM093342), the use of solute-binding proteins for transport systems to identify novel metabolic pathways. In a previous study, this strategy was applied to the tripartite ATP-independent periplasmic transporters. Here, we apply this strategy to the ATP-binding cassette transporters and report the discovery of novel catabolic pathways for d-altritol and galactitol in Agrobacterium tumefaciens C58. These efforts resulted in the description of three novel enzymatic reactions as follows: 1) oxidation of d-altritol to d-tagatose via a dehydrogenase in Pfam family PF00107, a previously unknown reaction; 2) phosphorylation of d-tagatose to d-tagatose 6-phosphate via a kinase in Pfam family PF00294, a previously orphan EC number; and 3) epimerization of d-tagatose 6-phosphate C-4 to d-fructose 6-phosphate via a member of Pfam family PF08013, another previously unknown reaction. The epimerization reaction catalyzed by a member of PF08013 is especially noteworthy, because the functions of members of PF08013 have been unknown. These discoveries were assisted by the following two synergistic bioinformatics web tools made available by the Enzyme Function Initiative: the EFI-Enzyme Similarity Tool and the EFI-Genome Neighborhood Tool. Innovations in the discovery of the functions of uncharacterized proteins/enzymes have become increasingly important as advances in sequencing technology flood protein databases with an exponentially growing number of open reading frames. This study documents one such innovation developed by the Enzyme Function Initiative (EFI; U54GM093342), the use of solute-binding proteins for transport systems to identify novel metabolic pathways. In a previous study, this strategy was applied to the tripartite ATP-independent periplasmic transporters. Here, we apply this strategy to the ATP-binding cassette transporters and report the discovery of novel catabolic pathways for d-altritol and galactitol in Agrobacterium tumefaciens C58. These efforts resulted in the description of three novel enzymatic reactions as follows: 1) oxidation of d-altritol to d-tagatose via a dehydrogenase in Pfam family PF00107, a previously unknown reaction; 2) phosphorylation of d-tagatose to d-tagatose 6-phosphate via a kinase in Pfam family PF00294, a previously orphan EC number; and 3) epimerization of d-tagatose 6-phosphate C-4 to d-fructose 6-phosphate via a member of Pfam family PF08013, another previously unknown reaction. The epimerization reaction catalyzed by a member of PF08013 is especially noteworthy, because the functions of members of PF08013 have been unknown. These discoveries were assisted by the following two synergistic bioinformatics web tools made available by the Enzyme Function Initiative: the EFI-Enzyme Similarity Tool and the EFI-Genome Neighborhood Tool. The rate at which protein sequences are added to the UniProt Knowledgebase (UniProtKB) 2The abbreviations used are: UniProtKBUniProt KnowledgebaseABCATP-binding cassette transporterAltSBPd- altritol binding ABC SBPAtuFKputative A. tumefaciens C58 fructokinaseAtuSorbDputative A. tumefaciens C58 sorbitol dehydrogenaseAtuTag6PKputative A. tumefaciens C58 tagatose-6-phosphate kinaseAtuZnDputative A. tumefaciens C58 zinc-binding dehydrogenaseDHAdihydroxyacetoneDHAPdihydroxyacetone phosphateECEnzyme CommissionEFIEnzyme Function InitiativeEFI-ESTEnzyme Function Initiative-Enzyme Similarity ToolEFI-GNTEnzyme Function Initiative-Genome Neighborhood Networks ToolGNNgenome neighborhood networkPfamprotein familyPPPpentose phosphate PathwaySBPsolute-binding proteinSSNsequence similarity networkqRTquantitative RTDSFdifferential scanning fluorimetryFORforwardREVreversePEPP-enolpyruvate. has created a daunting gap between the number of manually curated protein annotations (SwissProt database, ∼550,000 in Release 2015_06) and the total number of proteins (SwissProt and TrEMBL databases, ∼48 million in release 2015_06). Approximately 50% of the automated annotations made by sequence similarity (TrEMBL database) are thought to be misleading or erroneous (1.Devos D. Valencia A. Intrinsic errors in genome annotation.Trends Genet. 