DOCSLIB.ORG

Hydrolysing System of Pseudomonas Putida RU-KM3S

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Hydrolysing System of Pseudomonas Putida RU-KM3S Characterization of the hydantoin- hydrolysing system of Pseudomonas putida RU-KM3S Thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy of Rhodes University by Gwynneth Felicity Matcher April 2004 ABSTRACT The biocatalytic conversion of 5-monosubstituted hydantoin derivatives to optically pure amino acids involves two reaction steps: the hydrolysis of hydantoin to N-carbamylamino acid by an hydantoinase or dihydropyrimidinase enzyme, followed by conversion of the N- carbamylamino acid to the corresponding amino acid by an N-carbamoylase enzyme. This biocatalytic process has been successfully applied in several industrial processes for the production of enantiomerically pure amino acids used in the synthesis of pharmaceuticals, insecticides, hormones, and food additives. P. putida RU-KM3S was selected for study based on inherent high levels of hydantoinase and N-carbamoylase activity. Subsequent biocatalytic analysis of the enzyme activity within this strain revealed unique properties thus prompting further characterization. The main focus of this research was the isolation of the genes encoding the hydantoin-hydrolysing pathway in RU-KM3S. A genomic library was constructed and screened for heterologous expression of the hydantoin-hydrolysing enzymes. However, this approach was unsuccessful prompting the use of transposon mutagenesis in order to circumvent the drawbacks associated with complementation studies. The enzymes responsible for hydantoin-hydrolysis were identified by insertional inactivation as a dihydropyrimidinase and β-ureidopropionase encoded by dhp and bup respectively. A third open reading frame, encoding a putative transport protein, was identified between the dhp and bup genes and appeared to share a promoter with bup. Analysis of the amino acid sequence deduced from bup and dhp substantiated the distinctive properties and potential industrial application of the L-enantioselective β-ureidopropionase and provided targets for potential optimisation of the substrate-selectivity and activity of the dihydropyrimidinase by site directed mutagenesis. Several transposon-generated mutants with an altered phenotype for growth on minimal medium with hydantoin as the sole source of nitrogen were also isolated. Analysis of the insertion events in these mutants revealed disruptions of genes encoding key elements of the Ntr global regulatory pathway. However, inactivation of these genes had no effect on the dihydropyrimidinase and β-ureidopropionase activity levels. An additional mutant in which the gene coding for the dihydrolipoamide succinyltransferase, which is involved in the TCA cycle, was isolated with reduced levels of both dihydropyrimidinase and β-ureidopropionase activities. These results indicated that the hydantoin-hydrolysis pathway in RU-KM3S is regulated by carbon rather than nitrogen catabolite repression. This was confirmed by the reduction of hydantoin-hydrolysis in cells grown in excess carbon as opposed to nitrogen. Identification of a putative CRP-binding site within the promoter region of these enzymes further supported the regulatory role of carbon catabolite repression (CCR). As CCR in Pseudomonads is poorly understood, elucidation of the mechanism by which the hydantoin- hydrolysing pathway in RU-KM3S is regulated would provide valuable insight into this complex process. I TABLE OF CONTENTS List of Figures......................................................................................................................... II List of Tables V List of Abbreviations VII Acknowledgements............................................................................................................... VIII Research outputs IX Chapter 1: Literature review................................................................................... 1 Chapter 2: Identification and biocatalytic characterization of strain RU-KM3S 45 Chapter 3: Construction and screening of RU-KM3S genome library................... 64 Chapter 4: Mutagenesis of RU-KM3S .......................................................................................................... 89 Chapter 5: Structural and regulatory components of the hydantoin-hydrolysing pathway of RU-KM3S ...................................................................................................................... 107 Chapter 6: Analysis of the bup and dhp genes from RU-KM3S 127 Chapter 7: General conclusions 149 Appendices................................................................................................................ 158 References 174 II LIST OF FIGURES Figure 1.1 Enzymatic hydrolysis of hydantoin to form enantiomerically pure amino acids 5 Figure 1.2 Keto-enol-tautomerism of 5-monosubstituted hydantoins.......................................... 24 Figure 1.3 Ribbon representation of the Thermus sp. D-hydantoinase quaternary structure (A), monomer (B), and catalytic site (C)............................................................................. 26 Figure 1.4 Hydrogen bond formation between the D-hydantoinase and D-5-monosubstituted hydantoin substrate (A), and the steric clash occurring when an L-5-monosubstitued hydantoin substrate is modelled into this site (B)........................................................ 