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

Abstract

DmpFG is a bifunctional enzyme comprised of an aldolase subunit, DmpG, and a dehydrogenase subunit, DmpF. The aldehyde intermediate produced by the aldolase is channeled directly through a buried molecular channel in the protein structure from the aldolase to the dehydrogenase active site. In this study, we have investigated the binding of a series of progressively larger substrates to the aldolase, DmpG, using molecular dynamics. All substrates investigated are easily accommodated within the active site, binding with free energy values comparable to the physiological substrate 4-hydroxy-2-ketovalerate. Subsequently, umbrella sampling was utilized to obtain free energy surfaces for the aldehyde intermediates (which would be generated from the aldolase reaction on each of these substrates) to move through the channel to the dehydrogenase DmpF. Small substrates were channeled with limited barriers in an energetically feasible process. We show that the barriers preventing bulky intermediates such as benzaldehyde from moving through the wild-type protein can be removed by selective mutation of channel-lining residues, demonstrating the potential for tailoring this enzyme to allow its use for the synthesis of specific chemical products. Furthermore, positions of transient escape routes in this flexible channel were determined.

Does metabolite channeling accelerate enzyme-catalyzed cascade reactions?

“…This is realized through formation of enzyme-enzyme complexes, also called enzyme assemblies or metabolons. In these complexes the reaction intermediates are transfered from the active site of one enzyme to the active site of another enzyme either inside a physical tunnel [ 11 – 14 ] or along an ‘electrostatic highway’ [ 11 , 12 , 15 , 16 ] (both called direct channeling), or diffusionally, but along a shortened path, in which case it is termed ‘channeling by proximity’ [ 17 – 22 ].…”

In Silico Studies of Small Molecule Interactions with Enzymes Reveal Aspects of Catalytic Function

“…Furthermore, kinetics studies of the bifunctional Escherichia coli enzyme proline utilization A (PutA), which catalyzes the oxidation of L-proline to L-glutamate in two successive reactions, exhibits substrate channeling between the proline dehydrogenase (PRODH) and Δ 1 -pyrroline-5-carboxylate dehydrogenase (P5CDH) components of PutA [ 178 ]. In computational studies, MD simulations were used to investigate substrate channeling in DmpFG bifunctional enzymes from aldolase (DmpG) to dehydrogenase (DmpF) subunits using a series of progressively larger substrates [ 179 ]. Water molecules were found to specify a route for aldehyde products toward the channel.…”

Structural Characterization and Molecular Dynamics Study of the REPI Fusion Protein from Papaver somniferum L.

“…Although substrate channeling can play key roles in biological processes by enabling trafficking of intermediates that need to be shielded from the environment, the elucidation of structural details of ligand migration through tunnels or channels is particularly chal-lenging [30]. In this regard, complex computational methods such as molecular dynamics (MD) simulations with further metadynamics calculations have been used to explore mechanisms of substrate channeling [31,32]. However, different easier-to-use methods have been specifically designed to identify and characterize tunnels and channels in proteins [33][34][35][36][37], thus providing useful information to address possible channeling processes without the extremely high computational cost of MD studies.…”