2001; 17: 429-431Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar2.Jones C.E. Brown A.L. Baumann U. Estimating the annotation error rate of the curated GO database sequence annotations.BMC Bioinformatics. 2007; 8: 170Crossref PubMed Scopus (108) Google Scholar, 3.Schnoes A.M. Brown S.D. Dodevski I. Babbitt P.C. Annotation error in public databases: misannotation of molecular function in enzyme superfamilies.PLoS Comput. Biol. 2009; 5: e1000605Crossref PubMed Scopus (469) Google Scholar4.Gilks W.R. Audit B. De Angelis D. Tsoka S. Ouzounis C.A. Modeling the percolation of annotation errors in a database of protein sequences.Bioinformatics. 2002; 18: 1641-1649Crossref PubMed Scopus (129) Google Scholar). The presence of a large fraction of entries in UniProtKB that are functionally ambiguous or incorrect undermines efforts to 1) determine drug targets in pathogens affecting human health or the agricultural industry, 2) develop in vitro enzymatic pathways for biomanufacturing, 3) further investigate the relationship between human health and the gut microbiota, and 4) understand critical environmental issues such as global nutrient cycles. The Enzyme Function Initiative (EFI) (5.Gerlt J.A. Allen K.N. Almo S.C. Armstrong R.N. Babbitt P.C. Cronan J.E. Dunaway-Mariano D. Imker H.J. Jacobson M.P. Minor W. Poulter C.D. Raushel F.M. Sali A. Shoichet B.K. Sweedler J.V. The Enzyme Function Initiative.Biochemistry. 2011; 50: 9950-9962Crossref PubMed Scopus (137) Google Scholar) worked to address these challenges by the development of robust strategies for accurate and efficient assignment of functions to unknown or uncharacterized proteins discovered in genome projects via bioinformatics, computational modeling, in vitro experimentation, and in vivo verification (6.Erb T.J. Evans B.S. Cho K. Warlick B.P. Sriram J. Wood B.M. Imker H.J. Sweedler J.V. Tabita F.R. Gerlt J.A. A RubisCO-like protein links SAM metabolism with isoprenoid biosynthesis.Nat. Chem. Biol. 2012; 8: 926-932Crossref PubMed Scopus (61) Google Scholar7.Zhao S. Kumar R. Sakai A. Vetting M.W. Wood B.M. Brown S. Bonanno J.B. Hillerich B.S. Seidel R.D. Babbitt P.C. Almo S.C. Sweedler J.V. Gerlt J.A. Cronan J.E. Jacobson M.P. Discovery of new enzymes and metabolic pathways by structure and genome context guided analyses.Nature. 2013; 502: 698-702Crossref PubMed Scopus (100) Google Scholar, 8.Bouvier J.T. Groninger-Poe F.P. Vetting M. Almo S.C. Gerlt J.A. Galactaro δ-lactone isomerase: lactone isomerization by a member of the amidohydrolase superfamily.Biochemistry. 2014; 53: 614-616Crossref PubMed Scopus (14) Google Scholar, 9.Wichelecki D.J. Balthazor B.M. Chau A.C. Vetting M.W. Fedorov A.A. Fedorov E.V. Lukk T. Patskovsky Y.V. Stead M.B. Hillerich B.S. Seidel R.D. Almo S.C. Gerlt J.A. Discovery of function in the enolase superfamily: d-mannonate and d-gluconate dehydratases in the d-mannonate dehydratase subgroup.Biochemistry. 2014; 53: 2722-2731Crossref PubMed Scopus (23) Google Scholar, 10.Zhao S. Sakai A. Zhang X. Vetting M.W. Kumar R. Hillerich B. San Francisco B. Solbiati J. Steves A. Brown S. Akiva E. Barber A. Seidel R. Babbitt P.C. Almo S.C. Gerlt J.A. Jacobson M.P. Prediction and characterization of enzymatic activities guided by sequence similarity and genome neighborhood networks.eLife. 2014; 3: e03275Crossref Google Scholar11.Gerlt J.A. Bouvier J.T. Davidson D.B. Imker H.J. Sadkhin B. Slater D.R. Whalen K.L. Enzyme function initiative-enzyme similarity tool (EFI-EST): a web tool for generating protein sequence similarity networks.Biochim. Biophys. Acta. 2015; 1854: 1019-1037Crossref PubMed Scopus (468) Google Scholar). UniProt Knowledgebase ATP-binding cassette transporter d- altritol binding ABC SBP putative A. tumefaciens C58 fructokinase putative A. tumefaciens C58 sorbitol dehydrogenase putative A. tumefaciens C58 tagatose-6-phosphate kinase putative A. tumefaciens C58 zinc-binding dehydrogenase dihydroxyacetone dihydroxyacetone phosphate Enzyme Commission Enzyme Function Initiative Enzyme Function Initiative-Enzyme Similarity Tool Enzyme Function Initiative-Genome Neighborhood Networks Tool genome neighborhood network protein family pentose phosphate Pathway solute-binding protein sequence similarity network quantitative RT differential scanning fluorimetry forward reverse P-enolpyruvate. The most recent contribution from the EFI was the demonstration of the utility of solute-binding proteins (SBPs) for tripartite ATP-independent periplasmic transporters to guide functional discovery (12.Vetting M.W. Al-Obaidi N. Zhao S. San Francisco B. Kim J. Wichelecki D.J. Bouvier J.T. Solbiati J.O. Vu H. Zhang X. Rodionov D.A. Love J.D. Hillerich B.S. Seidel R.D. Quinn R.J. et al.Experimental strategies for functional annotation and metabolism discovery: targeted screening of solute-binding proteins and unbiased panning of metabolomes.Biochemistry. 