Substrate-bound Structures of Benzylsuccinate Synthase Reveal How Toluene Is Activated in Anaerobic Hydrocarbon Degradation

Michael A Funk,E Neil G Marsh,Catherine L Drennan

doi:10.1074/jbc.m115.670737

Abstract

Various bacteria perform anaerobic degradation of small hydrocarbons as a source of energy and cellular carbon. To activate non-reactive hydrocarbons such as toluene, enzymes conjugate these molecules to fumarate in a radical-catalyzed, C-C bond-forming reaction. We have determined x-ray crystal structures of the glycyl radical enzyme that catalyzes the addition of toluene to fumarate, benzylsuccinate synthase (BSS), in two oligomeric states with fumarate alone or with both substrates. We find that fumarate is secured at the bottom of a long active site cavity with toluene bound directly above it. The two substrates adopt orientations that appear ideal for radical-mediated C-C bond formation; the methyl group of toluene is positioned between fumarate and a cysteine that forms a thiyl radical during catalysis, which is in turn adjacent to the glycine that serves as a radical storage residue. Toluene is held in place by fumarate on one face and tight packing by hydrophobic residues on the other face and sides. These hydrophobic residues appear to become ordered, thus encapsulating toluene, only in the presence of BSSβ, a small protein subunit that forms a tight complex with BSSα, the catalytic subunit. Enzymes related to BSS are able to metabolize a wide range of hydrocarbons through attachment to fumarate. Using our structures as a guide, we have constructed homology models of several of these "X-succinate synthases" and determined conservation patterns that will be useful in understanding the basis for catalysis and specificity in this family of enzymes.

Highlights

Benzylsuccinate synthase (BSS) catalyzes the formation of a COC bond between toluene and fumarate by a radical mechanism
The two substrates adopt orientations that appear ideal for radical-mediated COC bond formation; the methyl group of toluene is positioned between fumarate and a cysteine that forms a thiyl radical during catalysis, which is in turn adjacent to the glycine that serves as a radical storage residue
We identified a channel that substrates could take to enter the active site; benzylsuccinate synthase (BSS)␤ appeared to block the channel entrance, suggesting either that substrates cannot bind in the presence of BSS␤ or that BSS␤ plays a role in gating active site accessibility

Summary

Background

Benzylsuccinate synthase (BSS) catalyzes the formation of a COC bond between toluene and fumarate by a radical mechanism. Our current study investigates how BSS binds to fumarate and toluene in both ␣␤␥ and ␣␥ complexes We find that both substrates can bind to the BSS␣␤␥ complex, demonstrating that the presence of BSS␤ does not prevent substrate access into the active site. Ordering of BSS␣ into the fully closed, catalytic state does not occur in this structure in the absence of BSS␤; only structures with BSS␣␤␥ depict the fully closed, catalytically competent state In this closed state, both substrates are bound at the bottom of the proposed channel in an orientation in which toluene is ideally positioned to undergo hydrogen atom abstraction by the putative, transient thiyl radical followed by COC bond formation between toluene and fumarate. This analysis will aid in characterization of the diverse communities of microbes known to cooperate in the degradation of hydrocarbons

Experimental Procedures

Results

Discussion