27 Figure 1.5 Ribbon representation of the D-carbamoylase quaternary structure (A), monomer (B), and catalytic site (C) ....................................................................................................30 Figure 1.6 Model of the catalytic binding pocket of the D-carbamoylase with hydroxyphenyl- hydantoin substrate (A) and the interactions of the substrate with the enzyme (B) 30 Figure 2.1 Rational development of a biocatalyst for production of industrially important organic compounds ....................................................................................................................45 Figure 2.2 Production of N-carbamylglycine and glycine from hydantoin by RU-KM3S at various phases of growth in complete medium......................................................................... 50 Figure 2.3 The effect of divalent cations on hydantoinase and N-carbamoylase activity after dialysis of cell free extracts of RU-KM3S against phosphate buffer containing Na2EDTA .......................................................................................................................54 Figure 2.4 Effect of ATP on hydantoinase and N-carbamoylase activities in resting whole cell biocatalytic assays of RU-KM3S............................................................................................................................ 55 Figure 2.5 DNA fragments generated by restriction endonuclease digestion of the 16S rRNA gene of RU-KM3S and separated electophoretically through an agarose gel 56 Figure 2.6 Restriction endonuclease map of the 16S rRNA gene from P. putida RU-KM3S constructed from sequence data obtained from the parental and deletion constructs of the PCR product in pUC18 57 Figure 2.7 Boxshade of the aligned 16S rRNA gene sequences of P. putida strains BH and RU-KM3S ...................................................................................................................................................................................60 Figure 3.1 Electrophoresis of the native plasmid extracted from RU-KM3S as well as RU-KM3S chromosomal DNA (A) and plasmid extraction supernatant for RU-KM3S and GMPpc (B) 71 Figure 3.2 Partial digestion of RU-KM3S chromosomal DNA by addition of varying concentrations of Sau 3A1..................................................................................................................... 72 Figure 3.3 Estimation of insertion frequency and average size of insert of the recombinant clones in the constructed genomic library of RU-KM3S....................................................................................... 73 Figure 3.4 Schematic diagram of the pBK-CMV vector in which the genomic library of RU-KM3S was constructed............................................................................................................. 76 Figure 3.5 Endonuclease digestion of plasmid preparations (A) probed with radioactive PCR product of the insert from pGMLib15 (B) 77 III Figure 3.6 Southern analysis of the chromosomal DNA flanking the sequence encoded by the insert in pGMLib15........................................................................................................ 79 Figure 3.7 Graphical representation of the strategy used to determine the nucleotide sequence of the RU-KM3S genomic fragment contained in pGMlib15........................................... 81 Figure 3.8 Restriction endonuclease digestion of pGMlib1 and pGMlib2 from initial culture of the recombinant directly from the NCG-MM plate (A), and subsequent to sub-culturing in Luria broth (B) 83 Figure 3.9 Comparison of the growth of E. coli DH5α transformants expressing pBK-CMV, pGMlib1, or pGMlib2 over time.................................................................................... 84 Figure 4.1 Schematic map of the plasposon pTnMod-OKm ........................................................90 Figure 4.2 Optimisation of EMS mutagenesis treatment of RU-KM3S cells................................ 94 Figure 4.3 Phenotypes of RU-KM3S wild type (WT) and corresponding chemically induced mutant strains GMP1 and GMP2 on nutrient agar plates supplemented with and without 0.005% 5-FU, in the presence and absence of hydantoin as
Load more

Recommended publications
  • The Regulation of Carbamoyl Phosphate Synthetase-Aspartate Transcarbamoylase-Dihydroorotase (Cad) by Phosphorylation and Protein-Protein Interactions
    THE REGULATION OF CARBAMOYL PHOSPHATE SYNTHETASE-ASPARTATE TRANSCARBAMOYLASE-DIHYDROOROTASE (CAD) BY PHOSPHORYLATION AND PROTEIN-PROTEIN INTERACTIONS Eric M. Wauson A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pharmacology. Chapel Hill 2007 Approved by: Lee M. Graves, Ph.D. T. Kendall Harden, Ph.D. Gary L. Johnson, Ph.D. Aziz Sancar M.D., Ph.D. Beverly S. Mitchell, M.D. 2007 Eric M. Wauson ALL RIGHTS RESERVED ii ABSTRACT Eric M. Wauson: The Regulation of Carbamoyl Phosphate Synthetase-Aspartate Transcarbamoylase-Dihydroorotase (CAD) by Phosphorylation and Protein-Protein Interactions (Under the direction of Lee M. Graves, Ph.D.) Pyrimidines have many important roles in cellular physiology, as they are used in the formation of DNA, RNA, phospholipids, and pyrimidine sugars. The first rate- limiting step in the de novo pyrimidine synthesis pathway is catalyzed by the carbamoyl phosphate synthetase II (CPSase II) part of the multienzymatic complex Carbamoyl phosphate synthetase, Aspartate transcarbamoylase, Dihydroorotase (CAD). CAD gene induction is highly correlated to cell proliferation. Additionally, CAD is allosterically inhibited or activated by uridine triphosphate (UTP) or phosphoribosyl pyrophosphate (PRPP), respectively. The phosphorylation of CAD by PKA and ERK has been reported to modulate the response of CAD to allosteric modulators. While there has been much speculation on the identity of CAD phosphorylation sites, no definitive identification of in vivo CAD phosphorylation sites has been performed. Therefore, we sought to determine the specific CAD residues phosphorylated by ERK and PKA in intact cells.
