Structure–function Analysis of Enoyl Thioester Reductase Involved in Mitochondrial Maintenance

Airenne, Tomi T.; Torkko, Juha M.; plas, Sam Van den; Sormunen, Raija; Kastaniotis, Alexander J.; Wierenga, Rik K.; Hiltunen, J. Kalervo

doi:10.1016/s0022-2836(03)00038-x

Cited by 41 publications

(41 citation statements)

References 56 publications

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“…In contrast, in bovine -crystallin, which shows minimal QOR activity, the corresponding residue is a His, suggesting that the Tyr is essential in the kinetic mechanism (51). In addition, mutagenesis and/or structural data support the participation of a Tyr in the mechanism of the related, Zn 2ϩ -lacking MDRs (Table 2) enoyl reductase (39,47), Arabidopsis NADP-dependent oxidoreductase P1, and leukotriene B 4 12-hydroxydehydrogenase (45,46). Thus, it was appropriate to propose a catalytic role for PIG3 Tyr-51, the only Tyr residue in the active-site cavity, further supported by a docking model (Fig.…”

Section: Discussionmentioning

confidence: 86%

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Three-dimensional Structure and Enzymatic Function of Proapoptotic Human p53-inducible Quinone Oxidoreductase PIG3

Porté

Valencia

Yakovtseva

et al. 2009

Journal of Biological Chemistry

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Tumor suppressor p53 regulates the expression of p53-induced genes (PIG) that trigger apoptosis. PIG3 or TP53I3 is the only known member of the medium chain dehydrogenase/reductase superfamily induced by p53 and is used as a proapoptotic marker. Although the participation of PIG3 in the apoptotic pathway is proven, the protein and its mechanism of action were never characterized. We analyzed human PIG3 enzymatic function and found NADPH-dependent reductase activity with ortho-quinones, which is consistent with the classification of PIG3 in the quinone oxidoreductase family. However, the activity is much lower than that of -crystallin, a better known quinone oxidoreductase. In addition, we report the crystallographic structure of PIG3, which allowed the identification of substrate-and cofactor-binding sites, with residues fully conserved from bacteria to human. Tyr-59 in -crystallin (Tyr-51 in PIG3) was suggested to participate in the catalysis of quinone reduction. However, kinetics of Tyr/Phe and Tyr/Ala mutants of both enzymes demonstrated that the active site Tyr is not catalytic but may participate in substrate binding, consistent with a mechanism based on propinquity effects. It has been proposed that PIG3 contribution to apoptosis would be through oxidative stress generation. We found that in vitro activity and in vivo overexpression of PIG3 accumulate reactive oxygen species. Accordingly, an inactive PIG3 mutant (S151V) did not produce reactive oxygen species in cells, indicating that enzymatically active protein is necessary for this function. This supports that PIG3 action is through oxidative stress produced by its enzymatic activity and provides essential knowledge for eventual control of apoptosis.

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Section: Discussionmentioning

confidence: 86%

“…Site-directed Mutagenesis of the Active Site Tyr-In several Zn 2ϩ -independent MDRs, evidence supports a Tyr residue acting as an acid/base catalyst (39,(45)(46)(47). The crystallographic structures suggest that Tyr-51 in PIG3 and Tyr-59 in -crystallin are candidates to perform this catalytic function.…”

Section: Nadpmentioning

confidence: 99%

Three-dimensional Structure and Enzymatic Function of Proapoptotic Human p53-inducible Quinone Oxidoreductase PIG3

Porté

Valencia

Yakovtseva

et al. 2009

Journal of Biological Chemistry

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“…The active site Tyr proposed for MDR homologs such as PDB ID codes 1GUF (35), 1QOR (36), and 2OBY (37) corresponds to F60 in LovC (Fig. S6A).…”

Section: Resultsmentioning

confidence: 99%

Crystal structure and biochemical studies of the trans-acting polyketide enoyl reductase LovC from lovastatin biosynthesis

