The UbiK protein is an accessory factor necessary for bacterial ubiquinone (UQ) biosynthesis and forms a complex with the UQ biogenesis factor UbiJ

Loiseau, Laurent; Fyfe, Cameron David; Aussel, Laurent; Chehade, Mahmoud Hajj; Hernández, Sara B.; Faivre, Bruno; Hamdane, Djemel; Mellot‐Draznieks, Caroline; Rascalou, Bérengère; Pélosi, Ludovic; Velours, Christophe; Cornu, David; Lombard, Murielle; Casadesús, Josep; Pierrel, Fabien; Fontecave, Marc; Barras, Frédéric

doi:10.1074/jbc.m117.789164

Cited by 36 publications

(77 citation statements)

References 40 publications

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“…SEC-MALS data analysis using the ''UV extinction from RI peak'' method in the ASTRA software (28) indicated an experimental extinction coefficient for the Nter-BsRNaseY/Fab RY79-90 complex of 1.09 mL , mg À1 cm À1 , close to the theoretical extinction coefficient for a complex with a stoichiometry of 1 Fab per one Nter-BsRNaseY monomer (1.103 mL , mg À1 cm À1 ). Moreover, given that the extinction coefficients for Nter-BsRNaseY and Fab RY79-90 are significantly different, the protein conjugate method could also be used to determine the diffusion signal corresponding to each protein (29). The deduced molar masses of Fab and Nter-BsRNaseY in the complex were 48.2 and 22.0 kDa, respectively, confirming the 1:1 stoichiometry (total mass of 70.2 kDa).…”

Section: One Fab Binds To One Nter-bsrnasey Monomermentioning

confidence: 78%

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Dissociation of the Dimer of the Intrinsically Disordered Domain of RNase Y upon Antibody Binding

Hardouin

Velours

Bou‐Nader

et al. 2018

Biophysical Journal

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Although RNase Y acts as the key enzyme initiating messenger RNA decay in Bacillus subtilis and likely in many other Gram-positive bacteria, its three-dimensional structure remains unknown. An antibody belonging to the rare immunoglobulin G (IgG) 2b lx isotype was raised against a 12-residue conserved peptide from the N-terminal noncatalytic domain of B. subtilis RNase Y (BsRNaseY) that is predicted to be intrinsically disordered. Here, we show that this domain can be produced as a stand-alone protein called Nter-BsRNaseY that undergoes conformational changes between monomeric and dimeric forms. Circular dichroism and size exclusion chromatography coupled with multiangle light scattering or with small angle x-ray scattering indicate that the Nter-BsRNaseY dimer displays an elongated form and a high content of a-helices, in agreement with the existence of a central coiled-coil structure appended with flexible ends, and that the monomeric state of Nter-BsRNaseY is favored upon binding the fragment antigen binding (Fab) of the antibody. The dissociation constants of the IgG/BsRNaseY, IgG/Nter-BsRNaseY, and IgG/peptide complexes indicate that the affinity of the IgG for Nter-BsRNaseY is in the nM range and suggest that the peptide is less accessible in BsRNaseY than in Nter-BsRNaseY. The crystal structure of the Fab in complex with the peptide antigen shows that the peptide adopts an elongated U-shaped conformation in which the unique hydrophobic residue of the peptide, Leu6, is completely buried. The peptide/Fab complex may mimic the interaction of a microdomain of the N-terminal domain of BsRNaseY with one of its cellular partners within the degradosome complex. Altogether, our results suggest that BsRNaseY may become accessible for protein interaction upon dissociation of its N-terminal domain into the monomeric form. FIGURE 1 Characterization of a monoclonal antibody induced against a 12-residue peptide of Nter-BsRNaseY. (A) Sequence alignment of the N-terminal domains of RNases Y from B. subtilis, Staphylococcus aureus, Streptococcus pyogenes, Listeria monocytogenes, Clostridium perfringens, Lactoccus lactis, Borrelia burgdorferi, and Leadbetterella byssophila was performed with Clustal Omega (67) and rendered with ESPript (68). The predicted N-terminal TM helix (residues 6-24) and the inducing peptide (residues 79-90) are indicated. (B) Recognition of BsRNaseY by IgG RY79-90 as detected by immunoblotting (legend continued on next page) Fab Binding Dissociates RNase Y IDD Biophysical Journal 115, 2102-2113, December 4, 2018 2103Protein stoichiometry analysis of the Nter-BsRNaseY/Fab RY79-90 complex (at a 1:2 ratio) was performed by SEC-MALS using the ''UV extinction from RI peak'' method (28) and the protein conjugate method (29) (see Supporting Materials and Methods). In the first method, the UV 280 extinction coefficients of the peaks are extracted from the SEC-MALS data and compared to the predicted extinction coefficients Fab Binding Dissociates RNase Y IDD (C) Structural models of Nter-BsRNaseY. Top: Two at...

