Poster Presentation The 42nd Lorne Conference on Protein Structure and Function 2017

Ancestral reconstruction of a thermostable quinolinate synthase (#103)

James P Antoney 1 , William H Zhang 1 , Nicholas Cox 2 , Carol Hartley 3 , Colin Scott 3 , Colin J Jackson 1
  1. Australian National University, Canberra, ACT, Australia
  2. Max Planck Institute für Chemische Energiekonversion, Mülheim an der Ruhr, Germany
  3. Land & Water Flagship, Black Mountain, CSIRO, Canberra

Nicotinamide cofactors NADH and NADPH are ubiquitous electron carriers in both primary and secondary metabolism.1 Nicotinamide cofactors are also used in a number of key biocatalytic processes2,3, however the high cost of these cofactors presents a challenge to their use in industrial processes.4 Quinolinate is the first common biological precursor to nicotinamide cofactors and in prokaryotes is produced via the condensation of iminoaspartate and dihydroxyacetone phosphate by quinolinate synthase.5 A phylogenetic analysis revealed that quinolinate synthase sequences form several clades divided between aerobic and anaerobic species. As quinolinate synthase activity requires an oxygen-labile [4Fe-4S] cluster6 it was reasoned that obligately aerobic organisms would face selection pressure for a more oxygen-tolerant quinolinate synthase than obligate or facultative anaerobes and thus be more amenable for biocatalysis. Five previously uncharacterised extant quinolinate synthases were expressed and characterised, three from obligate aerobes and two from obligate anaerobes. In addition, a reconstructed ancestral sequence from one of the aerobic clades was characterised which appears to show high thermostability.

  1. Pollak, N.; Dölle, C.; Ziegler, M. Biochemical Journal 2007, 402, 205.
  2. Kara, S.; Schrittwieser, J. H.; Hollmann, F.; Ansorge-Schumacher, M. B. Applied microbiology and biotechnology 2014, 98, 1517.
  3. Bornscheuer, U.; Huisman, G.; Kazlauskas, R.; Lutz, S.; Moore, J.; Robins, K. Nature 2012, 485, 185.
  4. Paul, C. E.; Gargiulo, S.; Opperman, D. J.; Lavandera, I.; Gotor-Fernández, V.; Gotor, V.; Taglieber, A.; Arends, I. W.; Hollmann, F. Organic letters 2012, 15, 180.
  5. Foster, J. W.; Moat, A. G. Microbiological reviews 1980, 44, 83.
  6. Saunders, A. H.; Griffiths, A. E.; Lee, K.-H.; Cicchillo, R. M.; Tu, L.; Stromberg, J. A.; Krebs, C.; Booker, S. J. Biochemistry 2008, 47, 10999.