    [Show full text]
  • Transcriptomic Characterization of Bradyrhizobium Diazoefficiens
    International Journal of Molecular Sciences Article Transcriptomic Characterization of Bradyrhizobium diazoefficiens Bacteroids Reveals a Post-Symbiotic, Hemibiotrophic-Like Lifestyle of the Bacteria within Senescing Soybean Nodules Sooyoung Franck 1, Kent N. Strodtman 1 , Jing Qiu 2 and David W. Emerich 1,* 1 Division of Biochemistry, University of Missouri, Columbia, MO 65211, USA; [email protected] (S.F.); [email protected] (K.N.S.) 2 Applied Economics and Statistics, University of Delaware, Newark, DE 19716, USA; [email protected] * Correspondence: [email protected]; Tel: +1-573-882-4252 Received: 8 October 2018; Accepted: 28 November 2018; Published: 7 December 2018 Abstract: The transcriptional activity of Bradyrhizobium diazoefficens isolated from soybean nodules was monitored over the period from symbiosis to late plant nodule senescence. The bacteria retained a near constant level of RNA throughout this period, and the variation in genes demonstrating increased, decreased, and/or patterned transcriptional activity indicates that the bacteria are responding to the changing environment within the nodule as the plant cells progress from an organized cellular structure to an unorganized state of internal decay. The transcriptional variation and persistence of the bacteria suggest that the bacteria are adapting to their environment and acting similar to hemibiotrophs, which survive both as saprophytes on live plant tissues and then as necrophytes on decaying plant tissues. The host plant restrictions of symbiosis make B. diazoefficiens a highly specialized, restricted hemibiotroph. Keywords: bradyrhizobium diazoefficiens; soybean; Glycine max; nitrogen fixation; senescence; transcriptomics; hemibiotroph 1. Introduction Soybean nodules are symbiotic organs that are formed on roots by the complex interaction between soybean plants and rhizobia, nitrogen-fixing bacteria, under nitrogen-limiting conditions.
    [Show full text]
  • General Physics
    GENERAL PHYSICS I. MOLECULE MICROSCOPY Academic and Research Staff Prof. John G. King Dr. Stanley R. Rosenthal Dr. John W. Peterson Dr. James C. Weaver Graduate Students H. Frederick Dylla Dusan G. Lysy Bruce R. Silver Joseph A. Jarrell Peter W. Stephens RESEARCH OBJECTIVES AND SUMMARY OF RESEARCH JSEP Joint Services Electronics Program (Contract DAAB07-74-C-0630) National Institutes of Health (Grants 1 PO1 HL14322-03 and 5 SO5 RR07047-08) John G. King, James C. Weaver Two basic types of microscopy, light and electron microscopy (and related ion versions), are well established, and are of importance to a diversity of applied and basic research problems. We are continuing our development of a new, third type, molecule microscopy, in which neutral molecules carry image-generating information from the sample. Neutral molecules are uniquely suited for probing interactions based on the chemistry of a sample; thus, molecule micrographs obtain contrast based on spatial variations of the permeability, diffusion, and binding of neutral molecules. For this reason, spatial variation in the permeation of molecules through a thin sample, dif- fusion along grain boundaries, binding of applied neutral molecules to a sample surface, and the existence of constituent molecules can all be revealed directly by some type of molecule microscope. Molecule microscopy should be useful in many fields of science and engineering, particularly in materials science and biology, since molecules carrying information from the sample interact through the same weak forces that are significant in deter- mining surface properties, and because the interactions are highly surface-specific (in contrast to photons and electrons which penetrate many atomic layers).
