Crystallization and preliminary X-ray diffraction studies of mitochondrial short-chain Δ3,Δ2-enoyl-CoA isomerase from rat liver

Jp, Zeelen; Pauptit, RA; Rk, Wierenga; Wh, Kunau; Jk, Hiltunen

doi:10.1016/0022-2836(92)90591-7

Cited by 12 publications

(5 citation statements)

References 16 publications

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“…Crystallization trials were carried out using in‐house screens and the experiments were done in IQ 96‐well sitting drop plates (TTP Labtech) using the mosquito LCP nanodispenser (TTP Labtech). Initial hits were obtained only at 4°C with PSB‐II in the absence of peptides using well solutions containing 100 m M MOPS, pH 7.5, 2.45 M ammonium sulfate and 10% DMSO.…”

Section: Methodsmentioning

confidence: 99%

Structural enzymology binding studies of the peptide‐substrate‐binding domain of human collagen prolyl 4‐hydroxylase (type‐II): High affinity peptides have a PxGP sequence motif

et al. 2018

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The peptide-substrate-binding (PSB) domain of collagen prolyl 4-hydroxylase (C-P4H, an α β tetramer) binds proline-rich procollagen peptides. This helical domain (the middle domain of the α subunit) has an important role concerning the substrate binding properties of C-P4H, although it is not known how the PSB domain influences the hydroxylation properties of the catalytic domain (the C-terminal domain of the α subunit). The crystal structures of the PSB domain of the human C-P4H isoform II (PSB-II) complexed with and without various short proline-rich peptides are described. The comparison with the previously determined PSB-I peptide complex structures shows that the C-P4H-I substrate peptide (PPG) , has at most very weak affinity for PSB-II, although it binds with high affinity to PSB-I. The replacement of the middle PPG triplet of (PPG) to the nonhydroxylatable PAG, PRG, or PEG triplet, increases greatly the affinity of PSB-II for these peptides, leading to a deeper mode of binding, as compared to the previously determined PSB-I peptide complexes. In these PSB-II complexes, the two peptidyl prolines of its central P(A/R/E)GP region bind in the Pro5 and Pro8 binding pockets of the PSB peptide-binding groove, and direct hydrogen bonds are formed between the peptide and the side chains of the highly conserved residues Tyr158, Arg223, and Asn227, replacing water mediated interactions in the corresponding PSB-I complex. These results suggest that PxGP (where x is not a proline) is the common motif of proline-rich peptide sequences that bind with high affinity to PSB-II.

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Section: Methodsmentioning

confidence: 99%

Structural enzymology binding studies of the peptide‐substrate‐binding domain of human collagen prolyl 4‐hydroxylase (type‐II): High affinity peptides have a PxGP sequence motif

et al. 2018

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“…The structure of the ∆ 3 -∆ 2 -enoyl-CoA isomerase has not yet been determined (24); therefore, the active site geometry of the isomerase is not known. From isomerase mutagenesis studies of the equivalent of Glu164, it is found that changing this glutamate to a glutamine almost completely abolished the catalytic activity (23).…”

mentioning

confidence: 99%

Mutagenic and Enzymological Studies of the Hydratase and Isomerase Activities of 2-Enoyl-CoA Hydratase-1

Schmitz

et al. 1999

Biochemistry

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Structural and enzymological studies have shown the importance of Glu144 and Glu164 for the catalysis by 2-enoyl-CoA hydratase-1 (crotonase). Here we report about the enzymological properties of the Glu144Ala and Glu164Ala variants of rat mitochondrial 2-enoyl-CoA hydratase-1. Size-exclusion chromatography and CD spectroscopy showed that the wild-type protein and mutants have similar oligomerization states and folding. The kcat values of the active site mutants Glu144Ala and Glu164Ala were decreased about 2000-fold, but the Km values were unchanged. For study of the potential intrinsic Delta3-Delta2-enoyl-CoA isomerase activity of mECH-1, a new assay using 2-enoyl-CoA hydratase-2 and (R)-3-hydroxyacyl-CoA dehydrogenase as auxiliary enzymes was introduced. It was demonstrated that rat wild-type mECH-1 is also capable of catalyzing isomerization with the activity ratio (isomerization/hydration) of 1/5000. The kcat values of isomerization in Glu144Ala and Glu164Ala were decreased 10-fold and 1000-fold, respectively. The data are in line with the proposal that Glu164 acts as a protic amino acid residue for both the hydration and the isomerization reaction. The structural factors favoring the hydratase over the isomerase reaction have been addressed by investigating the enzymological properties of the Gln162Ala, Gln162Met, and Gln162Leu variants. The Gln162 side chain is hydrogen bonded to the Glu164 side chain; nevertheless, these mutants have enzymatic properties similar to that of the wild type, indicating that catalytic function of the Glu164 side chain in the hydratase and isomerase reaction does not depend on the interactions with the Gln162 side chain.

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“…Crystallization trials were carried out in the crystallization facility of the Biocenter Oulu Structural Biology core facility by using the sitting drop vapor diffusion method. Initial screening was done with the wild-type MECR (19.5 mg/ml, in 100 mM sodium phosphate,150 mM NaCl, 1 mM EDTA, 1 mM NaN 3 , pH 7.4 and 10% glycerol) by using the in-house screen 55 and IQ 96-well sitting drop plates (SPT Labtech, Melbourn, Hertfordshire, U.K.) using the Mosquito LCP nanodispenser (SPT Labtech) at two different temperatures (at +4 °C and +22 °C). The plates were imaged by using Formulatrix Rock Imagers RI54, and the crystallization results were monitored with the IceBear software expert system 56 .…”

Section: Methodsmentioning

confidence: 99%

An engineered variant of MECR reductase reveals indispensability of long-chain acyl-ACPs for mitochondrial respiration

et al. 2023

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Mitochondrial fatty acid synthesis (mtFAS) is essential for respiratory function. MtFAS generates the octanoic acid precursor for lipoic acid synthesis, but the role of longer fatty acid products has remained unclear. The structurally well-characterized component of mtFAS, human 2E-enoyl-ACP reductase (MECR) rescues respiratory growth and lipoylation defects of a Saccharomyces cerevisiae Δetr1 strain lacking native mtFAS enoyl reductase. To address the role of longer products of mtFAS, we employed in silico molecular simulations to design a MECR variant with a shortened substrate binding cavity. Our in vitro and in vivo analyses indicate that the MECR G165Q variant allows synthesis of octanoyl groups but not long chain fatty acids, confirming the validity of our computational approach to engineer substrate length specificity. Furthermore, our data imply that restoring lipoylation in mtFAS deficient yeast strains is not sufficient to support respiration and that long chain acyl-ACPs generated by mtFAS are required for mitochondrial function.

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Crystallization and preliminary X-ray diffraction studies of mitochondrial short-chain Δ3,Δ2-enoyl-CoA isomerase from rat liver

Cited by 12 publications

References 16 publications

Structural enzymology binding studies of the peptide‐substrate‐binding domain of human collagen prolyl 4‐hydroxylase (type‐II): High affinity peptides have a PxGP sequence motif

Structural enzymology binding studies of the peptide‐substrate‐binding domain of human collagen prolyl 4‐hydroxylase (type‐II): High affinity peptides have a PxGP sequence motif

Mutagenic and Enzymological Studies of the Hydratase and Isomerase Activities of 2-Enoyl-CoA Hydratase-1

An engineered variant of MECR reductase reveals indispensability of long-chain acyl-ACPs for mitochondrial respiration

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