An Asymmetric Model for Na+-translocating Glutaconyl-CoA Decarboxylases

Dietmar Linder,Wolfgang Buckel,Daniel Kress,Daniela Brügel,Lars-Oliver Essen,Iris Schall

doi:10.1074/jbc.m109.037762

Dietmar Linder, Wolfgang Buckel + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m109.037762

Copy DOI

Abstract

Glutaconyl-CoA decarboxylase (Gcd) couples the biotin-dependent decarboxylation of glutaconyl-CoA with the generation of an electrochemical Na(+) gradient. Sequencing of the genes encoding all subunits of the Clostridium symbiosum decarboxylase membrane complex revealed that it comprises two distinct biotin carrier subunits, GcdC(1) and GcdC(2), which differ in the length of a central alanine- and proline-rich linker domain. Co-crystallization of the decarboxylase subunit GcdA with the substrate glutaconyl-CoA, the product crotonyl-CoA, and the substrate analogue glutaryl-CoA, respectively, resulted in a high resolution model for substrate binding and catalysis revealing remarkable structural changes upon substrate binding. Unlike the GcdA structure from Acidaminococcus fermentans, these data suggest that in intact Gcd complexes, GcdA is associated as a tetramer crisscrossed by a network of solvent-filled tunnels.

Highlights

In aerobic organisms and respiring anaerobes this decarboxylation represents the irreversible second half-reaction of the FAD-containing homotetrameric enzyme glutaryl-CoA dehydrogenase [1]
The enzyme complex comprises a hydrophilic ␣-subunit (GcdA, 65 kDa) that catalyzes the Naϩ-independent transfer of the carboxylate of glutaconyl-CoA to biotin, the latter being covalently bound to the ␥-subunit (GcdC, 14 kDa, Equation 2)
Evaluation using PROCHECK [40] revealed good stereochemistry for all four structures, with more than 98% of the Partial co-purification of subcomplexes of His6-gene coding for the carboxytransferase (GcdA) and residues in the most favored and allowed regions of the Rambiotinylated GcdC1 was demonstrated by Western blotting achandran plot and with RMSD values for bond distances and using an anti-Penta-His-HRP-conjugate and an avidin-peroxi- angles below 0.010 Å and 1.3°, respectively

Summary

Introduction

In aerobic organisms and respiring anaerobes this decarboxylation represents the irreversible second half-reaction of the FAD-containing homotetrameric enzyme glutaryl-CoA dehydrogenase [1]. GcdC1 was purified by avidin-biotin affinity chromatography analogous to the purification of the glutaconyl-CoA decarboxylase complex.

Results

Conclusion