FGAR Amidotransferase: A Purine Biosynthetic Enzyme with Potential as a Target for Antimicrobial Dru

1
FGAR Amidotransferase A Purine Biosynthetic
Enzyme with Potential as a Target for
Antimicrobial Drugs.

• Jeffrey D. Newman
• Lycoming College
• May 7, 1998

2
Overview

• Symbiosis Project
• Background - Graduate work
• Goal - Identification of genes associated with
  commitment to determinate nodule development.
• Purine Project
• Historical Background
• Purine Auxotroph Phenotypes - The Problem
• Identification of Purine Genes - The Hypothesis
• Support from Genome Sequences
• Recent Results - Heterologous complementation
• Future Directions

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Rhizobium-Legume Symbiosis Early Steps
Attachment, nod gene induction
Root hair curling, meristem initiation
Infection thread development
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Rhizobium-Legume Symbiosis Intermediate Steps
Infection thread development
Nodule Emergence and Release from Infection
Thread.
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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.

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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
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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.

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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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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
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An Intriguing Observation
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Complementation Analysis
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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

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pCOS110 Complementation Groups
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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
Partial sequence
Completed sequence
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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
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FGAR Amidotransferase
glutamine
5'-phosphoribosyl-N- formylglycinamide
5'-phosphoribosyl-N- formylglycinamidine
FGAR
FGAM
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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.
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FGAR AmidotransferasePhylogenetic Analysis
proteobacteria
?
?
?
?
?
cyanobacteria
plants
animals
Gram
Methanogens
Extreme thermophiles
Deinococci
fungi
Thermotoga
Type I
Aquifex
Type 2
unknown
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PurY as a Third Subunit
SalI
EcoRI
E.coli purL 3.9 kb
SalI
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PurY as a Third Subunit
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Future Work

• Finish Rhizobium purL sequence.
• Overexpression and purification of Rhizobium
  and/or Staphylococcus aureus PurY, PurQ, PurL and
  E.coli PurL polypeptides.
• FGAR Amidotransferase assays.
• Identification of Type 2 inhibitors.

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Lab Members

• Alumni
• Diana Burley
• Kevin Ferguson
• Jonathan Cook
• Kathy Roberts
• Matthew Georgy

• Current
• Mark McCleland
• Kim Mistiszyn
• Lori Schultz
• Rachel Lawton
• Laura Singer
• Tom Rombold