Poster Presentation The 43rd Lorne Conference on Protein Structure and Function 2018

Structural studies reveal the mechanism of nucleotide triphosphate promiscuity for Mycobacterium tuberculosis dethiobiotin synthetase: a novel antibiotic target (#254)

ANDREW THOMPSON , Wanisa Salaemae , Kwang J Lee , Andrew Abell , Grant W Booker , John B Bruning , Kate L Wegener , Steven W Polyak

Tuberculosis (TB) is one of the deadliest infectious diseases in the world. While the infection is treatable, there has been a rise of TB infections that are resistant to many currently employed antibiotics. To combat this, we investigate pathways that aren’t subject to current resistance mechanisms with the ultimate aim of developing novel antibiotics.

Mycobacterium tuberculosis (Mtb), the causative pathogen of TB, relies on biosynthesis to generate the essential nutrient biotin, making this a promising pathway for novel anti-TB antibiotics. Dethiobiotin synthetase (DTBS) catalyses the penultimate step of biotin synthesis – the nucleotide triphosphate (NTP) dependant conversion of diaminopelargonic acid (DAPA) to dethiobiotin.

Using surface plasmon resonance (SPR) binding and enzyme assays, we made the unexpected discovery that MtbDTBS can utilise all NTPs with a marked affinity increase for cytidine triphosphate (CTP). While CTP associates tightly and dissociates slowly, the other NTPs bind with fast kinetics. Our crystal structure of MtbDTBS in complex with substrates: Mg2+-CTP and DAPA-carbamate confirmed the CTP binding mode. 

To investigate the binding of other NTPs, we optimised a novel crystal soaking technique, which garnered four new NTP-bound structures. These structures demonstrate that the lower affinity NTPs bind exclusively to MtbDTBS via their triphosphate group, a distinct mechanism from that observed for CTP. These data provide the structural basis for the promiscuous utilisation of NTPs and an unconventional mechanism of enzyme catalysis. These findings enhance our overall understanding of the DTBS active site and will guide the rational design of inhibitors to target this essential TB enzyme.