    [Show full text]
  • Genome-Scale Fitness Profile of Caulobacter Crescentus Grown in Natural Freshwater
    Supplemental Material Genome-scale fitness profile of Caulobacter crescentus grown in natural freshwater Kristy L. Hentchel, Leila M. Reyes Ruiz, Aretha Fiebig, Patrick D. Curtis, Maureen L. Coleman, Sean Crosson Tn5 and Tn-Himar: comparing gene essentiality and the effects of gene disruption on fitness across studies A previous analysis of a highly saturated Caulobacter Tn5 transposon library revealed a set of genes that are required for growth in complex PYE medium [1]; approximately 14% of genes in the genome were deemed essential. The total genome insertion coverage was lower in the Himar library described here than in the Tn5 dataset of Christen et al (2011), as Tn-Himar inserts specifically into TA dinucleotide sites (with 67% GC content, TA sites are relatively limited in the Caulobacter genome). Genes for which we failed to detect Tn-Himar insertions (Table S13) were largely consistent with essential genes reported by Christen et al [1], with exceptions likely due to differential coverage of Tn5 versus Tn-Himar mutagenesis and differences in metrics used to define essentiality. A comparison of the essential genes defined by Christen et al and by our Tn5-seq and Tn-Himar fitness studies is presented in Table S4. We have uncovered evidence for gene disruptions that both enhanced or reduced strain fitness in lake water and M2X relative to PYE. Such results are consistent for a number of genes across both the Tn5 and Tn-Himar datasets. Disruption of genes encoding three metabolic enzymes, a class C β-lactamase family protein (CCNA_00255), transaldolase (CCNA_03729), and methylcrotonyl-CoA carboxylase (CCNA_02250), enhanced Caulobacter fitness in Lake Michigan water relative to PYE using both Tn5 and Tn-Himar approaches (Table S7).
    [Show full text]
  • Colletotrichum Graminicola</Em>
    University of Kentucky UKnowledge Plant Pathology Faculty Publications Plant Pathology 3-8-2016 A Colletotrichum graminicola Mutant Deficient in the Establishment of Biotrophy Reveals Early Transcriptional Events in the Maize Anthracnose Disease Interaction Maria F. Torres University of Kentucky, [email protected] Noushin Ghaffari Texas A&M University Ester A. S. Buiate University of Kentucky, [email protected] Neil Moore University of Kentucky, [email protected] Scott chS wartz Texas A&M University FSeoe nelloxtw pa thige fors aaddndition addal aitutionhorsal works at: https://uknowledge.uky.edu/plantpath_facpub Part of the Bioinformatics Commons, Genomics Commons, Integrative Biology Commons, and Right click to open a feedback form in a new tab to let us know how this document benefits oy u. the Plant Pathology Commons Repository Citation Torres, Maria F.; Ghaffari, Noushin; Buiate, Ester A. S.; Moore, Neil; Schwartz, Scott; Johnson, Charles D.; and Vaillancourt, Lisa J., "A Colletotrichum graminicola Mutant Deficient in the Establishment of Biotrophy Reveals Early Transcriptional Events in the Maize Anthracnose Disease Interaction" (2016). Plant Pathology Faculty Publications. 53. https://uknowledge.uky.edu/plantpath_facpub/53 This Article is brought to you for free and open access by the Plant Pathology at UKnowledge. It has been accepted for inclusion in Plant Pathology Faculty Publications by an authorized administrator of UKnowledge. For more information, please contact [email protected]. Authors Maria F. Torres, Noushin Ghaffari, Ester A. S. Buiate, Neil Moore, Scott chS wartz, Charles D. Johnson, and Lisa J. Vaillancourt A Colletotrichum graminicola Mutant Deficient in the Establishment of Biotrophy Reveals Early Transcriptional Events in the Maize Anthracnose Disease Interaction Notes/Citation Information Published in BMC Genomics, v.17, 202, p.
