An Opine on Opines: Characterizing Opine Metallophore Biosynthesis in Bacterial Pathogens

Abstract

An uncharacterized biosynthetic operon found in pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa and Yersinia pestis was proposed as the source of a novel metallophore in 2015. Growing evidence demonstrates the important role this operon plays in metal acquisition and virulence, including in lung and burn wound infections (P. aeruginosa) and in blood and heart infections (S. aureus). In 2016, the S. aureus metallophore product was elucidated by Ghssein et. al. and named staphylopine. In 2017, we identified the metallophore product in P. aeruginosa, naming it pseudopaline. Staphylopine and pseudopaline are produced in two biosynthetic steps by a nicotianamine synthase and an opine dehydrogenase. We have examined substrate specificity and determined steady-state kinetic parameters for both nicotianamine synthase and opine dehydrogenase enzymes from multiple species, and have generated X-ray crystal structures and collected transient state kinetic data for the opine dehydrogenases. Our work provides the first kinetic analysis of a nicotianamine synthase and reveals a non-processive mechanism that is dependent on S-adenosyl-L-methionine and either L- or D-histidine. We currently have promising nicotianamine synthase crystals for upcoming diffraction studies. Importantly, only one nicotianamine synthase X-ray crystal structure, from an archeal species, has been solved to date. Our studies of opine dehydrogenases suggest varied substrate specificity. For example, S. aureus opine dehydrogenase incorporates pyruvate as an α-keto acid substrate and uses NADPH as a co-substrate, while P. aeruginosa opine dehydrogenase uses α-ketoglutarate and either NADH or NADPH. We have solved X-ray crystal structures for the opine dehydrogenases ranging from 1.9–2.5 Å revealing a structural basis for the NAD(P)H specificity. We are currently completing a single turnover analysis of hydride transfer by the S. aureus opine dehydrogenase to complement our full steady-state analysis of both the forward and reverse reactions. This work contributes to our understanding of two enzymes involved in virulence that form a unique pathway generating a new metallophore class, the opine metallophores. Support or Funding Information NIH Chemical Biology Training Grant T32 GM08545; AHA Predoctoral Fellowship PRE33960374; NIH R01 GM127655-01 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.