Human Peroxisomal Multifunctional Enzyme Type 2

Self Cite

107

2-Enoyl-CoA hydratase 2, a part from multifunctional enzyme type 2, hydrates trans-2-enoyl-CoA to 3-hydroxyacyl-CoA in the (3R)-hydroxy-dependent route of peroxisomal ␤-oxidation of fatty acids. Unliganded and (3R)-hydroxydecanoyl coenzyme A-complexed crystal structures of 2-enoyl-CoA hydratase 2 from Candida tropicalis multifunctional enzyme type 2 were solved to 1.95-and 2.35-Å resolution, respectively. 2-Enoyl-CoA hydratase 2 is a dimeric, ␣؉␤ protein with a novel quaternary structure. The overall structure of the two-domain subunit of eukaryotic 2-enoyl-CoA hydratase 2 resembles the homodimeric, hot dog fold structures of prokaryotic (R)-specific 2-enoyl-CoA hydratase and ␤-hydroxydecanoyl thiol ester dehydrase. Importantly, though, the eukaryotic hydratase 2 has a complete hot dog fold only in its C-domain, whereas the N-domain lacks a long central ␣-helix, thus creating space for bulkier substrates in the binding pocket and explaining the observed difference in substrate preference between eukaryotic and prokaryotic enzymes. Although the N-and C-domains have an identity of <10% at the amino acid level, they share a 50% identity at the nucleotide level and fold similarly. We suggest that a subunit of 2-enoylCoA hydratase 2 has evolved via a gene duplication with the concomitant loss of one catalytic site. The hydrogen bonding network of the active site of 2-enoyl-CoA hydratase 2 resembles the active site geometry of mitochondrial (S)-specific 2-enoyl-CoA hydratase 1, although in a mirror image fashion. This arrangement allows the reaction to occur by similar mechanism, supported by mutagenesis and mechanistic studies, although via reciprocal stereochemistry.

Section: Resultsmentioning

confidence: 59%

Section: Multiple Sequence Alignment Of Eukaryotic Hydratase 2 Has Rementioning

confidence: 99%

Section: Multiple Sequence Alignment Of Eukaryotic Hydratase 2 Has Rementioning

confidence: 99%

See 1 more Smart Citation

A Two-domain Structure of One Subunit Explains Unique Features of Eukaryotic Hydratase 2

Koski

Haapalainen

Hiltunen

et al. 2004

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107

“…Hydration by both types of hydratases, hence, occurs with reciprocal stereochemistry due to mirror-image active centers (31). Multiple sequence alignments of the hydratase 2 domains from MFE-2 and eukaryotic and prokaryotic fatty acid synthases revealed the conserved consensus sequence (Y/F)X 1,2 (L/V/I/G)(S/T/G/ C)GDXNP(L/I/V)HX 5 (A/S) (35) with the catalytic residues shown in bold. The remaining conserved amino acids likely stabilize the structure with hydrophobic and hydrogen bond interactions (33).…”

Section: Discussionmentioning

confidence: 99%

Studies on the Mechanism of Ring Hydrolysis in Phenylacetate Degradation

Teufel¹,

Gantert²,

Voss³

et al. 2011

115

The widespread, long sought-after bacterial aerobic phenylalanine/phenylacetate catabolic pathway has recently been elucidated. It proceeds via coenzyme A (CoA) thioesters and involves the epoxidation of the aromatic ring of phenylacetylCoA, subsequent isomerization to an uncommon seven-membered C-O-heterocycle (oxepin-CoA), and non-oxygenolytic ring cleavage. Here we characterize the hydrolytic oxepin-CoA ring cleavage catalyzed by the bifunctional fusion protein PaaZ. The enzyme consists of a C-terminal (R)-specific enoyl-CoA hydratase domain (formerly MaoC) that cleaves the ring and produces a highly reactive aldehyde and an N-terminal NADP ؉ -dependent aldehyde dehydrogenase domain that oxidizes the aldehyde to 3-oxo-5,6-dehydrosuberyl-CoA. In many phenylacetate-utilizing bacteria, the genes for the pathway exist in a cluster that contains an NAD ؉ -dependent aldehyde dehydrogenase in place of PaaZ, whereas the aldehyde-producing hydratase is encoded outside of the cluster. If not oxidized immediately, the reactive aldehyde condenses intramolecularly to a stable cyclic derivative that is largely prevented by PaaZ fusion in vivo. Interestingly, the derivative likely serves as the starting material for the synthesis of antibiotics (e.g. tropodithietic acid) and other tropone/tropolone related compounds as well as for -cycloheptyl fatty acids. Apparently, bacteria made a virtue out of the necessity of disposing the dead-end product with ring hydrolysis as a metabolic branching point.Aromatics like phenylacetic acid constitute the second most abundant class of natural organic compounds that serve as substrates mainly for microorganisms. Oxygen availability is key to how bacteria utilize such inert substrates (1). Under aerobic conditions oxygen is used to hydroxylate and cleave the ring (2, 3). In contrast, under anaerobic conditions the inert substrates become activated to CoA-thioesters followed by shortening of the side chain and energy-driven ring reduction; furthermore, ring cleavage occurs hydrolytically rather than by oxygenation. This strategy also applies to anaerobic phenylacetate catabolism (Ref. 4 and literature cited therein) (Fig. 1A).Phenylacetate (I) is a key intermediate in the degradation of various substrates like phenylalanine, lignin-related aromatic compounds, or environmental contaminants (5, 6). The first studies from more than 20 years ago reported the induction of a phenylacetate-CoA ligase under aerobic conditions in Pseudomonas putida, suggesting an unconventional strategy for aerobic phenylacetic acid (Paa) 2 degradation (7, 8). A total of 14 genes in 3 transcriptional units were identified in the corresponding paa gene cluster (9). However, the underlying biochemical conversions remained obscure until they were recently elucidated in Escherichia coli K12 and Pseudomonas sp. strain Y2 (10). This novel metabolic strategy involves the usage of oxygen as well as CoA-thioester intermediates throughout the pathway, hence, showing typical features of aerobic as well as anaerobic strategies...