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Section: One Fab Binds To One Nter-bsrnasey Monomermentioning

confidence: 78%

“…as described previously (28,29). M w is the weight-averaged molar mass measured, M N is the theoretical molar mass of Nter-BsRNaseY (20,665 Da), and [M] T is the molar concentration of the monomer determined by the refractive index detector.…”

Section: Determination Of the Dissociation Constant Of The Nter-bsrnamentioning

confidence: 99%

Dissociation of the Dimer of the Intrinsically Disordered Domain of RNase Y upon Antibody Binding

Hardouin

Velours

Bou‐Nader

et al. 2018

Biophysical Journal

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“…Neither Coq10p nor Coq9p has a known homolog in E. coli . However, the recently discovered E. coli CoQ biosynthesis proteins UbiJ and UbiK, which physically bind each other and a lipid [143, 144], could be functional homologs of Coq10p or Coq9p.…”

Section: Uncharacterized Proteins In Coq Biosynthesismentioning

confidence: 99%

Biochemistry of Mitochondrial Coenzyme Q Biosynthesis

Stefely

Pagliarini

2017

Trends in Biochemical Sciences

262

290

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Coenzyme Q (CoQ, ubiquinone) is a redox active lipid produced across all domains of life that functions in electron transport and oxidative phosphorylation and whose deficiency causes human diseases. Yet, CoQ biosynthesis has not been fully defined in any organism. Several proteins with unclear molecular functions facilitate CoQ biosynthesis through unknown means, and multiple steps in the pathway are catalyzed by currently unidentified enzymes. Here we highlight recent progress toward filling these knowledge gaps through both traditional biochemistry and cutting-edge “omics” approaches. To help fill the remaining gaps, we present questions framed by the recently discovered CoQ biosynthetic complex and by putative biophysical barriers. Mapping CoQ biosynthesis, metabolism, and transport pathways has great potential to enhance treatment of numerous human diseases.

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“…We recently showed that the purified UbiJ-UbiK heterocomplex binds palmitoleic acid (Loiseau et al, 2017), one of the most abundant fatty acid in E. coli. Overexpression of UbiJ-K caused a decrease in cellular UQ8 levels and the accumulation of OPP and DMQ8 ( Figure 4C and S3C), suggesting the sequestration of these UQ8 biosynthetic intermediates by UbiJ-K. We then purified the UbiJ-K complex ( Figure S3D) using affinity chromatography and size-exclusion chromatography, and analyzed its quinone content by HPLC-ECD-MS ( Figure S3E).…”

Section: The Scp2 Domain Of Ubij Binds Isoprenoid Lipidsmentioning

confidence: 99%

A Soluble Metabolon Synthesizes the Isoprenoid Lipid Ubiquinone

et al. 2018

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Ubiquinone (UQ) is a polyprenylated lipid that is conserved from bacteria to humans and is crucial to cellular respiration. How the cell orchestrates the efficient synthesis of UQ, which involves the modification of extremely hydrophobic substrates by multiple sequential enzymes, remains an unresolved issue. Here, we demonstrate that seven Ubi proteins form the Ubi complex, a stable metabolon that catalyzes the last six reactions of the UQ biosynthetic pathway in Escherichia coli. The SCP2 domain of UbiJ forms an extended hydrophobic cavity that binds UQ intermediates inside the 1 MDa Ubi complex. We purify the Ubi complex from cytoplasmic extracts and demonstrate that UQ biosynthesis occurs in this fraction, challenging the current thinking of a membrane-associated biosynthetic process. Collectively, our results document a rare case of stable metabolon and highlight how the supramolecular organization of soluble enzymes allows the modification of hydrophobic substrates in a hydrophilic environment. Besides the Ubi enzymes that catalyze the chemical reactions of the pathway (Figure 1A), three Ubi proteins of unknown function are also required for efficient UQ biosynthesis, UbiB (Poon et al., 2000), UbiJ (Aussel et al., 2014a) and UbiK (Loiseau et al., 2017). Lately, we demonstrated that purified UbiJ and UbiK form a 1:2 heterocomplex that binds palmitoleic acid, one of E. coli most abundant fatty acid (Loiseau et al., 2017). This capacity to bind lipids suggested that UbiJ and UbiK may act as accessory factors of Ubi enzymes, but the role of these two proteins and of UbiB, although central for UQ

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The UbiK protein is an accessory factor necessary for bacterial ubiquinone (UQ) biosynthesis and forms a complex with the UQ biogenesis factor UbiJ

Cited by 36 publications

References 40 publications

Dissociation of the Dimer of the Intrinsically Disordered Domain of RNase Y upon Antibody Binding

Dissociation of the Dimer of the Intrinsically Disordered Domain of RNase Y upon Antibody Binding

Biochemistry of Mitochondrial Coenzyme Q Biosynthesis

A Soluble Metabolon Synthesizes the Isoprenoid Lipid Ubiquinone

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