Metabolic pathways are frequently transferred between bacterial strains in the environment through horizontal gene transfer (HGT), yet laboratory engineering to introduce new metabolic pathways often fails. Successful use of a pathway requires co-evolution of both pathway and host, and these interactions may be disrupted upon transfer to a new host. Here we show that two different pathways for catabolism of coumarate failed to function when initially transferred into Escherichia coli. Using laboratory evolution, we elucidated the factors limiting activity of the newly-acquired pathways and the modifications required to overcome these limitations. Both pathways required mutations to the host to enable effective growth with coumarate, but the necessary mutations differed depending on the chemistry and intermediates of the pathways. In one case, an intermediate inhibited purine nucleotide biosynthesis, and this inhibition was relieved by single amino acid mutations to IMP dehydrogenase. A strain that natively contains this coumarate catabolism pathway, Acinetobacter baumannii, is already resistant to inhibition by the relevant intermediate, suggesting that natural pathway transfers have faced and overcome similar challenges. These discoveries will aid in our understanding of HGT and ability to predictably engineer metabolism.
Inosine 5’‐monophosphate dehydrogenase (IMPDH) catalyzes the oxidation of IMP to XMP, with concomitant reduction of NAD+ to NADH. This is the pivotal step for de novo guanine nucleotide biosynthesis. The structural and functional differences between mammalian and bacterial IMPDHs make this a promising antibiotic target. Selective and potent inhibitors of bacterial IMPDHs with different scaffolds, like A110 and C91, bind in the cofactor site. Despite the high conservation of this site, some inhibitors display a surprising preference for certain bacterial IMPDHs. The structural basis for this selectivity is unclear even though 16 crystal structures of IMPDH‐inhibitor complexes have been solved. IMPDH contains an active site flap that is highly dynamic and disordered. Here, we hypothesize that transient interactions with the flap may account for varied inhibitor potencies. In this work, we report a mutagenesis study and pre‐steady‐state stopped‐flow kinetic investigation of Bacillus anthracis IMPDH (BaIMPDH) aimed to determine the role of the flap in inhibitor selectivity. We introduced a L413A flap mutation in BaIMPDH and measured how inhibitor potencies changed. The results show some inhibitors (eg. A110) have the same potency towards both wild type and mutant enzymes, but other inhibitors (eg. C91) were found to have different potencies. To determine if the mutation changes the relative amount of inhibitor binding intermediates in catalytic cycle, we performed stopped‐flow kinetic experiments to defined eleven microscopic rate constants and two equilibrium constants that characterize both the catalytic cycle and details of the inhibition mechanism. Together with steady‐state initial rate studies, the results obtained for wild type BaIMPDH show that both A110 and C91 binds with high affinity predominantly to the covalent intermediate (E‐XMP*) on the reaction pathway (Kd ≍ 50 nM for A110 and Kd ≍ 40 nM for C91). Only a weak binding interaction (Kd ≍ 1 μM) is observed between A110 and E•IMP, while C91 has a bit stronger interaction with E•IMP (Kd ≍ 0.5 μM). Further pre‐steady‐state stopped‐flow kinetic investigation for BaIMPDH/L413A are in progress to determine the effects of this particular mutation on the microscopic rate constants that characterize the catalytic cycle and on the details of the inhibition mechanism.
Many bacterial pathogens, including Staphylococcus aureus, require inosine 5′-monophosphate
dehydrogenase (IMPDH) for
infection, making this enzyme a promising new target for antibiotics.
Although potent selective inhibitors of bacterial IMPDHs have been
reported, relatively few have displayed antibacterial activity. Here
we use structure-informed design to obtain inhibitors of S.
aureus IMPDH (SaIMPDH) that have potent
antibacterial activity (minimal inhibitory concentrations less than
2 μM) and low cytotoxicity in mammalian cells. The physicochemical
properties of the most active compounds were within typical Lipinski/Veber
space, suggesting that polarity is not a general requirement for achieving
antibacterial activity. Five compounds failed to display activity
in mouse models of septicemia and abscess infection. Inhibitor-resistant S. aureus strains readily emerged in vitro. Resistance resulted from substitutions in the cofactor/inhibitor
binding site of SaIMPDH, confirming on-target antibacterial
activity. These mutations decreased the binding of all inhibitors
tested, but also decreased catalytic activity. Nonetheless, the resistant
strains had comparable virulence to wild-type bacteria. Surprisingly,
strains expressing catalytically inactive SaIMPDH
displayed only a mild virulence defect. Collectively these observations
question the vulnerability of the enzymatic activity of SaIMPDH as a target for the treatment of S. aureus infections, suggesting other functions of this protein may be responsible
for its role in infection.
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