2015; 54: 909-931Crossref PubMed Scopus (67) Google Scholar). The premise was that the small molecule ligands of SBPs represent the chemical “starting points” of metabolic pathways (i.e. the substrate for the first enzyme in a metabolic pathway). This information imposes significant constraints on the regions of chemical space and types of enzymatic transformations that must be considered for pathway deconvolution. In combination with annotated functions for the proteins encoded by the genes proximal to that encoding the SBP, these insights allow the facilitated prediction of new metabolic pathways and the discovery of novel enzymatic functions. In this study, we apply this strategy to an alternative class of transporters, the ATP-binding cassette (ABC) transporters. Most ABC transporters are composed of three components as follows: a transmembrane domain, a nucleotide binding domain, and an SBP (13.Jones P.M. George A.M. The ABC transporter structure and mechanism: perspectives on recent research.Cell. Mol. Life Sci. 2004; 61: 682-699Crossref PubMed Scopus (454) Google Scholar, 14.Davidson A.L. Dassa E. Orelle C. Chen J. Structure, function, and evolution of bacterial ATP-binding cassette systems.Microbiol. Mol. Biol. Rev. 2008; 72: 317-364Crossref PubMed Scopus (959) Google Scholar). Hydrolysis of ATP provides the energy needed to transport the solute across the cell membrane (15.Rees D.C. Johnson E. Lewinson O. ABC transporters: the power to change.Nat. Rev. Mol. Cell Biol. 2009; 10: 218-227Crossref PubMed Scopus (880) Google Scholar). ABC transporters are widely spread throughout all phyla of life. Applying the SBP-based strategy for pathway discovery to the ABC transporters enabled the discovery of a novel polyol metabolic pathway for catabolism of d-altritol (also known as d-talitol). For this pathway, the SBP-proximal genome neighborhood was found to contain three genes encoding enzymes that convert d-altritol to the central metabolite fructose 6-phosphate. Two enzymes perform novel reactions, and the third enzyme is responsible for a previously orphan Enzyme Commission (EC) number. Notably, one of these is a member of a family of ∼6,000 previously unannotated proteins. Together, these results demonstrate the effectiveness of this strategy for facilitating functional discovery and correcting errors in the protein databases. Rare sugars, such as d-altritol, have attracted attention because of their importance to the food, pharmaceutical, artificial sweetener, and nutrition industries (16.Zijie L. Yahui G. Hideki N. Xiaodong G. Li C. Biosynthesis of rare hexoses using microorganisms and related enzymes.Beilstein J. Org. Chem. 2013; 9: 2434-2445Crossref PubMed Scopus (69) Google Scholar, 17.Alajarin R. Garcia-Junceda E. Wong C. A short enzymic synthesis of l-glucose from dihydroxyacetone phosphate and l-glyceraldehyde.Beilstein J. Org. Chem. 1995; 9: 2434-2445Google Scholar). From a biomedical perspective, rare sugars and their nucleoside derivatives are candidates for anticancer and antiviral drugs (18.Beerens K. Desmet T. Soetaert W. Enzymes for the biocatalytic production of rare sugars.J. Ind. Microbiol. Biotechnol. 