    [Show full text]
  • Supplementary Table S4. FGA Co-Expressed Gene List in LUAD
    Supplementary Table S4. FGA co-expressed gene list in LUAD tumors Symbol R Locus Description FGG 0.919 4q28 fibrinogen gamma chain FGL1 0.635 8p22 fibrinogen-like 1 SLC7A2 0.536 8p22 solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 DUSP4 0.521 8p12-p11 dual specificity phosphatase 4 HAL 0.51 12q22-q24.1histidine ammonia-lyase PDE4D 0.499 5q12 phosphodiesterase 4D, cAMP-specific FURIN 0.497 15q26.1 furin (paired basic amino acid cleaving enzyme) CPS1 0.49 2q35 carbamoyl-phosphate synthase 1, mitochondrial TESC 0.478 12q24.22 tescalcin INHA 0.465 2q35 inhibin, alpha S100P 0.461 4p16 S100 calcium binding protein P VPS37A 0.447 8p22 vacuolar protein sorting 37 homolog A (S. cerevisiae) SLC16A14 0.447 2q36.3 solute carrier family 16, member 14 PPARGC1A 0.443 4p15.1 peroxisome proliferator-activated receptor gamma, coactivator 1 alpha SIK1 0.435 21q22.3 salt-inducible kinase 1 IRS2 0.434 13q34 insulin receptor substrate 2 RND1 0.433 12q12 Rho family GTPase 1 HGD 0.433 3q13.33 homogentisate 1,2-dioxygenase PTP4A1 0.432 6q12 protein tyrosine phosphatase type IVA, member 1 C8orf4 0.428 8p11.2 chromosome 8 open reading frame 4 DDC 0.427 7p12.2 dopa decarboxylase (aromatic L-amino acid decarboxylase) TACC2 0.427 10q26 transforming, acidic coiled-coil containing protein 2 MUC13 0.422 3q21.2 mucin 13, cell surface associated C5 0.412 9q33-q34 complement component 5 NR4A2 0.412 2q22-q23 nuclear receptor subfamily 4, group A, member 2 EYS 0.411 6q12 eyes shut homolog (Drosophila) GPX2 0.406 14q24.1 glutathione peroxidase
    [Show full text]
  • Uracil and Beta-Alanine Degradation in Saccharomyces Kluyveri - Discovery of a Novel Catabolic Pathway
    Downloaded from orbit.dtu.dk on: Sep 30, 2021 Uracil and beta-alanine degradation in Saccharomyces Kluyveri - discovery of a novel catabolic pathway Andersen, Gorm Publication date: 2006 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Andersen, G. (2006). Uracil and beta-alanine degradation in Saccharomyces Kluyveri - discovery of a novel catabolic pathway. Technical University of Denmark. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit-making activity or commercial gain You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Uracil and beta-alanine degradation in Saccharomyces kluyveri - Discovery of a novel catabolic pathway Ph.D. Thesis Gorm Andersen BioCentrum-DTU Technical University of Denmark May 2006 PREFACE The thesis presented here is a part of the requirements for the Ph.D. degree under the ”Chemistry and Biotechnology Programme” at the Technical University of Denmark (DTU). Since October 2002, I have been enrolled at BioCentrum-DTU. The study was financed by DTU and has been carried out under supervision of former Associate Professor at BioCentrum-DTU, (now Professor at Department of Cell and Organism Biology, Lund University, Sweden) Jure Piskur.
    [Show full text]
  • Study on Yeast Enzymes Urc1p and Urc4p in a Novel Uracil Catabolic Pathway (URC)
    Study on yeast enzymes Urc1p and Urc4p in a novel uracil catabolic pathway (URC) Dineshkumar Kandasamy Degree project in molecular biotechnology, 2012 Examensarbete i molekylär bioteknik 45 hp till masterexamen, 2012 Biology Education Centre, Uppsala University, and Department of Biology, Lund University Supervisors: Jure Piškur and Anna Rasmussen ABSTRACT Purine and pyrimidine bases are central precursors of DNA and RNA and their intracellular concentration is balanced by three pathways- de novo, salvage and catabolic pathways. Uracil catabolism pathway has been found in several bacteria and in some fungi (including yeast). Seven genes, URC1-7 have been found to be involved in this novel pathway. There are two “unknown genes” in the yeast Saccharomyces (Lachancea) kluyveri, namely URC1 and URC4, which play a central role in this pathway and their exact function remains a mystery. In this project, two S. kluyveri genes, URC1&URC4, were over-expressed in the bacterial system and successfully purified. Our preliminary functional assay showed that uridine monophosphate (UMP) is a likely substrate for Urc1p at pH 7, 25ºC. It was shown clearly that both uracil and uridine were not the substrate for Urc1p.We tried to phosphorylate chemically synthesized ribosylurea (RU) using Drosophila melanogaster deoxyribonucleoside kinase and compared the activity between phosphorylated and non- phosphorylated RU at different conditions. Phosphorylated ribosylurea seemed to be a likely substrate for Urc4p at pH 7, 37ºC. Keywords: Uridine monophosphate (UMP), ribosylurea (RU), uracil catabolism. Study on yeast enzymes Urc1p and Urc4p in a novel uracil catabolic pathway (URC) Popular science summary Dineshkumar Kandasamy Purines and pyrimidines are important constituent of nucleic acids and they are called building blocks of DNA and RNA.
