Molecular basis for metabolite channeling in a ring opening enzyme of the phenylacetate degradation pathway

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.

Five enzymes of the Arg/N-degron pathway form a targeting complex: The concept of superchanneling

“…Enzymes or assemblies of enzymes that catalyze sequential reactions have been observed to exhibit substrate channeling, in which a reaction intermediate can be transferred between active sites of interacting enzymes without release of intermediate to the bulk solution (77)(78)(79)(80)(81)(82)(83)(84)(85). It remains to be determined whether targeting complexes identified in the present work function to enable substrate channeling.…”

Bacterial Tropone Natural Products and Derivatives: Overview of their Biosynthesis, Bioactivities, Ecological Role and Biotechnological Potential

“…[14] Recently, this hypothesis was corroborated by structural studies of PaaZ including smallangle X-ray scattering and cryogenic electron microscopy. [31] Accordingly, the active site of a PaaZÀ ECH domain was found closest to the ALDH substrate-binding tunnel of its intertwined partner monomer rather than its own ALDH domain ( Figure 6). Presumably, substrate channeling is then achieved via a set of conserved positively charged residues that promote the electrostatic pivoting of the CoA moiety and thus the transfer of 6 between the ECH and ALDH active sites of the partner monomers.…”

“…[30] PaaZ from Escherichia coli K12 forms homohexamers with a trilobed architecture, in which each lobe consists of an intertwined PaaZ dimer with swapped ECH domains (all six ECH domains together form the inner core of the protein complex). [31] PaaZÀ ECH was shown to efficiently cleave oxepinÀ CoA, most likely via an acid-base mechanism ( Figure 6). [14,30] Ring cleavage by PaaZÀ ECH is accordingly initiated by attack of a hydroxyl ion (i. e., generated .…”

“…Presumably, substrate channeling is then achieved via a set of conserved positively charged residues that promote the electrostatic pivoting of the CoA moiety and thus the transfer of 6 between the ECH and ALDH active sites of the partner monomers. [31] However, many bacteria apparently found a more remarkable strategy for preventing CoA depletion and make use of 8 as precursor for tropone natural products (such as 15) and ω-cycloheptyl fatty acids.…”

“…CryoEM structures of bifunctional PaaZ from E. coli with distinct ECH and ALDH domains. [31] Overlay cartoon representation of hexameric, trilobed PaaZ (each lobe consists of an intertwined dimer). The substrate analogue crotonyl CoA bound in the active site of the ALDH domain was experimentally observed (green chains, PDB ID: 6JQN), whereas octanoyl CoA was modeled into the ECH active site (blue chains, PDB ID: 6JQO).…”