2012; 39: 823-834Crossref PubMed Scopus (140) Google Scholar). Specifically, siRNA made from d-altritol-modified nucleic acids has been used to inhibit hepatitis B virus replication (19.Hean J. Crowther C. Ely A. Ul Islam R. Barichievy S. Bloom K. Weinberg M.S. van Otterlo W.A. de Koning C.B. Salazar F. Marion P. Roesch E.B. Lemaitre M. Herdewijn P. Arbuthnot P. Inhibition of hepatitis B virus replication in vivo using lipoplexes containing altritol-modified antiviral siRNAs.Artif. DNA PNA XNA. 2010; 1: 17-26Crossref PubMed Scopus (24) Google Scholar) and the expression of the multidrug-resistant efflux pump MDR1 (20.Fisher M. Abramov M. Van Aerschot A. Xu D. Juliano R.L. Herdewijn P. Inhibition of MDR1 expression with altritol-modified siRNAs.Nucleic Acids Res. 2007; 35: 1064-1074Crossref PubMed Scopus (68) Google Scholar). Defining metabolic pathways involving d-altritol may enable the development of new low cost synthetic routes, which will further its impact in medicine and industry. d-Altritol has been isolated from brown algae of the order Fucales (21.Raven J.A. Beardall J. Chudek J.A. Scrimgeour C.M. Clayton M.N. McInroy S.G. Altritol synthesis by Notheia anomala.Phytochemistry. 2001; 58: 389-394Crossref PubMed Scopus (9) Google Scholar). Although d-altritol is not known to be a widespread plant metabolite, polyols are primary photosynthetic products; therefore, the existence of d-altritol may be phylogenetically widespread, albeit at low abundance (22.Fox T.C. Kennedy R.A. Loescher W.H. Developmental changes in photosynthetic gas exchange in the polyol-synthesizing species, Apium graveolens L. (Celery).Plant Physiol. 1986; 82: 307-311Crossref PubMed Google Scholar). Notably, Izumori and co-workers (23.Muniruzzaman S. Kobayashi H. Izumori K. Production of d-talitol from d-tagatose by Aureobasidium pullulans strain 113B.J. Fermentation Bioeng. 1994; 78: 346-350Crossref Scopus (13) Google Scholar24.Sasahara H. Mine M. Izumori K. Production of d-talitol from d-psicose by Candida famata R28.J. Fermentation Bioeng. 1998; 85: 84-88Crossref Scopus (27) Google Scholar, 25.Yoshihara K. Shinohara Y. Hirotsu T. Izumori K. Bioconversion of d-psicose to d-tagatose and d-talitol by mucoraceae fungi.J. Biosci. Bioeng. 2006; 101: 219-222Crossref PubMed Scopus (24) Google Scholar26.Poonperm W. Takata G. Izumori K. Polyol conversion specificity of Bacillus pallidus.Biosci. Biotechnol. Biochem. 2008; 72: 231-235Crossref PubMed Scopus (16) Google Scholar) have shown that various organisms, including fungi, yeast, and bacteria, have the capability to synthesize d-altritol, although the pathway has not been studied. In this study, d-altritol catabolism was predicted and verified in the tumor-producing plant pathogen Agrobacterium tumefaciens C58. Tumor production proceeds through insertion into the plant genome of a small segment of DNA from Agrobacterium's Ti plasmid (27.Chilton M.D. Drummond M.H. Merio D.J. Sciaky D. Montoya A.L. Gordon M.P. Nester E.W. Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis.Cell. 1977; 11: 263-271Abstract Full Text PDF PubMed Scopus (642) Google Scholar). This mode-of-action has made Agrobacterium an important tool in the genetic engineering of plants (28.Zambryski P. Joos H. Genetello C. Leemans J. Montagu M.V. Schell J. Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity.EMBO J. 1983; 2: 2143-2150Crossref PubMed Google Scholar). Additionally, A. tumefaciens can infect a wide variety of plants, making it of great interest to the agricultural industry (it has been identified as the third most important plant pathogen) (29.Mansfield J. Genin S. Magori S. Citovsky V. Sriariyanum M. Ronald P. Dow M. Verdier V. Beer S.V. Machado M.A. Toth I. Salmond G. Foster G.D. Top 10 plant pathogenic bacteria in molecular plant pathology.Mol. Plant Pathol. 2012; 13: 614-629Crossref PubMed Scopus (1148) Google Scholar, 30.Moore L.W. Chilton W.S. Canfield M.L. Diversity of opines and opine-catabolizing bacteria isolated from naturally occurring crown gall tumors.Appl. Environ. Microbiol. 1997; 63: 201-207Crossref PubMed Google Scholar). The fact that sugars are known to induce virulence factors in Agrobacterium (31.Cangelosi G.A. Ankenbauer R.G. Nester E.W. Sugars induce the Agrobacterium virulence genes through a periplasmic binding protein and a transmembrane signal protein.Proc. Natl. Acad. Sci. U.S.A. 1990; 87: 6708-6712Crossref PubMed Scopus (229) Google Scholar) has resulted in many studies on carbohydrate metabolism in A. tumefaciens (32.Arthur L.O. Bulla Jr., L.A. Julian G.S. Nakamura L.K. Carbohydrate metabolism in Agrobacterium tumefaciens.J. Bacteriol. 