Critical Residues for Structure and Catalysis in Short-chain Dehydrogenases/Reductases

Filling, Charlotta; Berndt, Kurt D.; Benach, J.; Knapp, Stefan; Prozorovski, Timour; Nordling, Erik; Ladenstein, Rudolf; Jörnvall, Hans; Oppermann, U.

doi:10.1074/jbc.m202160200

Cited by 521 publications

(559 citation statements)

References 25 publications

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“…family, containing a typical Rossmann-fold scaffold for NAD ϩ binding and a Ser-Tyr-Lys triad for catalysis (47). The hydrophobic cavity leading to the active site also appears to be a common feature among members of the short-chain alcohol dehydrogenase/reductase family.…”

Section: Discussionmentioning

confidence: 99%

Identification of a Substrate-binding Site in a Peroxisomal β-Oxidation Enzyme by Photoaffinity Labeling with a Novel Palmitoyl Derivative

Kashiwayama

Tomohiro

Narita

et al. 2010

Journal of Biological Chemistry

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Peroxisomes play an essential role in a number of important metabolic pathways including ␤-oxidation of fatty acids and their derivatives. Therefore, peroxisomes possess various ␤-oxidation enzymes and specialized fatty acid transport systems. However, the molecular mechanisms of these proteins, especially in terms of substrate binding, are still unknown. In this study, to identify the substrate-binding sites of these proteins, we synthesized a photoreactive palmitic acid analogue bearing a diazirine moiety as a photophore, and performed photoaffinity labeling of purified rat liver peroxisomes. As a result, an 80-kDa peroxisomal protein was specifically labeled by the photoaffinity ligand, and the labeling efficiency competitively decreased in the presence of palmitoyl-CoA. Mass spectrometric analysis identified the 80-kDa protein as peroxisomal multifunctional enzyme type 2 (MFE2), one of the peroxisomal ␤-oxidation enzymes. Recombinant rat MFE2 was also labeled by the photoaffinity ligand, and mass spectrometric analysis revealed that a fragment of rat MFE2 (residues Trp 249 to Arg 251 ) was labeled by the ligand. MFE2 mutants bearing these residues, MFE2(W249A) and MFE2(R251A), exhibited decreased labeling efficiency. Furthermore, MFE2(W249G), which corresponds to one of the disease-causing mutations in human MFE2, also exhibited a decreased efficiency. Based on the crystal structure of rat MFE2, these residues are located on the top of a hydrophobic cavity leading to an active site of MFE2. These data suggest that MFE2 anchors its substrate around the region from Trp 249 to Arg 251 and positions the substrate along the hydrophobic cavity in the proper direction toward the catalytic center.Peroxisomes are organelles bound by a single membrane, which are present in almost all eukaryotic cells. Peroxisomes are involved in a variety of important metabolic processes including the ␤-oxidation of fatty acids, synthesis of plasmalogen, and bile acids (1). A specialized set of enzymes responsible for the peroxisomal functions are compartmentalized in the organelle, and the deficiency of peroxisomal enzymes causes severe metabolic disease such as acyl-CoA oxidase deficiency and D-bifunctional protein deficiency (2-4). Fibroblasts from patients with these deficiencies exhibit the disturbed peroxisomal ␤-oxidation of very long-chain fatty acids (VLCFAs), 4 and VLCFAs are accumulated in the plasma and tissues of these patients (5). Analyses of these peroxisomal diseases reflect the importance of the peroxisomal ␤-oxidation in organisms along with disclosing the enzymatic organization of the peroxisomal ␤-oxidation system. Peroxisomes are involved in the ␤-oxidation of VLCFAs, branched chain fatty acids such as pristanic acid, and bile acid intermediates such as dihydroxycholestanoic acid and trihydroxycholestanoic acid, which are incompatible with mitochondrial ␤-oxidation (6). Peroxisomal ␤-oxidation of fatty acids proceeds via a four-step pathway as in mitochondria, and multiple enzymes are involved in each step of the...

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Section: Discussionmentioning

confidence: 99%

Identification of a Substrate-binding Site in a Peroxisomal β-Oxidation Enzyme by Photoaffinity Labeling with a Novel Palmitoyl Derivative

Kashiwayama

Tomohiro

Narita

et al. 2010

Journal of Biological Chemistry

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“…With the success of the aromatase inhibitors in treating hormone-sensitive breast tumors [21,23], the characterization of a new target enzyme in this biosynthetic pathway enhances our ability to develop new treatments for breast cancer. (A) Docking of trilostane with our structural model of human wild-type 3β-HSD1 shows the predicted interaction between the 2α-cyanogroup of trilostane and Ser124 residue of the wildtype enzyme (3.4 Å in yellow).…”

Section: Discussionmentioning

confidence: 99%

Structure/function of the inhibition of human 3β-hydroxysteroid dehydrogenase type 1 and type 2 by trilostane