    [Show full text]
  • University of London Thesis
    REFERENCE ONLY UNIVERSITY OF LONDON THESIS Degree Year^^0^ Name of Author C O P Y R IG H T This is a thesis accepted for a Higher Degree of the University of London. It is an unpublished typescript and the copyright is held by the author. All persons consulting the thesis must read and abide by the Copyright Declaration below. COPYRIGHT DECLARATION I recognise that the copyright of the above-described thesis rests with the author and that no quotation from it or information derived from it may be published without the prior written consent of the author. LOANS Theses may not be lent to individuals, but the Senate House Library may lend a copy to approved libraries within the United Kingdom, for consultation solely on the premises of those libraries. Application should be made to: Inter-Library Loans, Senate House Library, Senate House, Malet Street, London WC1E 7HU. REPRODUCTION University of London theses may not be reproduced without explicit written permission from the Senate House Library. Enquiries should be addressed to the Theses Section of the Library. Regulations concerning reproduction vary according to the date of acceptance of the thesis and are listed below as guidelines. A. Before 1962. Permission granted only upon the prior written consent of the author. (The Senate House Library will provide addresses where possible). B. 1962- 1974. In many cases the author has agreed to permit copying upon completion of a Copyright Declaration. C. 1975 - 1988. Most theses may be copied upon completion of a Copyright Declaration. D. 1989 onwards. Most theses may be copied.
    [Show full text]
  • Nucleotide Metabolism Pathway: the Achilles' Heel for Bacterial Pathogens
    REVIEW ARTICLES Nucleotide metabolism pathway: the achilles’ heel for bacterial pathogens Sujata Kumari1,2,* and Prajna Tripathi1,3 1National Institute of Immunology, New Delhi 110 067, India 2Present address: Department of Zoology, Magadh Mahila College, Patna University, Patna 800 001, India 3Present address: Institute of Molecular Medicine, Jamia Hamdard, New Delhi 110 062, India de novo pathway, the nucleotides are synthesized from Pathogens exploit their host to extract nutrients for their survival. They occupy a diverse range of host simple precursor molecules. In the salvage pathway, the niches during infection which offer variable nutrients preformed nucleobases or nucleosides which are present accessibility. To cause a successful infection a patho- in the cell or transported from external environmental gen must be able to acquire these nutrients from the milieu to the cell are utilized to form nucleotides. host as well as be able to synthesize the nutrients on its own, if required. Nucleotides are the essential me- tabolite for a pathogen and also affect the pathophysi- Purine biosynthesis pathway ology of infection. This article focuses on the role of nucleotide metabolism of pathogens during infection The purine biosynthesis pathway is universally conserved in a host. Nucleotide metabolism and disease pathoge- in living organisms (Figure 1). As an example, we here nesis are closely related in various pathogens. Nucleo- present the pathway derived from well-studied Gram- tides, purines and pyrimidines, are biosynthesized by positive bacteria Lactococcus lactis. In the de novo the de novo and salvage pathways. Whether the patho- pathway the purine nucleotides are synthesized from sim- gen will employ the de novo or salvage pathway dur- ple molecules such as phosphoribosyl pyrophosphate ing infection is dependent on various factors, like (PRPP), amino acids, CO2 and NH3 by a series of enzy- availability of nucleotides, energy condition and pres- matic reactions.