1973; 116: 304-313Crossref PubMed Google Scholar33.Van Keer C. Kersters K. De Ley J. l-Sorbos metabolism in Agrobacterium tumefaciens.Antonie van Leeuwenhoek. 1976; 42: 13-24Crossref PubMed Scopus (3) Google Scholar, 34.Groninger-Poe F.P. Bouvier J.T. Vetting M.W. Kalyanaraman C. Kumar R. Almo S.C. Jacobson M.P. Gerlt J.A. Evolution of enzymatic activities in the enolase superfamily: galactarate dehydratase III from Agrobacterium tumefaciens C58.Biochemistry. 2014; 53: 4192-4203Crossref PubMed Scopus (14) Google Scholar35.Zhao J. Binns A.N. GxySBA ABC transporter of Agrobacterium tumefaciens and its role in sugar utilization and vir gene expression.J. Bacteriol. 2014; 196: 3150-3159Crossref PubMed Scopus (12) Google Scholar) but none involving d-altritol. Sequence similarity networks (SSNs) for Pfam families PF01547 (bacterial extracellular solute-binding protein), PF01116 (fructose-bisphosphate aldolase), PF00294 (PfkB family carbohydrate kinase), and PF08013 (tagatose-6-phosphate kinase) were generated with Option B of the EFI-Enzyme Similarity Tool (EFI-EST), which uses one or more Pfam families as input (11.Gerlt J.A. Bouvier J.T. Davidson D.B. Imker H.J. Sadkhin B. Slater D.R. Whalen K.L. Enzyme function initiative-enzyme similarity tool (EFI-EST): a web tool for generating protein sequence similarity networks.Biochim. Biophys. Acta. 2015; 1854: 1019-1037Crossref PubMed Scopus (468) Google Scholar). The SSNs of PF00106 (short-chain dehydrogenase) and PF00107 (zinc-binding dehydrogenase) could not be generated via Option B because of the large number of sequences in these Pfam families; these SSNs were generated via Option A of EFI-EST, which uses a protein sequence as input and generates an SSN from the top 5,000 BLAST hits identified by the query sequence. All SSNs were visualized as 100% representative node networks generated with a minimum edge alignment score threshold corresponding to ∼35% sequence identity selected from the percent identity-alignment score quartile plot provided by EFI-EST. The alignment score thresholds for edge inclusion were then made increasingly stringent using Cytoscape to generate the networks used in pathway prediction. The genome neighborhood networks (GNNs) for PF01547 (bacterial extracellular solute-binding protein) and PF00294 (PfkB family carbohydrate kinase) were generated using the EFI-Genome Neighborhood Tool (EFI-GNT). The input was the xgmml file for the SSN cluster containing the d-altritol binding ABC SBPs (AltSBPs). The output was generated using the default parameters (neighborhood size = 10, co-occurrence lower limit = 20%). The genes encoding ABC SBPs were amplified from genomic DNAs by PCR using KOD Hot Start DNA polymerase (Novagen) and the primers listed in Table 1. The conditions were as follows: 2 min at 95 °C, followed by 40 cycles of 30 s at 95 °C, 30 s at 66 °C, and 30 s at 72 °C. The amplified fragments were cloned into the N-terminal tobacco etch virus-cleavable His6 tag containing vector, pNIC28-Bsa4 (36.Savitsky P. Bray J. Cooper C.D. Marsden B.D. Mahajan P. Burgess-Brown N.A. Gileadi O. High-throughput production of human proteins for crystallization: the SGC experience.J. Struct. Biol. 2010; 172: 3-13Crossref PubMed Scopus (218) Google Scholar), by ligation-independent cloning (37.Aslanidis C. de Jong P.J. Ligation-independent cloning of PCR products (LIC-PCR).Nucleic Acids Res. 1990; 18: 6069-6074Crossref PubMed Scopus (934) Google Scholar). The periplasmic signal sequences, as predicted by SignalP (38.Petersen T.N. Brunak S. von Heijne G. Nielsen H. SignalP 4.0: discriminating signal peptides from transmembrane regions.Nat. Methods. 2011; 8: 785-786Crossref PubMed Scopus (7094) Google Scholar), were not included in the cloned products. SBPs (selenomethionine-substituted) were expressed by autoinduction in a LEX48 airlift fermenter and purified by nickel-nitrilotriacetic acid and size exclusion chromatography. Details are as described previously (12.Vetting M.W. Al-Obaidi N. Zhao S. San Francisco B. Kim J. Wichelecki D.J. Bouvier J.T. Solbiati J.O. Vu H. Zhang X. Rodionov D.A. Love J.D. Hillerich B.S. Seidel R.D. Quinn R.J. et al.Experimental strategies for functional annotation and metabolism discovery: targeted screening of solute-binding proteins and unbiased panning of metabolomes.Biochemistry. 