“…2A presents the alignment of AtECH2 with the monofunctional enoyl-CoA hydratase 2 from A. punctata (ApPHAJ) and P. aeruginosa (PaPHAJ2), and the hydratase domain of the MFE-2 from human (HsMFE2), S. cerevisiae (ScFox2p), and Candida tropicalis (CtMFE2). Multiple sequence alignment of eukaryotic enoyl-CoA hydratase 2 has previously revealed a conserved region showing a motif (Y/F)X 1,2 (L/V/I/G)(S/T/G/C)GDX-NP(L/I/V)HX 5 (AS) (35), called the hydratase 2 motif. This motif was found in AtECH2 between amino acids 203 and 219 (upper bracket in Fig.…”

Section: Identification Of a Candidate Gene Encoding An Enoyl-coamentioning

confidence: 99%

Identification and Functional Characterization of a Monofunctional Peroxisomal Enoyl-CoA Hydratase 2 That Participates in the Degradation of Even cis-Unsaturated Fatty Acids in Arabidopsis thaliana

Goepfert

Hiltunen

Poirier

2006

A gene, named AtECH2, has been identified in Arabidopsis thaliana to encode a monofunctional peroxisomal enoyl-CoA hydratase 2. Homologues of AtECH2 are present in several angiosperms belonging to the Monocotyledon and Dicotyledon classes, as well as in a gymnosperm. In vitro enzyme assays demonstrated that AtECH2 catalyzed the reversible conversion of 2E-enoyl-CoA to 3R-hydroxyacyl-CoA. AtECH2 was also demonstrated to have enoyl-CoA hydratase 2 activity in an in vivo assay relying on the synthesis of polyhydroxyalkanoate from the polymerization of 3R-hydroxyacyl-CoA in the peroxisomes of Saccharomyces cerevisiae. AtECH2 contained a peroxisome targeting signal at the C-terminal end, was addressed to the peroxisome in S. cerevisiae, and a fusion protein between AtECH2 and a fluorescent protein was targeted to peroxisomes in onion cells. AtECH2 gene expression was strongest in tissues with high ␤-oxidation activity, such as germinating seedlings and senescing leaves. The contribution of AtECH2 to the degradation of unsaturated fatty acids was assessed by analyzing the carbon flux through the ␤-oxidation cycle in plants that synthesize peroxisomal polyhydroxyalkanoate and that were over-or underexpressing the AtECH2 gene. These studies revealed that AtECH2 participates in vivo to the conversion of the intermediate 3R-hydroxyacyl-CoA, generated by the metabolism of fatty acids with a cis (Z)-unsaturated bond on an even-numbered carbon, to the 2E-enoyl-CoA for further degradation through the core ␤-oxidation cycle.The peroxisome is the site of numerous important biochemical reactions in plants, including photorespiration, the ␤-oxidation cycle, and the glyoxylate cycle. Although several enzymes involved in these pathways have been identified, analysis of the plant proteome for proteins possessing putative peroxisome targeting sequences have identified numerous candidate peroxisomal proteins for which no functions have been assigned (1). Furthermore, prediction of peroxisomal proteins can be made difficult by the absence of recognizable signal peptide, particularly for peroxisomal membrane protein. Thus, our present knowledge of the complexity of the biochemical pathways present in the peroxisome is fragmented.The peroxisomal ␤-oxidation cycle is of primary importance during seedling establishment following germination, because it is responsible for the breakdown of fatty acids into acetylCoA, which is subsequently converted to glucose via the glyoxylate cycle and gluconeogenesis (2). Although fatty acid ␤-oxidation is very active during germination in oleaginous seed and during senescence, this cycle is also present in mature photosynthetic tissues, such as leaves, as well as in developing seeds (3).Degradation of saturated fatty acids in the peroxisome occurs via four enzyme activities located on three proteins that form the core ␤-oxidation pathway. The first step is mediated by an acyl-CoA oxidase, converting acyl-CoA to 2E-enoyl-CoA. This is followed by the hydration of the 2E-enoyl-CoA to 3-hydroxyacyl-CoA by an...