Title: The Purine Biosynthetic Enzyme FGAR Amidotransferase as a Potential Target for Antimicrobial Drugs
1The Purine Biosynthetic Enzyme FGAR
Amidotransferase as a Potential Target for
Antimicrobial Drugs
- Jeffrey D. Newman (88)
- Lycoming College
- October 16, 1998
2(No Transcript)
3Overview
- 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
4Molecular 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
5Molecular 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.
6The 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
7An Intriguing Observation
Analysis of Rhizobium purine auxotrophs
8Complementation Analysis
9Marker 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
10pCOS110 Complementation Groups
11pCOS110 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
12The 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
13FGAR Amidotransferase
glutamine
5'-phosphoribosyl-N- formylglycinamide
5'-phosphoribosyl-N- formylglycinamidine
FGAR
FGAM
14FGAR 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.
15FGAR AmidotransferasePhylogenetic Analysis
proteobacteria
?
?
?
?
?
cyanobacteria
plants
animals
Gram
Methanogens
Extreme thermophiles
Deinococci
fungi
Thermotoga
Type I
Aquifex
Type 2
unknown
16PurY as a Third Subunit
SalI
EcoRI
E.coli purL 3.9 kb
SalI
17PurY as a Third Subunit
18Current, 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
19Lab 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
20Visit My Web Pagehttp//lyco.lycoming.edu/newman
/index.html
21Rhizobium-Legume Symbiosis Early Steps
Attachment, nod gene induction
Root hair curling, meristem initiation
Infection thread development
22Rhizobium-Legume Symbiosis Intermediate Steps
Infection thread development
Nodule Emergence and Release from Infection
Thread.
23Symbiotic 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.
24The 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
25Hypotheses
- 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.
26Symbiosis 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.