Crystal Structures of Human Glutaryl-CoA Dehydrogenase with and without an Alternate Substrate:  Structural Bases of Dehydrogenation and Decarboxylation Reactions^,

Abstract

Acyl-CoA dehydrogenases (ACDs) are a family of flavoenzymes that metabolize fatty acids and some amino acids. Of nine known ACDs, glutaryl-CoA dehydrogenase (GCD) is unique: in addition to the alpha,beta-dehydrogenation reaction, common to all ACDs, GCD catalyzes decarboxylation of glutaryl-CoA to produce CO(2) and crotonyl-CoA. Crystal structures of GCD and its complex with 4-nitrobutyryl-CoA have been determined to 2.1 and 2.6 A, respectively. The overall polypeptide folds are the same and similar to the structures of other family members. The active site of the unliganded structure is filled with water molecules that are displaced when enzyme binds the substrate. The structure strongly suggests that the mechanism of dehydrogenation is the same as in other ACDs. The substrate binds at the re side of the FAD ring. Glu370 abstracts the C2 pro-R proton, which is acidified by the polarization of the thiolester carbonyl oxygen through hydrogen bonding to the 2'-OH of FAD and the amide nitrogen of Glu370. The C3 pro-R proton is transferred to the N(5) atom of FAD. The structures indicate a plausible mechanism for the decarboxylation reaction. The carbonyl polarization initiates decarboxylation, and Arg94 stabilizes the transient crotonyl-CoA anion. Protonation of the crotonyl-CoA anion occurs by a 1,3-prototropic shift catalyzed by the conjugated acid of the general base, Glu370. A tight hydrogen-bonding network involving gamma-carboxylate of the enzyme-bound glutaconyl-CoA, with Tyr369, Glu87, Arg94, Ser95, and Thr170, optimizes orientation of the gamma-carboxylate for decarboxylation. Some pathogenic mutations are explained by the structure. The mutations affect protein folding, stability, and/or substrate binding, resulting in inefficient/inactive enzyme.

Probing conformational states of glutaryl-CoA dehydrogenase by fragment screening

“…None of these structures report FAD as a crystallization additive, despite its presence in the final structure. This suggests that the cofactor is usually carried through purification, with sufficient binding affinity for GCDH to remain bound through multiple purification steps (Fu et al, 2004;Rao et al, 2007;Thorpe & Kim, 1995;Wischgoll et al, 2010). In our study, overexpression of BpGCDH in E. coli and standard protein-purification methods did not yield crystals with FAD bound (see Methods).…”

“…Like other ACDHs, GCDH from B. pseudomallei (BpGCDH) exists as a homotetramer with tetrahedral symmetry and possesses an overall fold similar to that of the human enzyme (Fu et al, 2004). Each protomer of BpGCDH consists of an N-terminal solvent-facing region of five -helices followed by a flattened seven-stranded pseudo--barrel and a C-terminal -helix core (Fig.…”

“…The C-terminal domain comprises much of the tetrameric binding surface and forms parts of both the FAD and the glutaryl-CoA binding pockets. 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).…”

Glutaric Acidemia Type 1-Clinico-Molecular Profile and Novel Mutations in GCDH Gene in Indian Patients

“…Crystal structure of wild-type human GCDH complexed with coenzyme FAD (PDB: 1SIQ) (Fu et al 2004) is available in PDB. Model structure of protein with novel missense mutation was built with the help of crystal structure of wild-type protein and "Build Mutant" protocol incorporated in MODELLER 8.2 program (Sali and Blundell 1993) in the modelling environment of Discovery Studio (DS 2.0) (Discovery Studio 2006, Accelrys Inc., San Diego, CA, USA).…”

“…Structural analysis reveals that the proper positioning of cofactor FAD (flavin adenine dinucleotide) and glutaryl-CoA is essential for decarboxylation reaction followed by dehydrogenation (Fu et al 2004). Sequence analysis shows that residues Arg294, Tyr413 and Glu414 are highly conserved amongst the dehydrogenase family.…”