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The Purine Biosynthetic Enzyme FGAR Amidotransferase as a Potential Target for Antimicrobial Drugs

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AICAR = 5-aminoimidazole-4-carboxamide. ribonucleotide. TPP ... Hypothesis 1: A derivative of AICAR serves as signal for infection thread development ... – PowerPoint PPT presentation

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Title: The Purine Biosynthetic Enzyme FGAR Amidotransferase as a Potential Target for Antimicrobial Drugs


1
The Purine Biosynthetic Enzyme FGAR
Amidotransferase as a Potential Target for
Antimicrobial Drugs
  • Jeffrey D. Newman (88)
  • Lycoming College
  • October 16, 1998

2
(No Transcript)
3
Overview
  • Teaching Activities
  • Molecular Modelling
  • Molecular Biology Across the Curriculum Project.
  • Purine Project
  • Historical Background
  • Purine Auxotroph Phenotypes - The Problem
  • Identification of Purine Genes - The Hypothesis
  • Support from Genome Sequences
  • Recent Results - Heterologous complementation
  • Current Activity - Expression cloning and
    purification.
  • Future Directions

4
Molecular Modelling
  • Rasmol - free, stand-alone 3-D rendering program
    - uses pdb formatted files (such as those from
    Brookhaven Natl Lab)
  • Chime - free, internet browser plug-in- allows
    viewing of interactive molecules in web pages.
  • Protein structure tutorials
  • Student protein papers

5
Molecular Biology Across the Curriculum
  • Freshman Bio - Mr. Green Genes
  • Plasmid Prep, restriction digest, agarose gel
    electrophoresis, transformation.
  • Microbiology - The Real Unknown Lab
  • PCR, agarose gel electrophoresis, DNA sequencing.
  • Genetics - Cloning of a PCR Product.
  • PCR, restriction digest, ligation, transformation
    (with blue/white screening), plasmid prep,
    agarose gel electrophoresis.
  • Common experience in basic techniques allows
    upper level Cell, Molecular, Biochem courses to
    be more aggressive.

6
The Purine Biosynthetic Pathway
TPP
purF purD purN purL(Q) purM
PRPP
AIR
purEK purC purB
AICAR
GMP
purH purH
PRPP 5'-Phosphoribosyl-1'-Pyrophosphate
guaA guaB
AIR 5-aminoimidazole ribonucleotide
AICAR 5-aminoimidazole-4-carboxamide
IMP
ribonucleotide
TPP Thiamine Pyrophosphate
purB purA
AMP
7
An Intriguing Observation
Analysis of Rhizobium purine auxotrophs
8
Complementation Analysis
9
Marker Exchange - Recovery of Mutations
  • In a complemented Rhizobium strain, some
    recombination between plasmid and chromosome will
    occur.
  • Plasmid transferred into E. coli, selection for
    Tn5-encoded KanR.
  • Cosmids containing pur gene mutations designated
    pJNXXXA.
  • e.g. - CE110 ? pJN110A

10
pCOS110 Complementation Groups
11
pCOS110 Map
  • Initial partial sequencing out from Tn5
    identified purY, purQ, and purL mutations.
  • Subsequent complete sequencing identified purC
    and YJII.

