Production and characterization of recombinant perdeuterated cholesterol oxidase

Golden, Emily; Attwood, Paul V.; Duff, Anthony P.; Meilleur, Flora; Vrielink, Alice

doi:10.1016/j.ab.2015.06.008

Cited by 11 publications

(6 citation statements)

References 51 publications

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“…Crystals appeared within approximately 7 days from drops containing 7 mg/ml cholesterol oxidase (in 20 mM Tris-HCl buffer, pH 7.5), 8% PEG 8 K, 100 mM MnSO 4 and 100 mM sodium cacodylate pH 5.2. Large single crystals of COx were obtained by an iterative macroseeding procedure as described previously57. Crystals were soaked in the crystallization mother liquor containing 100% D 2 O for 24 hours before being mounted in a 1 mm glass capillary and stored for approximately 6 months prior to data collection.…”

Section: Experimental Methodsmentioning

confidence: 99%

An extended N-H bond, driven by a conserved second-order interaction, orients the flavin N5 orbital in cholesterol oxidase

Golden

Meilleur

et al. 2017

Sci Rep

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The protein microenvironment surrounding the flavin cofactor in flavoenzymes is key to the efficiency and diversity of reactions catalysed by this class of enzymes. X-ray diffraction structures of oxidoreductase flavoenzymes have revealed recurrent features which facilitate catalysis, such as a hydrogen bond between a main chain nitrogen atom and the flavin redox center (N5). A neutron diffraction study of cholesterol oxidase has revealed an unusual elongated main chain nitrogen to hydrogen bond distance positioning the hydrogen atom towards the flavin N5 reactive center. Investigation of the structural features which could cause such an unusual occurrence revealed a positively charged lysine side chain, conserved in other flavin mediated oxidoreductases, in a second shell away from the FAD cofactor acting to polarize the peptide bond through interaction with the carbonyl oxygen atom. Double-hybrid density functional theory calculations confirm that this electrostatic arrangement affects the N-H bond length in the region of the flavin reactive center. We propose a novel second-order partial-charge interaction network which enables the correct orientation of the hydride receiving orbital of N5. The implications of these observations for flavin mediated redox chemistry are discussed.

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Section: Experimental Methodsmentioning

confidence: 99%

An extended N-H bond, driven by a conserved second-order interaction, orients the flavin N5 orbital in cholesterol oxidase

Golden

Meilleur

et al. 2017

Sci Rep

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“…There are several studies that have compared the properties, activities and X-ray crystal structures of unlabelled (H/H) and perdeuterated (D/D) versions of the same protein: cholesterol oxidase, haloalkane dehalogenase and arginase I (Golden et al, 2015;Liu et al, 2007;Di Costanzo et al, 2007). These studies all showed minimal structural effects owing to perdeuteration (D/D).…”

Section: Introductionmentioning

confidence: 99%

Structural comparison of protiated, H/D-exchanged and deuterated human carbonic anhydrase IX

Koruza

Lafumat

Nyblom

et al. 2019

Acta Crystallogr D Struct Biol

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Human carbonic anhydrase IX (CA IX) expression is upregulated in hypoxic solid tumours, promoting cell survival and metastasis. This observation has made CA IX a target for the development of CA isoform-selective inhibitors. To enable structural studies of CA IX–inhibitor complexes using X-ray and neutron crystallography, a CA IX surface variant (CA IXSV; the catalytic domain with six surface amino-acid substitutions) has been developed that can be routinely crystallized. Here, the preparation of protiated (H/H), H/D-exchanged (H/D) and deuterated (D/D) CA IXSV for crystallographic studies and their structural comparison are described. Four CA IXSV X-ray crystal structures are compared: two H/H crystal forms, an H/D crystal form and a D/D crystal form. The overall active-site organization in each version is essentially the same, with only minor positional changes in active-site solvent, which may be owing to deuteration and/or resolution differences. Analysis of the crystal contacts and packing reveals different arrangements of CA IXSV compared with previous reports. To our knowledge, this is the first report comparing three different deuterium-labelled crystal structures of the same protein, marking an important step in validating the active-site structure of CA IXSV for neutron protein crystallography.

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“…In pathogenic bacteria ChOx is a membrane‐damaging factor and contributes as a virulence factor in the pathogenicity of these bacteria . The enzyme belongs to the family of flavin‐specific oxidoreductases and is found in two different forms: one with the flavin adenine dinucleotide (FAD) cofactor covalently linked to the protein and one with a non‐covalently linked cofactor . ChOx from bacterial sources is used in a wide range of clinical and industrial applications, such as the clinical determination of cholesterol for the assessment of arteriosclerosis and other lipid disorders.…”

Section: Introductionmentioning

confidence: 99%

Spectroelectrochemical Investigation of Cholesterol Oxidase from Streptomyces lividans at Different pH Values

Heinelt

Nöll

2019

ChemElectroChem

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Cholesterol oxidase from Streptomyces lividans (SlChOx) has been investigated by spectroelectrochemistry in the pH range between pH 5 and pH 9. SlChOx is a monomeric flavoenzyme with a molar mass of about 58 kDa that contains a non‐covalently bound flavin adenine dinucleotide (FAD) cofactor. Spectroelectrochemical measurements were carried out in the presence of different one and two electron redox mediators and methylene blue only, as methylene blue does not contribute significantly to the spectral changes between 350 nm and 500 nm. During the stepwise reduction and reoxidation, the presence of a stable singly reduced flavosemiquinone radical anion was observed. The pH dependent midpoint potentials (Em, pH7≈0 mV) and the redox potentials for the first and second transition Fl(quinone)/Fl(semiquinone rad anion) and Fl(semiquinone rad anion)/Fl(hydroquinone anion) were determined. A pH dependency of about −30 mV and −50 mV per pH unit was determined for the first and second reduction, respectively.

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Production and characterization of recombinant perdeuterated cholesterol oxidase

Cited by 11 publications

References 51 publications

An extended N-H bond, driven by a conserved second-order interaction, orients the flavin N5 orbital in cholesterol oxidase

An extended N-H bond, driven by a conserved second-order interaction, orients the flavin N5 orbital in cholesterol oxidase

Structural comparison of protiated, H/D-exchanged and deuterated human carbonic anhydrase IX

Spectroelectrochemical Investigation of Cholesterol Oxidase from Streptomyces lividans at Different pH Values

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