Pig heart short chain L‐3‐hydroxyacyl‐CoA dehydrogenase revisited: Sequence analysis and crystal structure determination

Abstract

Short chain L-3-hydroxyacyl CoA dehydrogenase (SCHAD) is a soluble dimeric enzyme critical for oxidative metabolism of fatty acids. Its primary sequence has been reported to be conserved across numerous tissues and species with the notable exception of the pig heart homologue. Preliminary efforts to solve the crystal structure of the dimeric pig heart SCHAD suggested the unprecedented occurrence of three enzyme subunits within the asymmetric unit, a phenomenon that was thought to have hampered refinement of the initial chain tracing. The recently solved crystal coordinates of human heart SCHAD facilitated a molecular replacement solution to the pig heart SCHAD data. Refinement of the model, in conjunction with the nucleotide sequence for pig heart SCHAD determined in this paper, has demonstrated that the previously published pig heart SCHAD sequence was incorrect. Presented here are the corrected amino acid sequence and the high resolution crystal structure determined for pig heart SCHAD complexed with its NAD+ cofactor (2.8 A; R(cryst) = 22.4%, R(free) = 28.8%). In addition, the peculiar phenomenon of a dimeric enzyme crystallizing with three subunits contained in the asymmetric unit is described.

Structural Studies of MFE-1: the 1.9Å Crystal Structure of the Dehydrogenase Part of Rat Peroxisomal MFE-1

“…So far, crystallization experiments with this monomeric multidomain enzyme have been unsuccessful, and consequently attempts were initiated to dissect the full-length enzyme into stable, functional parts while retaining its enzymatic activity. Sequence comparisons, using sequences of enzymes in the hydratase/isomerase (crotonase) superfamily 21 and of mammalian mitochondrial L-3-hydroxyacyl-CoAdehydrogenases (HAD), 22,23 have suggested the presence of five domains in MFE-1, referred to as A, B, C, D, and E. 24 At the N terminus of MFE-1, the sequence analysis suggested a domain of the hydratase/isomerase fold (domain A), followed by a linker region (domain B) leading to a domain (domain C) that was predicted to be the dinucleotidebinding domain of the dehydrogenase part. This Rossmann-fold domain is followed by domain D, which corresponds to the dimerization domain of the human monofunctional dehydrogenase.…”

Organization of the multifunctional enzyme type 1: interaction between N- and C-terminal domains is required for the hydratase-1/isomerase activity

“…The middle part of the perMFE-1 sequence (HAD part of MFE-1, mfeHAD) is related to the mitochondrial NAD +dependent HADs [18], the sequence identity being 34 % with both pig and human-soluble mitochondrial HADs. Previously published structures of human and pig heart homodimeric HADs [19,20] show that they are composed of two identical subunits, each having two domains. The larger N-terminal domain contains the Rossmann fold [21] with the dinucleotide-binding βαβ-unit [22], whereas the smaller, mostly α-helical, domain mediates subunit-subunit interactions.…”

Crystal Structure of Liganded Rat Peroxisomal Multifunctional Enzyme Type 1

“…The founding member of this superfamily is the human mitochondrial (3S)-hydroxyacyl-CoA dehydrogenase, which is also referred to as the short chain hmHAD (27). Also, the structure of the porcine homologue is known (28). Interestingly, the crotonase fold and the HAD fold (domains C and D) are also present in the ␣-chain of the bacterial Pseudomonas fragi fatty acid oxidation multienzyme ␣ 2 ␤ 2 complex (pfFOM), whose crystal structure has been determined by Morikawa and co-workers (25).…”