EEcoRI
1 kb
BBamHI
CE110
CE391
CE382
CE170
E
E
B
E
E
B
purL
purY
purC
purQ
YJII
Sequenced regions
12
The Purine Biosynthetic Pathway
TPP
purF purD purN purLQ purM
PRPP
AIR
purEK purC purB
AICAR
GMP
purH purH
PRPP 5'-Phosphoribosyl-1'-Pyrophosphate
guaA guaB
AIR 5-aminoimidazole ribonucleotide
AICAR 5-aminoimidazole-4-carboxamide
IMP
ribonucleotide
TPP Thiamine Pyrophosphate
purB purA
AMP
13
FGAR Amidotransferase
glutamine
5'-phosphoribosyl-N- formylglycinamide
5'-phosphoribosyl-N- formylglycinamidine
FGAR
FGAM
14
FGAR Amidotransferase Organization
glutamine
amide transfer
ATP-binding, cleavage
Type I
Single subunit
PurL
- 1295 aa (E.coli)
Escherichia coli, Salmonella typhimurium,
Haemophilus influenzae, Pseudomonas aeruginosa,
Vibrio cholerae, Neisseria gonnorheae, Neisseria
meningitidis, Saccharomyces cerevisiae,
Schizosaccharomyces pombe, Caenorabditis elegans,
Drosophila melanogaster, Gallus gallus, Homo
sapiens.
Type II
PurL
- 742 aa (B.subtilis )
Multi-subunit
Rhizobium etli, Bacillus subtilis,
Lactobacillus caseii, Staphylococcus aureus,
Mycobacterium tuberculosis, Mycobacterium
leprae, Streptococcus pneumoniae, Enterococcus
faecaelis, Synechocystis spp., Deinococcus
radiodurans, Thermotoga maritima, Aquifex
aeolicus, Archaeoglobus fulgidus, Methanococcus
jannaschii, Methanobacterium thermoautotrophicum.
15
FGAR AmidotransferasePhylogenetic Analysis
proteobacteria
?
?
?
?
?
cyanobacteria
plants
animals
Gram
Methanogens
Extreme thermophiles
Deinococci
fungi
Thermotoga
Type I
Aquifex
Type 2
unknown
16
PurY as a Third Subunit
SalI
EcoRI
E.coli purL 3.9 kb
SalI
17
PurY as a Third Subunit
18
Current, Future Studies
  • Cloning, expression and purification of subunits
    for
  • reconstitution of enzyme activity - inhibitor
    studies
  • generation of antisera for studies of
    protein-protein interactions
  • 3-D structural analysis (I'm dreaming!)
  • Protein-protein interactions using two-hybrid
    system.
  • Finish Rhizobium etli purL sequence.
  • Watch genome sequences pile up.
  • Identify type of enzyme in Giardia,
    Microsporidia, proteobacteria

19
Lab Members
  • Alumni
  • Diana Burley, Kathy Roberts
  • Kevin Ferguson, Jonathan Cook
  • Matthew Georgy
  • Current
  • Rachel Lawton
  • Mark McCleland
  • Lillian McTernan
  • Kim Mistiszyn
  • Tom Rombold
  • Lori Schultz
  • Laura Singer

20
Visit My Web Pagehttp//lyco.lycoming.edu/newman
/index.html
21
Rhizobium-Legume Symbiosis Early Steps
Attachment, nod gene induction
Root hair curling, meristem initiation
Infection thread development
22
Rhizobium-Legume Symbiosis Intermediate Steps
Infection thread development
Nodule Emergence and Release from Infection
Thread.
23
Symbiotic phenotype of Rhizobium purine auxotrophs
  • Purine auxotrophs initiate nodule formation
    (Nod), but do not induce infection thread
    development (Inf -).
  • Lack of infection leads to formation of
    pseudonodules.
  • Addition of AICA riboside but not purines
    restores infection and enhances nodule
    development.

24
The Purine Biosynthetic Pathway
TPP
purF purD purN purLQ purM
PRPP
AIR
purEK purC purB
AICAR
GMP
purH purH
PRPP 5'-Phosphoribosyl-1'-Pyrophosphate
guaA guaB
AIR 5-aminoimidazole ribonucleotide
AICAR 5-aminoimidazole-4-carboxamide
IMP
ribonucleotide
TPP Thiamine Pyrophosphate
purB purA
AMP
25
Hypotheses
  • Hypothesis 1 A derivative of AICAR serves as
    signal for infection thread development
  • AICA riboside must be present until day 6 for
    true nodule development (Ndv).
  • Hypothesis 2 Infection through day 6 triggers
    nodule-specific developmental pathway.

26
Symbiosis Research Projects
  • Long Term Goal - Identification of genes
    associatedwith commitment to determinate nodule
    development.
  • Use Rhizobium expressing GFP to examine
    relationship between AICA riboside
    supplementation and infection thread development.
  • Transition to Rhizobium loti - Lotus model
    system.
  • Use transgenic Lotus to examine promoters induced
    at the time of commitment to nodule organogenesis.
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