Thomas¹,

Mack²,

Glow³

et al. 2008

The Journal of Steroid Biochemistry and Molecular Biology

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The human type 1 (placenta, breast tumors) and type 2 (gonads, adrenals) isoforms of 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD) are key enzymes in biosynthesis of all active steroid hormones. Human 3β-HSD1 is a critical enzyme in the conversion of DHEA to estradiol in breast tumors and may be a major target enzyme for the treatment of breast cancer. 3β-HSD2 participates in the production of cortisol and aldosterone in the human adrenal gland. The goals of this project are to evaluate the role of the 2α-cyano group on trilostane (2α-cyano-4α,5α-epoxy-17β-ol-androstane-3-one) and determine which amino acids may be critical for 3β-HSD1 specificity. Trilostane without the 2α-cyano group, 4α,5α-epoxy-testosterone, was synthesized. Using our structural model of 3β-HSD1, trilostane or 4α,5α-epoxy-testosterone was docked in the active site using Autodock 3.0, and the potentially critical residues (Met187 and Ser124) were identified. The M187T and S124T mutants of 3β-HSD1 were created, expressed and purified. Dixon analyses of the inhibition of wild-type 3β-HSD1, 3β-HSD2, M187T and S124T by trilostane and 4α,5α-epoxy-testosterone suggest that the 2α-cyano group of trilostane is anchored by Ser124 in both isoenzymes. Kinetic analyses of cofactor and substrate utilization as well as the inhibition kinetics of M187T and the wild-type enzymes suggest that the 16-fold higher-affinity inhibition of 3β-HSD1 by trilostane may be related to the presence of Met187 in 3β-HSD1 and Thr187 in 3β-HSD2. This structure/function information may lead to the production of more highly specific inhibitors of 3β-HSD1 to block the hormone-dependent growth of breast tumors.

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“…In both SDRs, the catalytic center is positioned in a single domain structure, represented by a triad of amino acid residues at Ser 138 , Tyr 151 and Lys 155 (Jörnvall et al, 1995(Jörnvall et al, , 1999Benach et al, 2001;Filling et al, 2002). In the deduced amino acid sequence of honey bee SDR we find such a constellation at Ser 148 , Tyr 162 and Lys 166 , within motif 1 detected by MEME/MAST algorithms (Grundy et al, 1997).…”

Section: Discussionmentioning

confidence: 86%

“…Besides the catalytic triad, we also detected the putative NAD(H) coenzyme binding site of Apis mellifera SDR at Thr 13 , Asp 65 , Asn 91 , Asn 92 and Ala 93 , corresponding to that of the bacterian SDR (Thr 12 , Asp 60 , Asn 86 , Asn 87 and Ala 88 ). In honey bee SDR, Thr 13 is located within motif 2 detected by MEME/ MAST analysis, just one position upstream from the GXXXGXG sequence which constitutes another conserved element in SDR sequences (Jörnvall et al, 1995(Jörnvall et al, , 1999Filling et al, 2002). In D. melanogaster ADH, the corresponding motif is GXGGXG, which is more glycin-rich and thus more similar to medium-chain (MDR) alcohol dehydrogenases ).…”

Section: Discussionmentioning

confidence: 99%

A member of the short-chain dehydrogenase/reductase (SDR) superfamily is a target of the ecdysone response in honey bee (Apis mellifera) caste development

2004

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-Many aspects in caste polyphenism result from hormonally controlled differential gene expression. A DDRT-PCR screen for ecdysteroid-regulated genes in ovaries revealed a set of ESTs coding for metabolic enzymes. For a cDNA encoding a short-chain dehydrogenase/reductase (SDR) we obtained the complete coding sequence (246 amino acids), revealing the protein motifs typical of insect SDRs. Its initially high expression in early fifth-instar larvae vanished in prepupae. Expression levels in worker larvae were higher than in queen larvae, suggesting negative regulation by the caste-specific ecdysteroid titer. This finding was confirmed by in vitro exposure of competent worker ovaries to makisterone A. In contrast to whole body RNA extracts, two SDR transcripts were detected in the ovaries. Both had their expression downregulated by makisterone A. Hormonal regulation and tissue-specific expression pattern makes this SDR an interesting enzyme for comparative molecular studies on social insect caste polyphenisms.Apis mellifera / differential display PCR / ecdysteroid / alcohol dehydrogenase / caste polyphenism

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Critical Residues for Structure and Catalysis in Short-chain Dehydrogenases/Reductases

Cited by 521 publications

References 25 publications

Identification of a Substrate-binding Site in a Peroxisomal β-Oxidation Enzyme by Photoaffinity Labeling with a Novel Palmitoyl Derivative

Identification of a Substrate-binding Site in a Peroxisomal β-Oxidation Enzyme by Photoaffinity Labeling with a Novel Palmitoyl Derivative

Structure/function of the inhibition of human 3β-hydroxysteroid dehydrogenase type 1 and type 2 by trilostane

A member of the short-chain dehydrogenase/reductase (SDR) superfamily is a target of the ecdysone response in honey bee (Apis mellifera) caste development

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