2015; 54: 909-931Crossref PubMed Scopus (67) Google Scholar). ABC SBPs were concentrated to 10–20 mg/ml, flash-frozen using liquid nitrogen, and stored at −20 °C.TABLE 1PCR primers used in this studyPurposePrimer nameSequenceCloningA6X5Q5_FOR5′-TACTTCCAATCCATGGAAGAGCTCACCATCGCGAC-3′CloningA6X5Q5_REV5′-GACGCAGTCCGGTTACATCAAGTAACAGTAAAGGTGGATA-3′CloningD3PTN0_FOR5′-TACTTCCAATCCATGCAGACCACCATCACCATCGC-3′CloningD3PTN0_REV5′-GATGAAAGAGGCTGGCTACATCAAGTAACAGTAAAGGTGGATA-3′CloningQ2K3Z9_FOR5′-TACTTCCAATCCATGGAGACGCTGACAATCGCGAC-3′CloningQ2K3Z9_REV5′-GATGACCAAAGCGGGCTACATTAAATAACAGTAAAGGTGGATA-3′CloningB9JRF8_FOR5′-TACTTCCAATCCATGGGCGTGACCTCTGCCGAAAC-3′CloningB9JRF8_REV5′-GAAGGCGGGTTATCCGAAGAAGTAACAGTAAAGGTGGATA-3′CloningAtuSorbD_FOR5′-CATGACGGAGATCCATATGAGATTGAACAACAAGGTCGCGCTG-3′CloningAtuSorbD_REV5′-GAATGGCATGCATGGTCGGATCCGGCTTTTCAGCTCATC-3′CloningAtuZnD_FOR5′-GCTGAAAAGCCGCATATGACCATGCATGCCATTCAATTCGTCG-3′CloningAtuZnD_REV5′-CCGGTCGAATGGGGATCCACGGCCAAACGATCATTC-3′CloningAtuFK_FOR5′-GAGCAGACCGAAGTGGATCCCGCAGGTTTGCCG-3′CloningAtuFK_REV5′-CACATGCTTATGGAGGAGCATATGAGGCAGGCATCCGTAC-3′CloningAtuTag6PK_FOR5′-GCAGGGGCATCCGGATCCTGATTGCCGTCAGTG-3′CloningAtuTag6PK_REV5′-GCAATTGTCGGAGGAAGTCATATGACCGCCATTTTGGAAAATCTCG-3′RT-PCRAtuSorbD_RTPCR_FOR5′-CAACGGCATTACCGAAGAGAG-3′RT-PCRAtuSorbD_RTPCR_REV5′-GCGCAATAAAGCGTCACCAG-3′RT-PCRAtuZnD_RTPCR_FOR5′-CTTTGCGGAGTTCAGTGTGG-3′RT-PCRAtuZnD_RTPCR_REV5′-GATTTCAGCGACAGGGCAAG-3′RT-PCRAtuFK_RTPCR_FOR5′-AGGCCGTTAAAAGCGTCAAAG-3′RT-PCRAtuFK_RTPCR_REV5′-TATCGCTGCTTCCTCATCGTC-3′RT-PCRAtuTag6PK_RTPCR_FOR5′-CCATTCGACCGATTACCAGAC-3′RT-PCRAtuTag6PK_RTPCR_REV5′-GCCATAGGTGCCGACCATTT-3′RT-PCRAtuLacI_RTPCR_FOR5′-CACCGTCACCTGGGATTTTG-3′RT-PCRAtuLacI_RTPCR_REV5′-CAACCTTGATGCCCTTCTGG-3′RT-PCRAtuHisoD_RTPCR_FOR5′-TCGGTCGTGTCACTCATGTC-3′RT-PCRAtuHisoD_RTPCR_REV5′-GGCAAAACCGCCGAGTAAAG-3′RT-PCRAtuHomoD_RTPCR_FOR5′-GAGCCGCGTTACCGTTATTC-3′RT-PCRAtuHomoD_RTPCR_REV5′-GCAATCCAGTCCTGTGCCATT-3′RT-PCRAtuRpoD_RTPCR_FOR5′-ATCATCGATCTCGAAACGACCT-3′RT-PCRAtuRpoD_RTPCR_REV5′-TCGTCGTCGTCCTCTTCTTC-3′Knock-outKO_AtuSorbD_BamHI_OE_FOR5′-CCAAGCCGACCAAGGATCCGGTGCCCTATACCG-3′Knock-outKO_AtuSorbD_BamHI_OExt_REV5′-GCATGGTCATCTCCGGCTTTGTCGATCTCCGTCATGC-3′Knock-outKO_AtuSorbD_EcoRI_OExt_FOR5′-GCATGACGGAGATCGACAAAGCCGGAGATGACCATGC-3′Knock-outKO_AtuSorbD_EcoRI_OE_REV5′-CGCAATGCCGCGTGAATTCAGCGACAGGGCAAGC-3′Knock-outKO_AtuZnD_BamHI_OE_FOR5′-CAAGGCCGTGAAGCTGGATCCGACCGACCTTGC-3′Knock-outKO_AtuZnD_BamHI_OExt_REV5′-GGTTATCAACGGCCAAACGAGGTCATCTCCGGCTTTTCAG-3′Knock-outKO_AtuZnD_HindIII_OExt_FOR5′-CTGAAAAGCCGGAGATGACCTCGTTTGGCCGTTGATAACC-3′Knock-outKO_AtuZnD_HindIII_OE_REV5′-CCCGACGCTCTGAAGCTTGTCGGTGCCGACAAAG-3′Knock-outKO_AtuFK_BamHI_OE_FOR5′-GCGCCGCCTGCGGATCCGTCGGTGCGAC-3′Knock-outKO_AtuFK_BamHI_OExt_REV5′-GCACATGCTTATGGAGGAGACGATGACCGCCATTTTGGAAAATC-3′Knock-outKO_AtuFK_HindIII_OExt_FOR5′-GATTTTCCAAAATGGCGGTCATCGTCTCCTCCATAAGCATGTGC-3′Knock-outKO_AtuFK_HindIII_OE_REV5′-CTCCCAGGCGCTGGAAAGCTTCTGCATGATGATCATGTC-3′Knock-outKO_AtuTag6PK_BamHI_OE_FOR5′-GATCGACAGCAGCGGATCCTGCGCAAACCAGTCG-3′Knock-outKO_AtuTag6PK_BamHI_OExt_REV5′-GCAATTGTCGGAGGAAGTCCTATGACGGCAATCATAATCCGG-3′Knock-outKO_AtuTag6PK_HindIII_OExt_FOR5′-CCGGATTATGATTGCCGTCATAGGACTTCCTCCGACAATTGC-3′Knock-outKO_AtuTag6PK_HindIII_OE_REV5′-GACGGATGAGGCGCAGAAGCTTATCGAAAAGGCGGG-3′ Open table in a new tab Ligand mixtures (1–6 compounds) were prepared as concentrated stock solutions in water (10 mm) and stored at −20 °C. In a 10-μl volume (100 mm HEPES, pH 7.5, 150 mm NaCl, 5 mm DTT), the ligand concentration was 1 mm; the protein concentration was 10 μm, and Sypro-Orange was at 5× (Thermo Fisher Scientific). The ligand library consists of 405 ligands and eight controls (protein without compound) in 192 duplicated wells (384 wells total) (supplemental Table S1). DSF data were measured using an Applied Biosystems 7900HT Fast Real Time PCR system with excitation at 490 nm and emission at 530 nm. The samples were heated from 22 to 99 °C at a rate of 3 °C min−1; the resulting curves were fit to a Boltzmann equation to determine the midpoint temperature of the melting transition (Tm). These values were compared with control wells to calculate the ΔTm. A ΔTm of greater than 5 °C is considered to indicate a significant interaction. Details are as described previously (12.Vetting M.W. Al-Obaidi N. Zhao S. San Francisco B. Kim J. Wichelecki D.J. Bouvier J.T. Solbiati J.O. Vu H. Zhang X. Rodionov D.A. Love J.D. Hillerich B.S. Seidel R.D. Quinn R.J. et al.Experimental strategies for functional annotation and metabolism discovery: targeted screening of solute-binding proteins and unbiased panning of metabolomes.Biochemistry. 