    [Show full text]
  • (12) United States Patent (10) Patent No.: US 9,689,046 B2 Mayall Et Al
    USOO9689046B2 (12) United States Patent (10) Patent No.: US 9,689,046 B2 Mayall et al. (45) Date of Patent: Jun. 27, 2017 (54) SYSTEM AND METHODS FOR THE FOREIGN PATENT DOCUMENTS DETECTION OF MULTIPLE CHEMICAL WO O125472 A1 4/2001 COMPOUNDS WO O169245 A2 9, 2001 (71) Applicants: Robert Matthew Mayall, Calgary (CA); Emily Candice Hicks, Calgary OTHER PUBLICATIONS (CA); Margaret Mary-Flora Bebeselea, A. et al., “Electrochemical Degradation and Determina Renaud-Young, Calgary (CA); David tion of 4-Nitrophenol Using Multiple Pulsed Amperometry at Christopher Lloyd, Calgary (CA); Lisa Graphite Based Electrodes', Chem. Bull. “Politehnica” Univ. Kara Oberding, Calgary (CA); Iain (Timisoara), vol. 53(67), 1-2, 2008. Fraser Scotney George, Calgary (CA) Ben-Yoav. H. et al., “A whole cell electrochemical biosensor for water genotoxicity bio-detection”. Electrochimica Acta, 2009, 54(25), 6113-6118. (72) Inventors: Robert Matthew Mayall, Calgary Biran, I. et al., “On-line monitoring of gene expression'. Microbi (CA); Emily Candice Hicks, Calgary ology (Reading, England), 1999, 145 (Pt 8), 2129-2133. (CA); Margaret Mary-Flora Da Silva, P.S. et al., “Electrochemical Behavior of Hydroquinone Renaud-Young, Calgary (CA); David and Catechol at a Silsesquioxane-Modified Carbon Paste Elec trode'. J. Braz. Chem. Soc., vol. 24, No. 4, 695-699, 2013. Christopher Lloyd, Calgary (CA); Lisa Enache, T. A. & Oliveira-Brett, A. M., "Phenol and Para-Substituted Kara Oberding, Calgary (CA); Iain Phenols Electrochemical Oxidation Pathways”, Journal of Fraser Scotney George, Calgary (CA) Electroanalytical Chemistry, 2011, 1-35. Etesami, M. et al., “Electrooxidation of hydroquinone on simply prepared Au-Pt bimetallic nanoparticles'. Science China, Chem (73) Assignee: FREDSENSE TECHNOLOGIES istry, vol.
    [Show full text]
  • Mammalian Dihydroorotase: Nucleotide Sequence, Peptide
    Proc. Natl. Acad. Sci. USA Vol. 87, pp. 174-178, January 1990 Biochemistry Mammalian dihydroorotase: Nucleotide sequence, peptide sequences, and evolution of the dihydroorotase domain of the multifunctional protein CAD (aspartate transcarbamylase/expression/pyrimidine biosynthesis/protein domains) JAMES P. SIMMER, RUTH E. KELLY*, AUSTIN G. RINKER, JR., BARBARA H. ZIMMERMANN, JOSHUA L. SCULLY, HYESOOK KIM, AND DAVID R. EVANS Department of Biochemistry, Wayne State University, Detroit, MI 48201 Communicated by Mary Ellen Jones, August 24, 1989 (received for review July 5, 1989) ABSTRACT Mammalian DHOase (S-dihydroorotate ami- Saul ull Ps, Puli P'jII KI)Er dohydrolase, EC 3.5.2.3) is part of a large multifunctional protein called CAD, which also has a carbamoyl-phosphate 3 8 40% 4 44 4 synthetase [carbon-dioxide:L-glutamine amido-ligase (ADP- forming, carbamate-phosphorylating), EC 6.3.5.5] and aspar- tate transcarbamoylase (carbamoyl-phosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) activities. We sequenced FIG. 1. Nucleotide sequencing strategy. The region of the selected restriction fragments of a Syrian hamster CAD cDNA. pCAD142 sequenced is shown schematically: the CPSase domain The deduced amino acid sequence agreed with the sequence of (stippled bar), the DHOase domain (shaded bar), and the DHOase- tryptic peptides and the amino acid composition ofthe DHOase ATCase linker (clear bar). Map units represent the distance in domain isolated by controlled proteolysis of CAD. Escherichia kilobases from the start of the cDNA insert. Clones sequenced are coli transformed with a recombinant plasmid containing the indicated by arrows. cDNA segment 5' to the aspartate transcarbamoylase coding respectively, may participate in catalysis.
    [Show full text]