Ames

Nguyen

Bruegger

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Lovastatin is an important statin prescribed for the treatment and prevention of cardiovascular diseases. Biosynthesis of lovastatin uses an iterative type I polyketide synthase (PKS). LovC is a trans-acting enoyl reductase (ER) that specifically reduces three out of eight possible polyketide intermediates during lovastatin biosynthesis. Such trans-acting ERs have been reported across a variety of other fungal PKS enzymes as a strategy in nature to diversify polyketides. How LovC achieves such specificity is unknown. The 1.9-Å structure of LovC reveals that LovC possesses a medium-chain dehydrogenase/reductase (MDR) fold with a unique monomeric assembly. Two LovC cocrystal structures and enzymological studies help elucidate the molecular basis of LovC specificity, define stereochemistry, and identify active-site residues. Sequence alignment indicates a general applicability to trans-acting ERs of fungal PKSs, as well as their potential application to directing biosynthesis.

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“…4). In general, enoyl thioester reductases constitute a heterologues group with regard to their amino acid sequence but the majority of known enoyl thioester reductases belong to the shortchain dehydrogenases/reductases superfamily of proteins (44). However, two enoyl thioester reductases recently characterized from Candida tropicalis (Etr1p) and Saccharomyces cerevisiae (Mrf1p) were identified that belong to the family of medium chain dehydrogenases/reductases (45,46).…”

Section: Discussionmentioning

confidence: 99%

Mitochondrial trans-2-Enoyl-CoA Reductase of Wax Ester Fermentation from Euglena gracilis Defines a New Family of Enzymes Involved in Lipid Synthesis

Hoffmeister¹,

Piotrowski

Nowitzki³

et al. 2005

Journal of Biological Chemistry

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Under anaerobiosis, Euglena gracilis mitochondria perform a malonyl-CoA independent synthesis of fatty acids leading to accumulation of wax esters, which serve as the sink for electrons stemming from glycolytic ATP synthesis and pyruvate oxidation. An important enzyme of this unusual pathway is trans-2-enoyl-CoA reductase (EC 1.3.1.44), which catalyzes reduction of enoyl-CoA to acyl-CoA. Trans-2-enoyl-CoA reductase from Euglena was purified 1700-fold to electrophoretic homogeneity and was active with NADH and NADPH as the electron donor. The active enzyme is a monomer with molecular mass of 44 kDa. The amino acid sequence of tryptic peptides determined by electrospray ionization mass spectrometry were used to clone the corresponding cDNA, which encoded a polypeptide that, when expressed in Escherichia coli and purified by affinity chromatography, possessed trans-2-enoyl-CoA reductase activity close to that of the enzyme purified from Euglena. Trans-2-enoyl-CoA reductase activity is present in mitochondria and the mRNA is expressed under aerobic and anaerobic conditions. Using NADH, the recombinant enzyme accepted crotonyl-CoA (k m ‫؍‬ 68 M) and trans-2-hexenoyl-CoA (k m ‫؍‬ 91 M). In the crotonyl-CoA-dependent reaction, both NADH (k m ‫؍‬ 109 M) or NADPH (k m ‫؍‬ 119 M) were accepted, with 2-3-fold higher specific activities for NADH relative to NADPH. Trans-2-enoyl-CoA reductase homologues were not found among other eukaryotes, but are present as hypothetical reading frames of unknown function in sequenced genomes of many proteobacteria and a few Gram-positive eubacteria, where they occasionally occur next to genes involved in fatty acid and polyketide biosynthesis. Trans-2-enoyl-CoA reductase assigns a biochemical activity, NAD(P)H-dependent acyl-CoA synthesis from enoylCoA, to one member of this gene family of previously unknown function.

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Structure–function Analysis of Enoyl Thioester Reductase Involved in Mitochondrial Maintenance

Cited by 41 publications

References 56 publications

Three-dimensional Structure and Enzymatic Function of Proapoptotic Human p53-inducible Quinone Oxidoreductase PIG3

Three-dimensional Structure and Enzymatic Function of Proapoptotic Human p53-inducible Quinone Oxidoreductase PIG3

Crystal structure and biochemical studies of the trans-acting polyketide enoyl reductase LovC from lovastatin biosynthesis

Mitochondrial trans-2-Enoyl-CoA Reductase of Wax Ester Fermentation from Euglena gracilis Defines a New Family of Enzymes Involved in Lipid Synthesis

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