2015; 54: 909-931Crossref PubMed Scopus (67) Google Scholar). The genes annotated to encode the sorbitol dehydrogenase (AtuSorbD, PF00106, UniProt ID A9CES4), the zinc-binding dehydrogenase (AtuZnD, PF00107, UniProt ID A9CES3), the fructokinase (AtuFK, PF00294, Uniprot ID A9CES5), and the tagatose-6-phosphate kinase (AtuTag6PK, PF08013, UniProt ID A9CES6) from A. tumefaciens C58 (ATCC 33970) were PCR-amplified from genomic DNA using the cloning primers in Table 1. The PCRs (30 μl) contained 50 ng of genomic DNA, 1 mm MgCl2, 1× Pfx Amp Buffer, 0.33 mm dNTP, 0.33 μm of each primer, and 1.25 units of Pfx polymerase (Invitrogen Platinum Pfx DNA polymerase kit). Amplification was performed according to the manufacturer's guidelines. The amplified products were digested with NdeI/BamHI (New England Biolabs) and ligated into NdeI/BamHI-digested pET15b (Novagen). The recombinant plasmids were transformed into Escherichia coli BL21 (DE3) for expression. AtuSorbD, AtuZnD, AtuFK, and AtuTag6PK were purified from a 1-liter culture using a chelating Sepharose Fast Flow (Amersham Biosciences) column charged with Ni2+ as described previously (9.Wichelecki D.J. Balthazor B.M. Chau A.C. Vetting M.W. Fedorov A.A. Fedorov E.V. Lukk T. Patskovsky Y.V. Stead M.B. Hillerich B.S. Seidel R.D. Almo S.C. Gerlt J.A. Discovery of function in the enolase superfamily: d-mannonate and d-gluconate dehydratases in the d-mannonate dehydratase subgroup.Biochemistry. 2014; 53: 2722-2731Crossref PubMed Scopus (23) Google Scholar). The proteins were concentrated (AtuSorbD, 2 mg/ml; AtuZnD, 7.5 mg/ml; AtuFK, 6.7 mg/ml; AtuTag6PK, 7.3 mg/ml), flash-frozen using liquid nitrogen, and stored at −80 °C. A. tumefaciens C58 was grown to an OD of 0.4–0.5 in AB minimal media (1 liter contains 1 g of NH4Cl, 0.3 g of MgSO4·7H2O, 0.15 g of KCl, 0.01 g of CaCl·2H2O, 0.0025 g of FeSO4·7H2O, 3 g of K2HPO4, and 1 g of NaH2PO4) and 10 mm galactitol (Sigma)/d-altritol (Carbosynth) or 10 mm d-glucose (Sigma). Cells (500 μl) were incubated in 1 ml of RNA Protect (Qiagen) for 5 min at room temperature and pelleted at 15,000 rpm; the supernatant was removed. mRNA was then purified from the cells using an RNeasy mini kit (Qiagen). The RNA was further processed using RNase-free DNase (Qiagen) following the manufacturer's protocol. Purity was verified with 30-μl PCRs containing 50 ng of mRNA, 1 mm MgCl2, 1× Pfx amplification buffer, 2× PCR enhancer, 0.33 mm dNTP, 0.33 μm FOR/REV primers (AtuSorbD_RTPCR_FOR and AtuSorbD_RTPCR_REV), and 1.25 units of Pfx polymerase (Invitrogen Platinum Pfx DNA polymerase kit). The PCRs were analyzed on agarose gels to confirm amplification. Concentrations were determined spectrophotometrically at 280 nm. cDNA was generated using Protoscript First Strand (New England Biolabs) and 1 μg of mRNA following the manufacturer's protocol. The qRT-PCRs were performed using the Light Cycler 480 SYBR Green I Master kit (Roche Applied Science) and a Light Cycler 480 II (Roche Applied Science) using the manufacturer's protocol. Table 1 contains the sequences of the qRT-PCR primers (labeled as RT-PCR). The RpoD (RNA polymerase σ factor) primers were included to normalize the results based on the amount of cDNA added. AtuSorbD was screened for oxidation activity in a reaction containing 50 mm Tris, pH 9, 10 mm MgCl2, 1.5 mm NAD+, 1 μm AtuSorbD, and 5 mm polyol (as well as blanks without enzyme). Polyols tested include d-arabitol (Sigma), galactitol, d-mannitol (Sigma), d-sorbitol (Sigma), d-altritol and l-fucitol (Sigma), and volemitol (d-glycero-d-mannoheptitol, Sigma). The reactions were incubated at 25 °C for 60" @default.
• W2284865753 created "2016-06-24" @default.
