Biological Channeling of a Reactive Intermediate in the Bifunctional Enzyme DmpFG

Abstract

It has been hypothesized that the bifunctional enzyme DmpFG channels its intermediate, acetaldehyde, from one active site to the next using a buried intermolecular channel identified in the crystal structure. This channel appears to switch between an open and a closed conformation depending on whether the coenzyme NAD(+) is present or absent. Here, we applied molecular dynamics and metadynamics to investigate channeling within DmpFG in both the presence and absence of NAD(+). We found that substrate channeling within this enzyme is energetically feasible in the presence of NAD(+) but was less likely in its absence. Tyr-291, a proposed control point at the channel's entry, does not appear to function as a molecular gate. Instead, it is thought to orientate the substrate 4-hydroxy-2-ketovalerate in DmpG before reaction occurs, and may function as a proton shuttle for the DmpG reaction. Three hydrophobic residues at the channel's exit appear to have an important role in controlling the entry of acetaldehyde into the DmpF active site.

Evidence for Hysteretic Substrate Channeling in the Proline Dehydrogenase and Δ1-Pyrroline-5-carboxylate Dehydrogenase Coupled Reaction of Proline Utilization A (PutA)

“…Other conformational changes not yet known may also contribute to the activation of EcPutA. For instance, in the aldolase-dehydrogenase coupled reaction, conformational changes were found in DmpFG that contribute to allosteric communication and increase channeling activity between the two active sites (50,51).…”

Binding and Channeling of Alternative Substrates in the Enzyme DmpFG: a Molecular Dynamics Study

“…An alternative route to the second active site has been proposed from examination of the crystal structure of DmpFG, which revealed a 29 Å long water-filled channel linking the aldolase and dehydrogenase active sites (5). It has previously been shown by Smith et al (8) using molecular dynamics simulations that it is energetically feasible for acetaldehyde to move from one active site to the other within DmpFG when NAD þ , a coenzyme, is bound to DmpF. A hydrophobic triad (HT), observed in the crystal structure of DmpFG, is of interest in the context of channeling (5).…”

“…The DmpFG-substrate system with NAD þ bound to DmpF was utilized as described by Smith et al (8) The initial holo-enzyme coordinates were obtained from the Protein Data Bank (PDB:1NVM) and HKV was positioned in the active site of DmpG. Before collecting data, a series of equilibration steps were performed.…”

“…All simulations were performed using NAMD (18) and the CHARMM27 force field (19) using 2-fs timesteps with bonds to hydrogen kept rigid with the RATTLE algorithm. Additional parameters were required for the central Mn 2þ , enolate, and HKV and these were determined as described previously in Smith et al (8). Constant temperature (310 K) and pressure (1 atm) were maintained using Langevin dynamics and a Langevin piston.…”

“…In recent years, DmpFG and orthologous aldolase-dehydrogenases have begun to feature more extensively in the literature (8)(9)(10)(11)(12)(13)(14)(15)(16). While structures of MhpEF (15) and HsaF-HsaG (16) have recently been determined, DmpFG has the best-characterized structure so far (5).…”