Structural Basis for Promoting and Preventing Decarboxylation in Glutaryl-Coenzyme A Dehydrogenases

Abstract

Glutaryl-coenzyme A dehydrogenases (GDHs) involved in amino acid degradation were thought to catalyze both the dehydrogenation and decarboxylation of glutaryl-coenzyme A to crotonyl-coenzyme A and CO(2). Recently, a structurally related but nondecarboxylating, glutaconyl-coenzyme A-forming GDH was characterized in the obligately anaerobic bacteria Desulfococcus multivorans (GDH(Des)) which conserves the free energy of decarboxylation by a Na(+)-pumping glutaconyl-coenzyme A decarboxylase. To understand the distinct catalytic behavior of the two GDH types on an atomic basis, we determined the crystal structure of GDH(Des) with and without glutaconyl-coenzyme A bound at 2.05 and 2.1 A resolution, respectively. The decarboxylating and nondecarboxylating capabilities are provided by complex structural changes around the glutaconyl carboxylate group, the key factor being a Tyr --> Val exchange strictly conserved between the two GDH types. As a result, the interaction between the glutaconyl carboxylate and the guanidinium group of a conserved arginine is stronger in GDH(Des) (short and planar bidentate hydrogen bond) than in the decarboxylating human GDH (longer and monodentate hydrogen bond), which is corroborated by molecular dynamics studies. The identified structural changes prevent decarboxylation (i) by strengthening the C4-C5 bond of glutaconyl-coenzyme A, (ii) by reducing the leaving group potential of CO(2), and (iii) by increasing the distance between the C4 atom (negatively charged in the dienolate transition state) and the adjacent glutamic acid.

Probing conformational states of glutaryl-CoA dehydrogenase by fragment screening

“…Residues that are critical for enzymatic activity in the human enzyme are conserved in the bacterial sequence, including the catalytic glutamate Glu374 (Glu370 in human) which abstracts an proton from the substrate (Fu et al, 2004;Rao et al, 2007). At the time of writing, all GCDH structures in the PDB possess FAD bound in the cofactor-binding pocket (PDB entries 1siq, 1sir, 2r0m, 2r0n, 2eba, 3mpi and 3mpj; Fu et al, 2004;Rao et al, 2007; T. S. Kumarevel, P. Karthe, S. Kuramitsu & S. Yokoyama, unpublished work; Wischgoll et al, 2010). None of these structures report FAD as a crystallization additive, despite its presence in the final structure.…”

“…At the time of writing, seven structures of GCDH are available in the Protein Data Bank (PDB) from the human, Thermus thermophilus and Desulfococcus multivorans gene products, all of which contain the bound cofactor FAD (Fu et al, 2004;Rao et al, 2007;Thorpe & Kim, 1995;Wischgoll et al, 2010). Here, we report the first structures of GCDH from B. pseudomallei (BpGCDH), including the first apo structure of any GCDH characterized in the absence of FAD.…”

Identification of 3-Sulfinopropionyl Coenzyme A (CoA) Desulfinases within the Acyl-CoA Dehydrogenase Superfamily

“…2). This arginine was expected to be involved in binding of the sulfino group of 3SP-CoA (1), in analogy to glutaryl-CoA dehydrogenases, where an arginine residue is involved in binding of the terminal carboxyl group (44)(45)(46)(47)(48) prior to decarboxylation. Three residues (E87, S95, and Y369, according to the numbering for human glutaryl-CoA dehydrogenase) which probably enable the decarboxylating reaction are absent from nondecarboxylating glutaryl-CoA dehydrogenases and from all 3SP-CoA desulfinases.…”

A Novel 3-Sulfinopropionyl Coenzyme A (3SP-CoA) Desulfinase from Advenella mimigardefordensis Strain DPN7 T Acting as a Key Enzyme during Catabolism of 3,3′-Dithiodipropionic Acid Is a Member of the Acyl-CoA Dehydrogenase Superfamily

“…Recently, a structurally related but nondecarboxylating GCD in the obligately anaerobic bacterium Desulfococcus multivorans (60) was characterized. The results led to the assumption that decarboxylating and nondecarboxylating capabilities are provided by distinct structurally conserved amino acid residues that surround the carboxylate group of the glutaconyl-CoA intermediate (61). In all GCDs, decarboxylating and nondecarboxylating, an invariant arginine residue (R94 in GCD Human ) is involved in binding of the terminal carboxylate and right substrate positioning (60)(61)(62)(63)(64).…”

“…The results led to the assumption that decarboxylating and nondecarboxylating capabilities are provided by distinct structurally conserved amino acid residues that surround the carboxylate group of the glutaconyl-CoA intermediate (61). In all GCDs, decarboxylating and nondecarboxylating, an invariant arginine residue (R94 in GCD Human ) is involved in binding of the terminal carboxylate and right substrate positioning (60)(61)(62)(63)(64). Three residues (E87, S95, and Y369, according to GCD Human numbering), conserved only in decarboxylating GCDs, weaken the binding between R94 and the substrate carboxylate and enable the decarboxylating reaction.…”