• W2284865753 creator A5006013523 @default.
• W2284865753 creator A5025342046 @default.
• W2284865753 creator A5043037580 @default.
• W2284865753 creator A5048964187 @default.
• W2284865753 creator A5077472503 @default.
• W2284865753 creator A5089273362 @default.
• W2284865753 creator A5091252875 @default.
• W2284865753 date "2015-11-01" @default.
• W2284865753 modified "2023-10-10" @default.
• W2284865753 title "ATP-binding Cassette (ABC) Transport System Solute-binding Protein-guided Identification of Novel d-Altritol and Galactitol Catabolic Pathways in Agrobacterium tumefaciens C58" @default.
• W2284865753 cites W1524294403 @default.
• W2284865753 cites W1915578613 @default.
• W2284865753 cites W193914404 @default.
• W2284865753 cites W1964767165 @default.
• W2284865753 cites W1970114563 @default.
• W2284865753 cites W1970554741 @default.
• W2284865753 cites W1972885508 @default.
• W2284865753 cites W1976127881 @default.
• W2284865753 cites W1976851124 @default.
• W2284865753 cites W1987370810 @default.
• W2284865753 cites W1999420933 @default.
• W2284865753 cites W2006231140 @default.
• W2284865753 cites W2011025549 @default.
• W2284865753 cites W2020618100 @default.
• W2284865753 cites W2025410059 @default.
• W2284865753 cites W2032838501 @default.
• W2284865753 cites W2042656486 @default.
• W2284865753 cites W2043815249 @default.
• W2284865753 cites W2045180672 @default.
• W2284865753 cites W2046920360 @default.
• W2284865753 cites W2048188867 @default.
• W2284865753 cites W2052074731 @default.
• W2284865753 cites W2054225211 @default.
• W2284865753 cites W2055109879 @default.
• W2284865753 cites W2060797027 @default.
• W2284865753 cites W2061664287 @default.
• W2284865753 cites W2066800745 @default.
• W2284865753 cites W2072866908 @default.
• W2284865753 cites W2074430711 @default.
• W2284865753 cites W2080347382 @default.
• W2284865753 cites W2092435069 @default.
• W2284865753 cites W2093064281 @default.
• W2284865753 cites W2094201044 @default.
• W2284865753 cites W2094476648 @default.
• W2284865753 cites W2107251251 @default.
• W2284865753 cites W2121938728 @default.
• W2284865753 cites W2122632025 @default.
• W2284865753 cites W2133691154 @default.
• W2284865753 cites W2151007476 @default.
• W2284865753 cites W2151389728 @default.
• W2284865753 cites W2164673267 @default.
• W2284865753 cites W2165335121 @default.
• W2284865753 cites W2166406784 @default.
• W2284865753 cites W2167624940 @default.
• W2284865753 cites W2169765861 @default.
• W2284865753 cites W2170158942 @default.
• W2284865753 cites W2313469408 @default.
• W2284865753 cites W2319039376 @default.
• W2284865753 cites W2330905307 @default.
• W2284865753 cites W2384179156 @default.
• W2284865753 doi "https://doi.org/10.1074/jbc.m115.686857" @default.
• W2284865753 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/4661409" @default.
• W2284865753 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/26472925" @default.
• W2284865753 hasPublicationYear "2015" @default.
• W2284865753 type Work @default.
• W2284865753 sameAs 2284865753 @default.
• W2284865753 citedByCount "29" @default.
• W2284865753 countsByYear W22848657532016 @default.
• W2284865753 countsByYear W22848657532017 @default.
• W2284865753 countsByYear W22848657532018 @default.
• W2284865753 countsByYear W22848657532019 @default.
• W2284865753 countsByYear W22848657532020 @default.
• W2284865753 countsByYear W22848657532021 @default.
• W2284865753 countsByYear W22848657532022 @default.
• W2284865753 countsByYear W22848657532023 @default.
• W2284865753 crossrefType "journal-article" @default.
• W2284865753 hasAuthorship W2284865753A5006013523 @default.
• W2284865753 hasAuthorship W2284865753A5025342046 @default.
• W2284865753 hasAuthorship W2284865753A5043037580 @default.
• W2284865753 hasAuthorship W2284865753A5048964187 @default.
• W2284865753 hasAuthorship W2284865753A5077472503 @default.
• W2284865753 hasAuthorship W2284865753A5089273362 @default.
• W2284865753 hasAuthorship W2284865753A5091252875 @default.
• W2284865753 hasBestOaLocation W22848657531 @default.
• W2284865753 hasConcept C104317684 @default.
• W2284865753 hasConcept C149011108 @default.
• W2284865753 hasConcept C185592680 @default.
• W2284865753 hasConcept C204241405 @default.
• W2284865753 hasConcept C2776162087 @default.
• W2284865753 hasConcept C2776241147 @default.
• W2284865753 hasConcept C2778173381 @default.
• W2284865753 hasConcept C44312359 @default.
• W2284865753 hasConcept C55493867 @default.
• W2284865753 hasConcept C62231903 @default.
• W2284865753 hasConcept C86803240 @default.
• W2284865753 hasConcept C96942376 @default.

• next