Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2016

Bacterial metabolism of thiopurines: evidence for an alternative purine nucleotide salvage pathway (#43)

Ramya Movva 1 2 , Iulia Oancea 1 3 , John A Duley 1 4 , Paraic Ó Cuív 5 , Timothy H J Florin 1 3 6
  1. Mater Research Institute-University of Queensland, Translational Research Institute, Woolloongabba, Queensland, Australia
  2. School of Pharmacy, Griffith University, Gold Coast, Queensland, Australia
  3. Department of Medicine, University of Queensland, Brisbane , Queensland, Australia
  4. School of Pharmacy, University of Queensland, Woolloongabba, Queensland, Australia
  5. University of Queensland Diamantina Institute, Translational Research Institute, Woolloongabba, Queensland , Australia
  6. Department of Gastroenterology, Mater Health Services Adult Hospital, South Brisbane, Queensland, Australia

Introduction:

Immunomodulating thiopurine pro-drugs thioguanine (TG) and mercaptopurine (MP) are metabolised to active thioguanine nucleotides (TGNs) via the purine salvage enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT), which is highly conserved in bacteria. We confirmed that E.coli can metabolise TG or MP to TGNs and we presumed that this metabolism was driven by HGPRT. In order to further explore the role of HGPRT enzyme in bacteria for metabolising thiopurine pro-drugs, we have generated an E.coli BL21 HGPRT knockout.

Hypothesis:

We hypothesised that in the absence of HGPRT enzyme TG or MP should not be metabolised to TGN.

Aim:

To generate an HGPRT gene knockout in E.coli and measure metabolism of thiopurine pro-drugs.

Methods:

E.coli BL21 HGPRT mutant was constructed by Group II intron targeted mutagenesis. TGNs were measured by HPLC-UV.

Results:

Successful generation of a BL21 HGPRT mutant was confirmed using PCR. Surprisingly, the mutant strain converted TG or MP to TGNs. Furthermore, the production of TGNs was higher in the mutant strain compared to control.

Discussion and conclusion:

The generation of TGNs in the absence of HGPRT enzyme is strong evidence for an alternative purine nucleotide salvage pathway. We are investigating purine nucleoside phosphorylase, a reversible enzyme that interconverts thiopurine base and riboside, as a candidate enzyme. This enzyme pathway is not active in eukaryotes, as HGPRT-deficient cells are TG resistant.