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FGAR Amidotransferase: A Purine Biosynthetic Enzyme with Potential as a Target for Antimicrobial Drugs. Jeffrey D. Newman Lycoming College May 7, 1998 Overview • Symbiosis Project – Background - Graduate work – Goal - Identification of genes associated with commitment to determinate nodule development. • Purine Project – – – – – – 5/23/2017 Historical Background Purine Auxotroph Phenotypes - The Problem Identification of Purine Genes - The Hypothesis Support from Genome Sequences Recent Results - Heterologous complementation Future Directions Slide #2 Rhizobium-Legume Symbiosis Early Steps Attachment, nod gene induction Root hair curling, meristem initiation Infection thread development 5/23/2017 Slide #3 Rhizobium-Legume Symbiosis Intermediate Steps Infection thread development Nodule Emergence and Release from Infection Thread. 5/23/2017 Slide #4 Symbiotic phenotype of Rhizobium purine auxotrophs • Purine auxotrophs initiate nodule formation (Nod+), but do not induce infection thread development (Inf -). Pur - + purine • Lack of infection leads to formation of pseudonodules. Pur - + AICAr • Addition of AICA riboside but not purines restores infection and enhances nodule development. 5/23/2017 WT Slide #5 The Purine Biosynthetic Pathway TPP purF purD purN purLQ purM PRPP AIR AICAR GMP PRPP = 5'-Phosphoribosyl-1'-Pyrophosphate AIR = 5-aminoimidazole ribonucleotide AICAR = 5-aminoimidazole-4-carboxamide ribonucleotide TPP = Thiamine Pyrophosphate 5/23/2017 IMP AMP Slide #6 Hypotheses • Hypothesis 1: A derivative of AICAR serves as signal for infection thread development NH2 NH2 C N N 5/23/2017 N H2N N O N N • AICA riboside must be present until day 6 for true nodule development (Ndv+). • Hypothesis 2: Infection through day 6 triggers nodule-specific developmental pathway. C AMP RP AICAR RP NH2 C N N N Cytokinin N RP Slide #7 Symbiosis Research Projects • Long Term Goal - Identification of genes associated with 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. 5/23/2017 Slide #8 The Purine Biosynthetic Pathway TPP purF purD purN purLQ purM PRPP AIR AICAR GMP PRPP = 5'-Phosphoribosyl-1'-Pyrophosphate AIR = 5-aminoimidazole ribonucleotide AICAR = 5-aminoimidazole-4-carboxamide ribonucleotide TPP = Thiamine Pyrophosphate 5/23/2017 IMP AMP Slide #9 An Intriguing Observation Mutant Strains Pur? Thi? Accum? Explanation CE106, CE115, CE170, CE292, CE382 - - - CE124, CE290, CE384 - + + AIR CE110, CE390, CE391 5/23/2017 - Blocked before AIR Blocked between AIR and AICAR ??????? Slide #10 Complementation Analysis 5/23/2017 Cosmid Strains Complemented pCOS106 CE106 pCOS110 CE110, CE170, CE382, CE390, CE391 pCOS115 CE115, CE292 pCOS124 CE124, CE290, CE384 Slide #11 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. pur gene • Cosmids containing pur gene mutations designated pJNXXXA. – e.g. - CE110 pJN110A 5/23/2017 Slide #12 pCOS110 Complementation Groups CE110 CE390 pCOS110 pJN110A pJN390A pJN391A pJN170A pJN382A 5/23/2017 + + + + + + + + + CE391 CE170 CE382 + + + + + + + + - + + + + Slide #13 pCOS110 Map • Initial partial sequencing out from Tn5 identified purY, purQ, and purL mutations. • Subsequent complete sequencing identified purC and YJII. E=EcoRI B=BamHI B Completed sequence E E CE170 CE382 CE110 E 5/23/2017 CE391 1 kb E B Partial sequence Slide #14 The Purine Biosynthetic Pathway TPP purF purD purN purLQ purM PRPP AIR AICAR GMP PRPP = 5'-Phosphoribosyl-1'-Pyrophosphate AIR = 5-aminoimidazole ribonucleotide AICAR = 5-aminoimidazole-4-carboxamide ribonucleotide TPP = Thiamine Pyrophosphate 5/23/2017 IMP AMP Slide #15 FGAR Amidotransferase +H N 3 CH CH +H N 3 COO CH 2 CH 2 NH 2 + N N H O O C H O O O N H HN OH2 C C H O O O OH OH 5'-phosphoribosyl-N- formylglycinamide FGAR 5/23/2017 P H N O O OH2 C + O glutamate O P 2 C H glutamine O COO CH 2 C O CH OH OH 5'-phosphoribosyl-N- formylglycinamidine FGAM Slide #16 FGAR Amidotransferase Organization Type I Single subunit ATP-binding, cleavage FGAR glutamine enolization amide transfer 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 Multi-subunit PurL - 742 aa (B.subtilis ) PurQ - 227 aa 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. 5/23/2017 Slide #17 FGAR Amidotransferase Phylogenetic Analysis proteobacteria cyanobacteria plants Gram + Methanogens Deinococci animals Extreme thermophiles fungi Thermotoga Aquifex 5/23/2017 Type I Type 2 unknown Slide #18 PurY as a Third Subunit SalI EcoRI EcoRI E.coli purL 3.9 kb Mob CmR SalI SalI pTB93F (8.0 kb) GFP SalI pTrp from Daniel Gage of Sharon Long’s Lab Rep EcoRI SpcR 5/23/2017 Slide #19 PurY as a Third Subunit Complementation Strain Mutation by E.coli purL 5/23/2017 CE106 PurF::Tn5 ? CE110 PurY::Tn5 + CE170 PurL::Tn5 + CE390 PurQ::Tn5 ? Slide #20 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. 5/23/2017 Slide #21 Lab Members • Alumni – Diana Burley – Kevin Ferguson – Jonathan Cook – Kathy Roberts – Matthew Georgy 5/23/2017 • Current – Mark McCleland – Kim Mistiszyn – Lori Schultz – Rachel Lawton – Laura Singer – Tom Rombold Slide #22