Abstract
Substrate channeling is a mechanism for the internal transfer of hydrophobic, unstable or toxic intermediates from the active site of one enzyme to another. Such transfer has previously been described to be mediated by a hydrophobic tunnel, the use of electrostatic highways or pivoting and by conformational changes. The enzyme PaaZ is used by many bacteria to degrade environmental pollutants. PaaZ is a bifunctional enzyme that catalyzes the ring opening of oxepin-CoA and converts it to 3-oxo-5,6-dehydrosuberyl-CoA. Here we report the structures of PaaZ determined by electron cryomicroscopy with and without bound ligands. The structures reveal that three domain-swapped dimers of the enzyme form a trilobed structure. A combination of small-angle X-ray scattering (SAXS), computational studies, mutagenesis and microbial growth experiments suggests that the key intermediate is transferred from one active site to the other by a mechanism of electrostatic pivoting of the CoA moiety, mediated by a set of conserved positively charged residues.
Highlights
Substrate channeling is a mechanism for the internal transfer of hydrophobic, unstable or toxic intermediates from the active site of one enzyme to another
CryoEM grids of PaaZ were made on holey-carbon grids on ice without any support material
Using graphene oxide on Ultrafoil gold grids and low protein concentration yielded a particle distribution that was used to collect higher resolution data with a Falcon 3 detector in counting mode (Supplementary Fig. 1b)
Summary
Substrate channeling is a mechanism for the internal transfer of hydrophobic, unstable or toxic intermediates from the active site of one enzyme to another. Such transfer has previously been described to be mediated by a hydrophobic tunnel, the use of electrostatic highways or pivoting and by conformational changes. Substrate channeling is the direct transfer of an intermediate between the catalytic sites of a two-step reaction without its release to the bulk solvent. In the case of the bacterial fatty acid βoxidation multi-enzyme complex, Ishikawa et al, described a mechanism where Coenzyme A (CoA) binding to a single site acts as a pivot to transfer the intermediate from 2-enoyl-CoA hydratase (ECH) to L-3-hydroxyacyl-CoA dehydrogenase (HACD) active sites, while the transfer to the third component 3-. The substrate is first activated by covalent linkage to CoA to form a CoA-
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